WO2022126721A1 - Method for detecting target nucleic acid sequence at high specificity - Google Patents

Method for detecting target nucleic acid sequence at high specificity Download PDF

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WO2022126721A1
WO2022126721A1 PCT/CN2020/140053 CN2020140053W WO2022126721A1 WO 2022126721 A1 WO2022126721 A1 WO 2022126721A1 CN 2020140053 W CN2020140053 W CN 2020140053W WO 2022126721 A1 WO2022126721 A1 WO 2022126721A1
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sequence
mediator
probe
nucleic acid
hairpin
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PCT/CN2020/140053
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French (fr)
Chinese (zh)
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李庆阁
杜琛
廖逸群
许晔
周淑娟
宋甲宝
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厦门大学
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Publication of WO2022126721A1 publication Critical patent/WO2022126721A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6832—Enhancement of hybridisation reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present application relates to multiplex detection of nucleic acid molecules.
  • the present application provides a method for detecting a target nucleic acid sequence, which is capable of simultaneously detecting the presence of multiple target nucleic acid sequences in a sample with high specificity.
  • the present application also provides a probe set, and a kit comprising one or more of the probe sets, which can be used to implement the methods of the present invention.
  • Real-time fluorescent PCR is a common method for nucleic acid detection. It is easy to operate and widely used. At the same time, as a closed-tube detection mode, the chance of contamination of amplification products is low. Multiplex real-time PCR can simultaneously detect multiple target sequences in a single reaction tube. , not only the detection efficiency is improved, but the cost is further reduced.
  • the detection principle of real-time fluorescent PCR is based on the specific binding of fluorescently labeled oligonucleotide probes to target sequences between primers, which can avoid interference signals generated by non-specific amplification such as primer dimers, and ensure the specificity of detection results.
  • different fluorescent groups are used to label probes that specifically bind to target sequences, and the corresponding target sequences can be identified by detecting different fluorescent signals of the probes.
  • detection mode selection the real-time detection mode has no other steps except amplification, which is simple and direct, but the maximum number of target sequences that can be detected in this mode is limited by the number of fluorescence detection channels of the fluorescence real-time PCR instrument, generally no more than 6.
  • Another detection mode is the melting curve analysis after amplification.
  • a temperature change process is added after amplification.
  • the maximum number of target sequences that can be detected is equal to the increase of the number of probe and target sequences on the basis of the number of fluorescence detection channels. Compared with the real-time detection mode, the dimension of melting point is greatly improved.
  • CN108823287A discloses a multiplex real-time PCR detection system for detecting target nucleic acid sequences, which is characterized in that the number of detection probes used is less than the target sequence.
  • the above-mentioned multiplex real-time PCR detection system adopts a unique detection system, that is, for each target sequence, a non-fluorescently labeled target sequence-specific mediator probe is designed, and mediator sequences on multiple mediator probes are designed. It can be combined and extended with the same fluorescent detection probe, and the extension products have different melting points. Since each mediator probe corresponds to one target sequence, these target sequences are actually detected simultaneously by one fluorescent probe.
  • non-specific signals ie, non-specific melting peaks
  • the inventors of the present application have noticed that in some cases, non-specific signals (ie, non-specific melting peaks) may appear in the detection results obtained by this method, which may affect the interpretation of the results to a certain extent. Therefore, there is a need to improve the multiplex real-time PCR detection system and detection method to further reduce or even eliminate the generation of non-specific signals (ie, non-specific melting peaks) without affecting the detection sensitivity.
  • non-specific signals ie, non-specific melting peaks
  • the detection probe binds non-specifically to non-target sequences, so that the detection probe emits signal (eg, a self-quenching fluorescent probe nonspecifically binds to a non-target nucleic acid molecule such that the fluorophore it carries is separated from the quencher group and thus fluoresces).
  • the detection probe can usually be modified, such as designing a hairpin structure on the detection probe to inhibit the non-specific binding of the detection probe to non-target nucleic acid molecules.
  • the present application provides a method of detecting the presence of n target nucleic acid sequences in a sample, wherein n is an integer > 1 (eg, n is 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or larger integers), and the method includes the following step:
  • an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence;
  • the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator subsequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes comprise The mediator subsequences of are different from each other;
  • At least one mediator probe itself can form a hairpin structure
  • the at least one mediator probe has characteristics selected from the group consisting of:
  • the mediator probe further comprises a first hairpin-forming sequence downstream or at the 3' end of its target-specific sequence, the first hairpin-forming sequence and the mediator sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the mediator sequence and the first hairpin-forming sequence;
  • the mediator probe further comprises a second hairpin-forming sequence upstream or 5' of its mediator sequence, the second hairpin-forming sequence and the target-specific sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the second hairpin-forming sequence and the target-specific sequence;
  • the mediator probe further comprises a third hairpin-forming sequence upstream or 5' end of its mediator sequence, and further comprises a fourth hairpin-forming sequence downstream or 3' end of its target-specific sequence , and the third hairpin-forming sequence or part thereof is complementary to the fourth hairpin-forming sequence or part thereof, whereby the mediator probe can pass through the third hairpin-forming sequence and the third hairpin-forming sequence Four hairpin forming sequences form a hairpin structure;
  • step (2) (2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
  • step (3) providing m kinds of detection probes, and contacting the product of step (2) with the m kinds of detection probes under conditions that allow nucleic acid hybridization, wherein m is an integer greater than 0,
  • each detection probe independently comprises, from the 3' to 5' direction, one or more capture sequences complementary to one or more mediator subsequences or portions thereof, and a templating sequence;
  • the m detection probes comprise at least n capture sequences, which are respectively complementary to the mediator sequences of the n mediator probes provided in step (1) or parts thereof;
  • Each detection probe is independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the reporter group emitting and, each detection probe emits a different signal when hybridized to its complementary sequence than when not hybridized to its complementary sequence; and,
  • step (3) (4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
  • step (4) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
  • m is an integer less than n and greater than zero. In such embodiments, the number of detection probes used is less than the number of mediator probes. Thus, at least one detection probe comprises two or more capture sequences. In certain embodiments, m equals n. In such embodiments, the number of detection probes used is equal to the number of mediator probes. Therefore, the detection probes can be in a one-to-one correspondence with the mediator probes.
  • m is an integer >1, >2, >3, >4, >5, >6, >8, >10 (eg, m is 1, 2, 3, 4, 5, or 6 ); preferably, when m ⁇ 2, each of the m detection probes is labeled with a different reporter group.
  • step (1) provides at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 mediator probes; and, step (3) provides at least 1, at least 2, at least 3, at least 4 , at least 5, at least 6, at least 8, or at least 10 detection probes.
  • the mediator probes are each independently capable of forming a hairpin structure, for example each independently having features (i), (ii) or (iii) as defined in claim 1 .
  • each mediator probe is independently capable of forming a hairpin structure, eg, each independently has features (i), (ii) or (iii) as defined in claim 1 .
  • the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the m detection probes comprise the same reporter group; and, in step (5), melting curve analysis is performed on the product of step (4), and according to the obtained melting curve the melting peak (melting point) to determine the presence of a certain target nucleic acid sequence.
  • the reporter groups contained in the m detection probes are different from each other; and, in step (5), when the melting curve analysis is performed on the product of step (4), each real-time monitoring A signal of a reporter group, thereby obtaining a plurality of melting curves corresponding to the signal of a reporter group; the presence of a target nucleic acid sequence.
  • n is an integer > 1 (eg, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or larger integers).
  • the method includes the steps of:
  • an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence; and, the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator sequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes The included mediator subsequences differ from each other;
  • the at least one mediator probe itself is capable of forming a hairpin structure; for example, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1;
  • step (2) (2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
  • step (3) contacting the product of step (2) with a detection probe comprising from the 3' to 5' direction, under conditions allowing nucleic acid hybridization, with each of the mediator subsequences or portions thereof a complementary capture sequence, and a templating sequence; and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of signaling, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and, the detection probe emits a signal when hybridized to its complementary sequence that is different from the signal emitted when it is not hybridized to its complementary sequence;
  • step (3) (4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
  • step (4) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
  • the sample can be any sample to be detected.
  • the sample comprises or is a mixture of DNA, or RNA, or nucleic acids.
  • the target nucleic acid sequence is DNA or RNA; and/or, the target nucleic acid sequence is single-stranded or double-stranded.
  • the sample or target nucleic acid sequence is obtained from a source selected from prokaryotes, eukaryotes, viruses, or viroids.
  • the mediator probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the mediator probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the mediator probes comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine pyrimidine.
  • the mediator probe comprises non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the mediator probe is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80 -90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • the target-specific sequence in the mediator probe is 10-140nt in length, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the length of the mediator sequence in the mediator probe can be 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt , 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the inventors of the present application believe that although the detection probe has been designed to have a stable hairpin structure, non-specific binding of the mediator probe to the detection probe may still occur, which can make the hairpin of the detection probe possible.
  • the clip structure is opened, resulting in the separation of the fluorophore and the quencher group it carries, which in turn leads to the generation of a non-specific signal.
  • the mediator probe ie, adding a first hairpin-forming sequence downstream or 3' of its target-specific sequence, or adding a second hairpin upstream or 5' of its mediator sequence forming sequence, or adding a third hairpin forming sequence upstream or 5' end of its mediator sequence, and adding a fourth hairpin forming sequence downstream or 3' end of its target-specific sequence
  • making the mediator probe Capable of forming a hairpin structure by itself (by means of complementary base pairing between an intermediary subsequence and a first hairpin forming sequence, or base pairing between a second hairpin forming sequence and a target-specific sequence, or a third
  • the complementary base pairing between the hairpin-forming sequence and the fourth hairpin-forming sequence can further reduce or even eliminate the non-specific binding between the mediator probe and the detection probe, thereby further reducing or even eliminating the generation of non-specific signals.
  • the first, second, third or fourth hairpin-forming sequence can be of any length without limitation as long as it enables the mediator probe to form a hairpin structure.
  • the stability of the hairpin structure formed by the mediator probe will decrease, which may affect (eg increase the ) non-specific binding of the mediator probe to the detection probe.
  • the stability of the hairpin structure formed by the mediator probe will increase, which may affect (eg reduce) the mediator. Specific binding of probes to target nucleic acid sequences.
  • the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • mediator probes with hairpin structures may bring additional potential benefits: the ability to enhance the intensity of positive signals and improve the sensitivity of detection.
  • the inventors of the present application believe that the use of the mediator probe with a hairpin structure reduces the non-specific binding between the mediator probe and the detection probe, which allows more detection probes in the reaction system. The needle can bind to the cleaved mediator fragment, resulting in a stronger positive signal for the same positive sample.
  • a linker is also included between the target-specific sequence of the mediator probe and the first hairpin-forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is also included between the third hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the target-specific sequence of the mediator probe and the fourth hairpin-forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • the first hairpin-forming sequence is fully or partially complementary to the mediator sequence.
  • the mediator probe is capable of forming a hairpin structure through the mediator sequence and the first hairpin-forming sequence, wherein the arms of the hairpin structure have blunt ends (ie, do not with an overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang (eg, , its 3' end has at least 1, at least 2, or more free bases).
  • the arm consists of a fully complementary first hairpin-forming sequence and a mediator subsequence.
  • the arms are symmetrical or have blunt ends.
  • the arm consists of a partially complementary first hairpin-forming sequence and a mediator subsequence.
  • the arms are asymmetric or have 5' or 3' overhangs.
  • the first hairpin-forming sequence is the same length as the mediator subsequence.
  • the arms may have blunt ends.
  • the first hairpin-forming sequence is different in length from the mediator subsequence.
  • the length of the first hairpin forming sequence and the length of the mediator subsequence may differ by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt.
  • the arms may have 5' or 3' overhangs.
  • the length of the first hairpin-forming sequence is 1-10 nt longer than the length of the mediator subsequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt .
  • the length of the mediator subsequence is 1-10 nt longer than the length of the first hairpin forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10nt.
  • the second hairpin-forming sequence is fully or partially complementary to the target-specific sequence.
  • the mediator probe is capable of forming a hairpin structure via the second hairpin-forming sequence and the target-specific sequence, wherein the arms of the hairpin structure have blunt ends (ie, no overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang ( For example, it has at least 1, at least 2, or more free bases at its 3' end).
  • the arm consists of a fully complementary second hairpin-forming sequence and a target-specific sequence.
  • the arms are symmetrical or have blunt ends.
  • the arm consists of a partially complementary second hairpin-forming sequence and a target-specific sequence.
  • the arms are asymmetric or have 5' or 3' overhangs.
  • the second hairpin-forming sequence is the same length as the target-specific sequence.
  • the arms may have blunt ends.
  • the second hairpin-forming sequence is not the same length as the target-specific sequence.
  • the length of the second hairpin forming sequence may differ from the length of the target-specific sequence by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt.
  • the arms may have 5' or 3' overhangs.
  • the length of the second hairpin-forming sequence is 1-10 nt longer than the length of the target-specific sequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the length of the target-specific sequence is 1-10 nt longer than the length of the second hairpin forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the third hairpin-forming sequence is fully or partially complementary to the fourth hairpin-forming sequence.
  • the mediator probe is capable of forming a hairpin structure via the third hairpin-forming sequence and the fourth hairpin-forming sequence, wherein the arms of the hairpin structure have blunt ends ( That is, no overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang Overhang (eg, having at least 1, at least 2, or more free bases at its 3' end).
  • the arms are comprised of fully complementary third and fourth hairpin-forming sequences.
  • the arms are symmetrical or have blunt ends.
  • the arms are comprised of partially complementary third and fourth hairpin-forming sequences.
  • the arms are asymmetric or have 5' or 3' overhangs.
  • the third hairpin-forming sequence is the same length as the fourth hairpin-forming sequence.
  • the arms may have blunt ends.
  • the third hairpin-forming sequence is different in length from the fourth hairpin-forming sequence.
  • the length of the third hairpin forming sequence may differ from the length of the fourth hairpin forming sequence by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt.
  • the arms may have 5' or 3' overhangs.
  • the length of the third hairpin-forming sequence is 1-10 nt longer than the length of the fourth hairpin-forming sequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the length of the fourth hairpin-forming sequence is 1-10 nt longer than the length of the third hairpin-forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
  • the mediator probe has a 3'-OH terminus, or its 3'-terminus is blocked. In certain preferred embodiments, the 3'-terminus of the mediator probe is blocked to inhibit its extension.
  • the 3'-terminus of nucleic acids can be blocked by various methods. For example, the 3'-terminus of the mediator probe can be blocked by modifying the 3'-OH of the last nucleotide of the mediator probe. In certain embodiments, the 3'-terminus of the mediator probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the mediator probe . In certain embodiments, the mediator probe can be blocked by removing the 3'-OH of the last nucleotide of the mediator probe, or by replacing the last nucleotide with a dideoxynucleotide 3'-end.
  • a chemical moiety eg, biotin or an alkyl group
  • the upstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the upstream oligonucleotide sequence comprises or consists of natural nucleotides such as deoxyribonucleotides or ribonucleotides.
  • the upstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethyl base cytosine.
  • the upstream oligonucleotide sequence comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the upstream oligonucleotide sequence is not limited by its length, as long as it can specifically hybridize to the target nucleic acid sequence.
  • the upstream oligonucleotide sequence is 15-150nt in length, eg, 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • the upstream oligonucleotide sequence after hybridization to the target nucleic acid sequence, is located at the upstream distal end of the mediator probe, or is located upstream adjacent to the mediator probe, or is adjacent to the mediator probe.
  • Target-specific sequences have partially overlapping sequences.
  • the upstream oligonucleotide sequence is a primer specific for a target nucleic acid sequence or a probe specific for a target nucleic acid sequence.
  • step (2) the enzyme with 5' nuclease activity cleaves the mediator probe hybridized to the target nucleic acid sequence, and releases the entire mediator sequence or a portion of the mediator sequence (5'-end portion) of the mediator fragment.
  • the enzyme having 5' nuclease activity is a nucleic acid polymerase (eg, a DNA polymerase, particularly a thermostable) having 5' nuclease activity (eg, 5' exonuclease activity) DNA polymerase).
  • a nucleic acid polymerase eg, a DNA polymerase, particularly a thermostable
  • 5' nuclease activity eg, 5' exonuclease activity
  • the DNA polymerase is obtained from a bacterium selected from the group consisting of: Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima,Thermotoga bacterium
  • step (2) the nucleic acid polymerase having 5' nuclease activity catalyzes the extension of the upstream oligonucleotide sequence and induces cleavage of the mediator probe.
  • a nucleic acid polymerase is used to catalyze the extension of the upstream oligonucleotide sequence using the target nucleic acid sequence as a template, and then the enzyme having 5' nuclease activity is bound to the upstream An extension product of an oligonucleotide sequence and catalyzes cleavage of the mediator probe.
  • the sample in steps (1) and/or (2), is also contacted with a downstream oligonucleotide sequence (or downstream primer) specific for the target nucleic acid sequence.
  • a downstream oligonucleotide sequence or downstream primer
  • the use of nucleic acid polymerases and downstream oligonucleotide sequences (or downstream primers) is particularly advantageous.
  • the nucleic acid polymerase can use the target nucleic acid sequence as a template and the upstream oligonucleotide sequence and the downstream oligonucleotide sequence as primers to generate additional target nucleic acid sequences, thereby improving the sensitivity of the method of the present invention.
  • step (1) in addition to the upstream oligonucleotide sequences and mediator probes as defined above, for each target nucleic acid sequence to be detected , also provides a downstream oligonucleotide sequence; wherein, the downstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized with the target nucleic acid sequence, the downstream oligonucleotide sequence The nucleotide sequence is located downstream of the target-specific sequence; the sample is then contacted with the provided upstream oligonucleotide sequence, mediator probe, and downstream oligonucleotide sequence under conditions that allow nucleic acid hybridization .
  • step (2) the product of step (1) is contacted with a nucleic acid polymerase having 5' nuclease activity under conditions that allow nucleic acid amplification.
  • the downstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the downstream oligonucleotide sequence comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the downstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethyl base cytosine.
  • the downstream oligonucleotide sequence comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the downstream oligonucleotide sequence is not limited by its length, as long as it can specifically hybridize to the target nucleic acid sequence.
  • the downstream oligonucleotide sequence is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • all upstream and downstream oligonucleotide sequences provided in step (1) have an identical stretch of oligonucleotide sequence at the 5' end.
  • step (1) in addition to the upstream oligonucleotide sequence, the mediator probe and the downstream oligonucleotide sequence, a universal primer is provided, the universal primer has a sequence complementary to the same oligonucleotide sequence; the sample is then combined with the provided upstream oligonucleotide sequence, mediator probe, downstream oligonucleotide sequence under conditions that allow nucleic acid hybridization Contact with universal primers.
  • the identical oligonucleotide sequences are 8-50 nt in length, eg, 8-15 nt, 15-20 nt, 20-30 nt, 30-40 nt, or 40-50 nt.
  • the length of the universal primer may be 8-50nt, eg, 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
  • universal primers may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the universal primers comprise or consist of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the universal primers comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine.
  • the universal primers comprise non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole , 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole , 5-nitroindole or locked nucleic acid (LNA).
  • the universal primer is not limited by its length, as long as it can specifically hybridize to the same oligonucleotide sequence contained in the upstream and downstream oligonucleotide sequences.
  • a universal primer can be 8-50nt in length, such as 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
  • the detection probe comprises a plurality of capture sequences; and, the plurality of capture sequences are arranged adjacently, separated by linker sequences, or overlapping.
  • the detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the detection probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides).
  • the detection probes comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine .
  • the detection probe comprises non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro Pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy- ⁇ -D-ribofuranosyl)-3-nitro Pyrrole, 5-nitroindole or locked nucleic acid (LNA).
  • the detection probe is 15-1000nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80- 90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000nt.
  • the capture sequence in the detection probe can be of any length as long as it can specifically hybridize to the mediator fragment.
  • the capture sequence in the detection probe is 10-500nt in length, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-nt 80nt, 80-90nt, 90-100nt, 100-150nt, 150-200nt, 200-250nt, 250-300nt, 300-350nt, 350-400nt, 400-450nt, 450-500nt.
  • the template sequence in the detection probe can be of any length as long as it can be used as a template for extending the mediator subfragment.
  • the length of the template sequence in the detection probe is 1-900nt, such as 1-5nt, 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-nt 60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt.
  • the detection probe has a 3'-OH terminus, or its 3'-terminus is blocked to inhibit its extension.
  • the 3'-terminus of nucleic acids can be blocked by various methods.
  • the 3'-terminus of the detection probe can be blocked by modifying the 3'-OH of the last nucleotide of the detection probe.
  • the 3'-terminus of the detection probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the detection probe.
  • the 3'-OH of the detection probe can be blocked by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide. '-end.
  • the detection probe is a self-quenching probe; eg, the detection probe is labeled with a reporter group at its 5' end or upstream and labeled with a quencher at its 3' end or downstream group, or a reporter group is labeled at its 3' end or downstream and a quencher group is labeled at its 5' end or upstream.
  • the reporter group and the quencher group are separated by a distance of 10-80 nt or more.
  • the reporter group in the detection probe is a fluorophore (eg ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and a quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).
  • a quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).
  • the detection probes are modified or not.
  • the detection probe is resistant to nuclease activity (eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity);
  • the backbone of the detection probe comprises nuclease resistant Reactive modifications such as phosphorothioate bonds, alkyl phosphotriester bonds, aryl phosphotriester bonds, alkyl phosphonate bonds, aryl phosphonate bonds, hydrophosphate bonds, alkyl phosphoramidate bonds , aryl phosphoramidate linkages, 2'-O-aminopropyl modifications, 2'-O-alkyl modifications, 2'-O-allyl modifications, 2'-O-butyl modifications, and 1-( 4'-thio-PD-ribofuranosyl) modification.
  • nuclease activity eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity
  • the detection probe is linear, or has a hairpin structure. In certain preferred embodiments, the detection probe is linear. In certain preferred embodiments, the detection probe has a hairpin structure. Hairpin structures can be natural or artificially introduced.
  • the nucleic acid polymerase uses the detection probe as a template to extend the mediator fragment hybridized to the detection probe , and thus form a duplex.
  • the enzyme with 5' nuclease activity used is a nucleic acid polymerase with 5' nuclease activity, and is the same as the nucleic acid polymerase used in step (4) .
  • steps (1)-(5) are carried out by a scheme comprising the following steps (I)-(VII):
  • step (II) of the method the sample is mixed with the upstream oligonucleotide sequences, the mediator probe and downstream oligonucleotide sequences, and a nucleic acid polymerase, And carry out the PCR reaction, then, after the PCR reaction is completed, add the detection probe to the product of step (VI), and carry out melting curve analysis; or, in step (II), the sample and the upstream
  • the oligonucleotide sequence, the mediator probe, the downstream oligonucleotide sequence, the detection probe, and the nucleic acid polymerase are mixed and subjected to a PCR reaction, and then, after the PCR reaction is completed, a melting curve analysis is performed.
  • step (II) of the method the sample is combined with the upstream oligonucleotide sequences, the mediator probe and downstream oligonucleotide sequences, a nucleic acid polymerase, and a universal
  • the primers are mixed, and a PCR reaction is performed, and then, after the PCR reaction is completed, the detection probe is added to the product of step (VI), and a melting curve analysis is performed; or, in step (II), the sample is mixed with.
  • the upstream oligonucleotide sequence, the mediator probe, the downstream oligonucleotide sequence and the detection probe, nucleic acid polymerase, and universal primer are mixed, and a PCR reaction is performed, and then, after the PCR reaction is completed, a PCR reaction is performed. Melting curve analysis.
  • step (III) the product of step (II) is incubated at a temperature of 80-105°C, thereby denaturing the nucleic acid.
  • step (III) the product of step (II) is incubated for 10-20s, 20-40s, 40-60s, 1-2 min, or 2-5 min.
  • step (IV) at 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, or 65-70°C
  • the product of step (III) is incubated at a temperature to allow nucleic acid annealing or hybridization.
  • step (IV) the product of step (III) is incubated for 10-20s, 20-40s, 40-60s, 1-2 min, or 2-5 min.
  • step (V) at 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, 65-70°C, The product of step (IV) is incubated at temperatures of 70-75°C, 75-80°C, 80-85°C, thereby allowing nucleic acid extension.
  • step (V) the product of step (IV) is incubated for 10-20s, 20-40s, 40-60s, 1-2min, 2-5min, 5-10min, 10-20min or 20-30min.
  • steps (IV) and (V) are performed at the same or different temperatures.
  • steps (III)-(V) are repeated at least once, eg, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. In certain embodiments, when steps (III)-(V) are repeated one or more times, the conditions used in each cycle of steps (III)-(V) are each independently the same or different.
  • step (VII) the product of step (VI) is gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining each A plot of the signal intensity of the reporter group as a function of temperature.
  • the obtained curve is derived to obtain a melting curve for the product of step (VI).
  • the presence of a mediator subfragment corresponding to the melting peak (melting point) is determined based on the melting peak (melting point) in the melting curve; then, the mediator subsequence in the mediator subfragment and the target nucleic acid sequence are determined The corresponding relationship is determined to determine the existence of the target nucleic acid sequence corresponding to the mediator fragment.
  • the application provides a probe set comprising a detection probe, and one or more (eg, at least two) mediator probes, wherein,
  • the mediator probes each independently comprise, in the 5' to 3' direction, a mediator sequence comprising a sequence complementary to a target nucleic acid sequence and a target-specific sequence comprising a non-target nucleic acid sequence. a sequence complementary to the target nucleic acid sequence, and the mediator sequences contained in all mediator probes are different from each other;
  • the at least one mediator probe itself is capable of forming a hairpin structure; preferably, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1; and
  • the detection probe comprises, from 3' to 5', a capture sequence complementary to each mediator subsequence or a portion thereof, and a templating sequence; and the detection probe is labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the detection probe is hybridized to its complementary sequence The signal emitted is different from the signal emitted in the absence of hybridization to its complementary sequence.
  • the probe set comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 mediator probes.
  • each mediator probe is independently capable of forming a hairpin structure, eg, each independently has features (i), (ii) or (iii) as defined in claim 1 .
  • the mediator probe is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80 -90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
  • the target-specific sequence in the mediator probe is 10-140nt in length, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the length of the mediator sequence in the mediator probe can be 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt , 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
  • the mediator probe has a 3'-OH terminus, or its 3'-terminus is blocked. In certain preferred embodiments, the 3'-terminus of the mediator probe is blocked to inhibit its extension.
  • the 3'-terminus of nucleic acids can be blocked by various methods. For example, the 3'-terminus of the mediator probe can be blocked by modifying the 3'-OH of the last nucleotide of the mediator probe. In certain embodiments, the 3'-terminus of the mediator probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the mediator probe . In certain embodiments, the mediator probe can be blocked by removing the 3'-OH of the last nucleotide of the mediator probe, or by replacing the last nucleotide with a dideoxynucleotide 3'-end.
  • a chemical moiety eg, biotin or an alkyl group
  • a linker is also included between the target-specific sequence of the mediator probe and the first hairpin forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is also included between the third hairpin-forming sequence of the mediator probe and the mediator sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • a linker is further included between the target-specific sequence of the mediator probe and the fourth hairpin-forming sequence.
  • the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
  • all mediator probes comprise target-specific sequences that differ from each other.
  • all mediator probes each target a different target nucleic acid sequence.
  • the probe set comprises a mediator probe as defined above.
  • the probe set comprises detection probes as defined above.
  • the application provides a kit comprising one or more probe sets as previously defined.
  • the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 probe sets.
  • all mediator sequences in the kit each target a different target nucleic acid sequence.
  • all the mediator probes in the kit contain mediator sequences that are different from each other.
  • all mediator probes in the kit comprise target-specific sequences that differ from each other.
  • all detection probes in the kit are each independently labeled with the same or different reporter groups.
  • kits can be used to practice the methods of the invention described in detail above. Therefore, the various technical features described in detail above for the mediator probe and the detection probe can also be applied to the mediator probe and the detection probe in the kit. Furthermore, such kits may also contain other reagents required for carrying out the methods of the present invention.
  • the kit may further comprise upstream oligonucleotide sequences, downstream oligonucleotide sequences, universal primers, enzymes with 5' nuclease activity, nucleic acids as defined above A polymerase, or any combination thereof.
  • the kit may further comprise, reagents for nucleic acid hybridization, reagents for mediator probe cleavage, reagents for nucleic acid extension, nucleic acid amplification reagents, or any combination thereof.
  • Such reagents can be routinely determined by one of skill in the art, and include, but are not limited to, working buffers for enzymes (eg, nucleic acid polymerases), dNTPs, water, solutions containing ions (eg, Mg 2+ ), single-stranded DNA binding proteins (Single Strand DNA-Binding Protein, SSB), or any combination thereof.
  • enzymes eg, nucleic acid polymerases
  • dNTPs eg, dNTPs
  • water solutions containing ions
  • solutions ions eg, Mg 2+
  • Single-stranded DNA binding proteins Single Strand DNA-Binding Protein
  • the term “mediator probe” refers to a single-stranded chain containing a mediator sequence and a targeting sequence (ie, a target-specific sequence) from the 5' to 3' direction A nucleic acid molecule, which optionally may also comprise a first hairpin-forming sequence downstream or 3' of the targeting sequence, or a second hairpin-forming sequence upstream or 5' of the mediator sequence, or at the mediator A third hairpin-forming sequence is included upstream or 5' of the sequence, and a fourth hairpin-forming sequence is included downstream or 3' of the target-specific sequence.
  • the mediator probe When the mediator probe contains a first, second, third or fourth hairpin-forming sequence, the mediator probe can pass through the mediator sequence and the first hairpin-forming sequence, or the second hairpin-forming sequence
  • the clip-forming sequence and the target-specific sequence, or the third hairpin-forming sequence and the fourth hairpin-forming sequence form a hairpin structure.
  • the mediator subsequence does not contain a sequence complementary to the target nucleic acid sequence
  • the target specific sequence comprises a sequence complementary to the target nucleic acid sequence.
  • the mediator probe hybridizes or anneals (ie, forms a double-stranded structure) to the target nucleic acid sequence through the target-specific sequence under conditions that allow nucleic acid hybridization, annealing, or amplification, and the mediator sequence in the mediator probe Does not hybridize to the target nucleic acid sequence.
  • targeting sequence and “target-specific sequence” refer to those capable of selectively/specifically hybridizing or annealing to a target nucleic acid sequence under conditions that permit hybridization, annealing, or amplification of the nucleic acid.
  • a sequence comprising a sequence complementary to a target nucleic acid sequence.
  • targeting sequence and “target-specific sequence” have the same meaning and are used interchangeably. It is readily understood that a targeting sequence or target-specific sequence is specific for a target nucleic acid sequence.
  • a targeting sequence or target-specific sequence only hybridizes or anneals to a specific target nucleic acid sequence, and not to other nucleic acid sequences, under conditions that allow nucleic acid hybridization, annealing, or amplification.
  • the term "mediator sequence” refers to a stretch of oligonucleotide sequence in the mediator probe that is not complementary to the target nucleic acid sequence and is located upstream (5' end) of the target-specific sequence.
  • a unique mediator probe is designed or provided, which has a unique mediator sequence (in other words, the mediator sequences in all mediator probes used are different from each other)
  • each target nucleic acid sequence corresponds to a unique mediator probe (unique mediator sequence).
  • upstream oligonucleotide sequence refers to an oligonucleotide sequence comprising a sequence complementary to a target nucleic acid sequence under conditions that allow nucleic acid hybridization (or annealing) or amplification , is capable of hybridizing (or annealing) to the target nucleic acid sequence and, when hybridized to the target nucleic acid sequence, is located upstream of the mediator probe.
  • the term “complementary” means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the principles of base pairing (Waston-Crick principle), and thereby forming duplexes.
  • the term “complementary” includes “substantially complementary” and “completely complementary”.
  • the term “completely complementary” means that every base in one nucleic acid sequence is capable of pairing with bases in another nucleic acid strand without mismatches or gaps.
  • the term "substantially complementary” means that a majority of bases in one nucleic acid sequence are capable of pairing with bases in the other nucleic acid strand, which allows for mismatches or gaps (eg, one or mismatches or gaps of several nucleotides).
  • two nucleic acid sequences that are "complementary” eg, substantially complementary or fully complementary
  • non-complementary means that two nucleic acid sequences cannot hybridize or anneal under conditions that permit hybridization, annealing, or amplification of the nucleic acids to form a duplex.
  • not perfectly complementary means that bases in one nucleic acid sequence cannot perfectly pair with bases in another nucleic acid strand, at least one mismatch or gap exists.
  • hybridization and “annealing” mean the process by which complementary single-stranded nucleic acid molecules form a double-stranded nucleic acid.
  • hybridization and “annealing” have the same meaning and are used interchangeably.
  • two nucleic acid sequences that are completely complementary or substantially complementary can hybridize or anneal.
  • the complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, in particular the temperature.
  • PCR reaction has the meaning commonly understood by those skilled in the art, which refers to a reaction (polymerase chain reaction) that amplifies a target nucleic acid using a nucleic acid polymerase and primers.
  • multiplex amplification refers to the amplification of multiple target nucleic acids in the same reaction system.
  • asymmetric amplification means that in the amplification product obtained by amplifying a target nucleic acid, the amount of two complementary nucleic acid strands is different, and the amount of one nucleic acid strand is greater than that of the other. nucleic acid strands.
  • detection probe is labeled with a reporter group and a quencher group.
  • detection probes are capable of forming duplexes with their complementary sequences through base pairing.
  • the reporter group such as a fluorophore
  • the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. to the strongest signal (e.g. fluorescence signal).
  • the two strands of the duplex begin to dissociate (ie, the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe is in a single-stranded free coil state.
  • the reporter group (eg, fluorophore) and the quencher group on the dissociated detection probe are in close proximity to each other, whereby the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescent signal) gradually becomes weaker.
  • the two strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, all the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the detection probe is absorbed by the quencher group.
  • the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) is substantially undetectable.
  • melting curve analysis has the meaning commonly understood by those skilled in the art and refers to the analysis of the presence or identity of a double-stranded nucleic acid molecule by determining the melting curve of the double-stranded nucleic acid molecule. method, which is commonly used to assess the dissociation characteristics of double-stranded nucleic acid molecules during heating. Methods for performing melting curve analysis are well known to those skilled in the art (see, eg, The Journal of Molecular Diagnostics 2009, 11(2):93-101). In this application, the terms “melting curve analysis” and “melting analysis” have the same meaning and are used interchangeably.
  • melting curve analysis can be performed by using detection probes labeled with reporter and quencher groups.
  • detection probes are capable of forming duplexes with their complementary sequences through base pairing.
  • the reporter group such as a fluorophore
  • the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. to the strongest signal (e.g. fluorescence signal).
  • the two strands of the duplex begin to dissociate (ie, the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe assumes a single-stranded free coil state.
  • the reporter group (eg, fluorophore) and the quencher group on the dissociated detection probe are in close proximity to each other, whereby the signal (eg, fluorescence signal) emitted by the reporter group (eg, fluorophore) absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescent signal) gradually becomes weaker.
  • the two strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, all the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the detection probe is absorbed by the quencher group.
  • the signal eg, fluorescent signal
  • the reporter group eg, fluorophore
  • the hybridization and dissociation process of the detection probe and its complementary sequence can be observed, and the signal intensity changes with temperature. changing curve.
  • a curve with the change rate of signal intensity as the ordinate and the temperature as the abscissa ie, the melting curve of the duplex
  • the peak in the melting curve is the melting peak
  • the corresponding temperature is the melting point (T m value) of the duplex.
  • the more closely the detection probe matches the complementary sequence eg, fewer bases are mismatched and more bases are paired
  • the terms "melting peak”, “melting point” and “ Tm value” have the same meaning and are used interchangeably.
  • the method of the present application can effectively reduce (or even eliminate) the non-specific signal of the multiplex real-time PCR detection method using the mediator probe and the detection probe, and significantly improve the specificity of the detection method.
  • the method of the present application can also increase the intensity of the positive signal to a certain extent, thereby improving the detection sensitivity. Therefore, the methods of the present application are particularly advantageous, particularly suitable for multiplex detection of target nucleic acid sequences.
  • Figure 1 shows the structures of three hairpin mediator probes of the present invention.
  • 1A shows that when the mediator probe contains the mediator sequence, the target-specific sequence and the first hairpin forming sequence, the mediator subsequence is in a hairpin structure before being cleaved.
  • Figure IB shows that when the mediator probe contains a mediator sequence, a target-specific sequence and a second hairpin forming sequence, the mediator sequence is in a hairpin structure before the mediator sequence is cleaved.
  • Figure 1C shows that the mediator probe assumes a hairpin structure before the mediator sequence is cleaved when the mediator probe contains a mediator sequence, a target-specific sequence, a third hairpin-forming sequence, and a fourth hairpin-forming sequence.
  • Figure 2 schematically shows the principle of the system of the present invention.
  • Figure 2A shows that when the mediator probe contains a mediator sequence and a target-specific sequence, before the mediator sequence is cleaved, it has a linear structure, which is easy to non-specifically bind to the detection probe.
  • Figure 2B shows that when the mediator probe contains a mediator sequence, a target-specific sequence and a first hairpin-forming sequence, the mediator sequence is in a hairpin structure before the mediator sequence is cleaved, significantly reducing non-specificity with the detection probe combine.
  • Figure 3 shows the detection results of the reaction system of Example 1, wherein Figure 3A is the detection result produced by the Gp8wzx detection system using the linear mediator probe; Figure 3B is the Gp8wzx detection system using the hairpin mediator probe The detection results generated; Figure 3C shows the detection results generated by the O52wzm detection system using the linear mediator probes; Figure 3D shows the detection results generated by the O52wzm detection system using the hairpin mediator probes.
  • the solid line in the figure represents the detection result using the plasmid (4 samples) as the template; the dotted line represents the detection result (negative control) using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template.
  • Figure 4 shows the detection results of the Gp8wzx detection system, wherein Figure 4A is the detection result using the linear mediator probe; Figure 4B is the detection result using the hairpin mediator probe with 6 complementary bases; Figure 4C is the detection result. The detection result using the hairpin mediator probe with 8 complementary bases; Fig. 4D is the detection result using the hairpin mediator probe with 10 complementary bases.
  • the solid line in the figure represents the detection result using Escherichia coli Gp8wzx plasmid as the template; the dotted line represents the detection result (negative control) using TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template.
  • Figure 5 shows the detection results of the reaction system of Example 3, wherein Figure 5A is the detection system using the linear mediator probe; Figure 5B is the detection system using the hairpin mediator probe.
  • the solid line in the figure represents the detection result using the plasmid containing the target gene 1-6 as the template; the dotted line represents the detection result using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template (negative control) .
  • Figure 6 shows the detection results of the reaction system of Example 4, wherein Figure 6A is the detection system using the linear mediator probe; Figure 6B is the detection system using the hairpin mediator probe.
  • the solid line in the figure represents the detection result using the plasmid containing the target gene 7-12 as the template; the dashed line represents the detection result using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template (negative control) .
  • the hairpin mediator probe can be used for dual-probe real-time PCR detection, in this example, a linear mediator probe and a hairpin mediator probe are used for comparison, and the detection probe is the same.
  • the experiment used Escherichia coli O antigen-specific genes Gp8wzx and O52wzm as detection objects, and each 25 ⁇ L PCR reaction system included 1 ⁇ PCR buffer (Zhishan Biotechnology Co., Ltd., Xiamen), 7.0 mM MgCl 2 , 0.2 mM dNTPs, 3 U Taq01 enzyme ( Zhishan Biotechnology Co., Ltd., Xiamen), primers and probes (see Table 1 for dosage), and 5 ⁇ L of templates (the templates of positive control are E.
  • coli Gp8wzx plasmid and O52wzm plasmid (10000 copies/ul), respectively, will be as SEQ ID NO:
  • the nucleotide sequences shown in 56 and SEQ ID NO: 57 were inserted into the multi-cloning site of the puc57 vector to obtain the Escherichia coli Gp8wzx plasmid and the O52wzm plasmid respectively; the template of the negative control was TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5).
  • the PCR reaction program is: incubation at 50 °C for 2 min, denaturation at 95 °C for 5 min, then 40 cycles of 20 s at 95 °C and 1 min at 60 °C, fluorescence collection at 60 °C, and the procedure for melting analysis after PCR is as follows: Hybridization at 35 °C Extend for 20min, then denature at 95°C for 2min, keep at 45°C for 2min, then heat up from 45°C to 95°C at a heating rate of 0.04°C/s, and collect the fluorescence signal of the CY5 channel.
  • the experimental instrument is a SLAN 96S real-time PCR instrument (Hongshi Medical Technology Co., Ltd., Shanghai), primers and probes were synthesized by Shanghai Bioengineering Co., Ltd.
  • Reaction A is a Gp8wzx singleplex detection system, using a linear mediator probe
  • Reaction B is a Gp8wzx singleplex detection system, using a hairpin mediator probe.
  • the results showed that the negative control (dotted line) of reaction A (Fig. 3A) produced an obvious non-specific melting peak at 52°C, while the negative control (dotted line) of reaction B (Fig. 3B) had no obvious non-specific melting peak.
  • the experimental results show that the hairpin mediator probe can significantly reduce the non-specific melting signal compared with the linear mediator probe.
  • Reaction C is an O52wzm singleplex detection system, using a linear mediator probe
  • Reaction D is an O52wzm singleplex detection system, using a hairpin mediator probe.
  • the universal primer Tag is used in both the Gp8 wzx singleplex detection system and the O52 wzm singleplex detection system.
  • Example 2 the Gp8 wzx singleplex detection system described in Example 1 was used to investigate the use of the linear mediator probe and the mediator probe with hairpin structure complementary bases of 6, 8, and 10 bases, respectively, for melting curves. Feasibility of analysis.
  • the composition of the reaction system and the PCR reaction conditions are the same as those in Example 1.
  • the linear mediator probes used in this example are also the same as those in Example 1.
  • Table 2 shows the hairpin mediator probes.
  • a six-fold PCR melting curve analysis system was used to detect and distinguish 6 different target sequences.
  • Five target genes (target genes 1-5) and one positive quality control gene (target gene 6) were used as detection objects.
  • the nucleotide sequences shown in SEQ ID NO: 58 to SEQ ID NO: 63 were inserted into the multiple cloning sites of the puc57 vector, respectively, to obtain plasmids containing target genes 1-6.
  • reaction system A was a control system, using 6 linear mediator probes and 2 fluorescent probes.
  • Reaction system B is an experimental system, using 4 linear mediator probes, 2 hairpin mediator probes and 2 fluorescent probes, wherein the 4 linear mediator probes correspond to target genes 1, 2, and 3 respectively And 5, 2 hairpin mediator probes correspond to positive quality control gene and target gene 4.
  • the composition of the reaction system and the PCR reaction conditions were the same as those in Example 1, and the detected templates were 6 positive plasmids (concentration: 1000 copies/ul) carrying target genes or quality control genes.
  • the primers and probes used in this example are specifically shown in Table 3.
  • reaction system A Fig. 5A
  • reaction system B Fig. 5B
  • the melting peak signal of the sub-probe is generally higher than that of the reaction system A (all of which are linear mediator sub-probes).
  • the non-specific melting signal of the reaction system B at 45-50 °C was significantly reduced.
  • a six-fold PCR melting curve analysis system is used to detect and distinguish the target genes of 6 different microorganisms.
  • the target gene 7-12 was used as the detection object.
  • the nucleotide sequences shown in SEQ ID NO: 64 to SEQ ID NO: 69 were inserted into the multiple cloning sites of the puc57 vector, respectively, to obtain plasmids containing target genes 7-12.
  • reaction system A was a control system, using 6 linear mediator probes and 2 fluorescent probes
  • reaction system B was an experimental system, using 5 linear mediator probes, 1 hairpin mediator probe and 2 fluorescent probes.
  • the composition of the reaction system and the PCR reaction conditions were the same as those in Example 1, and the melting curve analysis system used the ROX fluorescence channel.
  • the detected templates were 6 positive plasmids (concentration of 1000 copies/ul) carrying the target gene.
  • the primers and probes used in this example are specifically shown in Table 4.
  • reaction system A (Fig. 6A) and reaction system B (Fig. 6B) detected melting peaks with the same Tm value for each target gene, however, reaction system B (with a hairpin medium)
  • the melting peak signal of the sub-probe is generally higher than that of the reaction system A (all of which are linear mediator sub-probes).
  • the non-specific melting signal of the reaction system B at 45-50°C was completely eliminated.

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Abstract

Provided is a method for detecting a target nucleic acid sequence. The method can simultaneously detect the presence of multiple target nucleic acid sequences in a sample at a high specificity. Further provided is a probe set and a kit comprising one or more of the probe sets. The probe set and the kit can be used to implement the method of the present invention.

Description

一种高特异性的检测靶核酸序列的方法A highly specific method for detecting target nucleic acid sequences 技术领域technical field
本申请涉及核酸分子的多重检测。特别地，本申请提供了一种检测靶核酸序列的方法，所述方法能够以高特异性同时检测多种靶核酸序列在样品中的存在。此外，本申请还提供了一种探针组，和包含一种或多种所述探针组的试剂盒，所述探针组和试剂盒可用于实施本发明的方法。The present application relates to multiplex detection of nucleic acid molecules. In particular, the present application provides a method for detecting a target nucleic acid sequence, which is capable of simultaneously detecting the presence of multiple target nucleic acid sequences in a sample with high specificity. In addition, the present application also provides a probe set, and a kit comprising one or more of the probe sets, which can be used to implement the methods of the present invention.
背景技术Background technique
实时荧光PCR是核酸检测的一种常用方法，其操作简便，应用广泛，同时作为一种闭管检测模式，扩增产物污染机会低，利用多重实时PCR可在单个反应管内同时检测多个靶序列，不仅检测效率提高，成本也进一步降低。Real-time fluorescent PCR is a common method for nucleic acid detection. It is easy to operate and widely used. At the same time, as a closed-tube detection mode, the chance of contamination of amplification products is low. Multiplex real-time PCR can simultaneously detect multiple target sequences in a single reaction tube. , not only the detection efficiency is improved, but the cost is further reduced.
实时荧光PCR的检测原理基于荧光标记的寡核苷酸探针特异地与引物之间的靶序列结合，能避免引物二聚体等非特异扩增产生的干扰信号，确保检测结果的特异性。在多重实时PCR中，使用不同荧光基团标记与靶序列特异结合的探针，就可以通过检测探针的不同荧光信号识别相应的靶序列。在检测模式选择上，实时检测模式除扩增外无其他步骤，简便直接，但此种模式能检测的最大靶序列数目受限于荧光实时PCR仪器的荧光检测通道数，一般不超过6个。另一种检测模式是扩增后的熔解曲线分析，该模式是在扩增后增加一个变温过程，可检测的最大靶序列数目等于在荧光检测通道数目的基础上增加了探针与靶序列之间的熔点这个维度，相对于实时检测模式大大提高。The detection principle of real-time fluorescent PCR is based on the specific binding of fluorescently labeled oligonucleotide probes to target sequences between primers, which can avoid interference signals generated by non-specific amplification such as primer dimers, and ensure the specificity of detection results. In multiplex real-time PCR, different fluorescent groups are used to label probes that specifically bind to target sequences, and the corresponding target sequences can be identified by detecting different fluorescent signals of the probes. In terms of detection mode selection, the real-time detection mode has no other steps except amplification, which is simple and direct, but the maximum number of target sequences that can be detected in this mode is limited by the number of fluorescence detection channels of the fluorescence real-time PCR instrument, generally no more than 6. Another detection mode is the melting curve analysis after amplification. In this mode, a temperature change process is added after amplification. The maximum number of target sequences that can be detected is equal to the increase of the number of probe and target sequences on the basis of the number of fluorescence detection channels. Compared with the real-time detection mode, the dimension of melting point is greatly improved.
现阶段，无论采用何种检测模式，基于荧光探针检测的多重实时PCR都存在一些问题，一方面，荧光标记探针的制备涉及复杂的化学修饰和纯化过程，其成本大大高于非标记探针，其中，双标记探针(或多标记探针)的成本又高于单标记探针，内部标记探针的成本高于末端标记探针，因此，使用多个荧光标记探针就会导致成本增加。另一方面，在多重实时PCR中，多个荧光标记探针共存使反应体系的背景荧光增加，引起检测信号的水平降低，而最终导致多重实时PCR检测灵敏度的下降。At this stage, no matter what detection mode is used, multiplex real-time PCR based on fluorescent probe detection has some problems. On the one hand, the preparation of fluorescently labeled probes involves complex chemical modification and purification processes, and the cost is much higher than that of non-labeled probes needles, where the cost of dual-labeled probes (or multi-labeled probes) is higher than that of single-labeled probes, and the cost of internally labeled probes is higher than that of end-labeled probes, so the use of multiple fluorescently labeled probes will result in Increased costs. On the other hand, in multiplex real-time PCR, the coexistence of multiple fluorescently labeled probes increases the background fluorescence of the reaction system, resulting in a decrease in the level of detection signals, which ultimately leads to a decrease in the detection sensitivity of multiplex real-time PCR.
CN108823287A公开了一种检测靶核酸序列的多重实时PCR检测***，其特点是使用的检测探针数目少于靶序列。上述多重实时PCR检测***，采用了一种独特的检测体系，即针对每一个靶序列，都设计一个非荧光标记的靶序列特异的媒介子探针，多个媒介子探针上的媒介子序列可以和同一条荧光检测探针结合并延伸，并且延伸产物具有不同的 熔点。由于每种媒介子探针对应一种靶序列，这些靶序列实际上通过一个荧光探针实现了同时检测。但是，本申请发明人注意到，在某些情况下，通过该方法获得的检测结果中可能会出现非特异性信号(即非特异性熔解峰)，这在一定程度上可能影响结果的判读。因此，需要对该多重实时PCR检测***和检测方法进行改进，在不影响检测灵敏度的情况下进一步减少甚至消除非特异性信号(即非特异性熔解峰)的产生。CN108823287A discloses a multiplex real-time PCR detection system for detecting target nucleic acid sequences, which is characterized in that the number of detection probes used is less than the target sequence. The above-mentioned multiplex real-time PCR detection system adopts a unique detection system, that is, for each target sequence, a non-fluorescently labeled target sequence-specific mediator probe is designed, and mediator sequences on multiple mediator probes are designed. It can be combined and extended with the same fluorescent detection probe, and the extension products have different melting points. Since each mediator probe corresponds to one target sequence, these target sequences are actually detected simultaneously by one fluorescent probe. However, the inventors of the present application have noticed that in some cases, non-specific signals (ie, non-specific melting peaks) may appear in the detection results obtained by this method, which may affect the interpretation of the results to a certain extent. Therefore, there is a need to improve the multiplex real-time PCR detection system and detection method to further reduce or even eliminate the generation of non-specific signals (ie, non-specific melting peaks) without affecting the detection sensitivity.
发明内容SUMMARY OF THE INVENTION
在PCR扩增后的探针熔解曲线分析方法中，非特异性信号(即非特异性熔解峰)通常是由于下述原因而导致的：检测探针与非目标序列非特异性结合，使得检测探针发出了信号(例如，自淬灭荧光探针与非目标核酸分子非特异性结合，使得其携带的荧光基团与淬灭基团分开，并因此发出荧光)。为了减少此类非特异性信号的产生，通常可对检测探针进行改造，例如在检测探针上设计发夹结构，以抑制检测探针与非目标核酸分子的非特异性结合。然而，已发现，在CN108823287A描述的方法中，使用具有发夹结构的检测探针在某些情况下并不能完全消除非特异性信号。为解决这一问题，本申请的发明人通过大量的实验和创造性劳动，首次发现通过对媒介子探针进行独特设计，可以进一步减少(甚至消除)非特异性信号，并且甚至在一定程度上提升阳性信号的强度，由此完成了本发明。In the probe melting curve analysis method after PCR amplification, non-specific signals (ie, non-specific melting peaks) are usually caused by the following reasons: the detection probe binds non-specifically to non-target sequences, so that the detection probe emits signal (eg, a self-quenching fluorescent probe nonspecifically binds to a non-target nucleic acid molecule such that the fluorophore it carries is separated from the quencher group and thus fluoresces). In order to reduce the generation of such non-specific signals, the detection probe can usually be modified, such as designing a hairpin structure on the detection probe to inhibit the non-specific binding of the detection probe to non-target nucleic acid molecules. However, it has been found that in the method described in CN108823287A, the use of a detection probe with a hairpin structure does not completely eliminate the non-specific signal in some cases. In order to solve this problem, the inventors of the present application, through a lot of experiments and creative work, discovered for the first time that through the unique design of the mediator probe, the non-specific signal can be further reduced (or even eliminated), and the positive signal can even be improved to a certain extent. signal strength, thus completing the present invention.
因此，在一个方面，本申请提供了一种检测n种靶核酸序列在样品中的存在的方法，其中，n为≥1的整数(例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40或更大的整数)，并且，所述方法包括以下步骤：Accordingly, in one aspect, the present application provides a method of detecting the presence of n target nucleic acid sequences in a sample, wherein n is an integer > 1 (eg, n is 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or larger integers), and the method includes the following step:
(1)针对待检测的每一种靶核酸序列，提供一种上游寡核苷酸序列和一种媒介子探针；其中，所述上游寡核苷酸序列包含与所述靶核酸序列互补的序列；(1) For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence;
并且，所述媒介子探针从5'至3'方向包含媒介子序列和靶特异性序列，所述媒介子序列包含不与所述靶核酸序列互补的序列，并且，所述靶特异性序列包含与所述靶核酸序列互补的序列；并且，当与所述靶核酸序列杂交时，所述上游寡核苷酸序列位于所述靶特异性序列的上游；并且，所有媒介子探针所包含的媒介子序列彼此不同；Also, the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator subsequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes comprise The mediator subsequences of are different from each other;
其中，至少一种媒介子探针自身能够形成发夹结构；Wherein, at least one mediator probe itself can form a hairpin structure;
优选地，所述至少一种媒介子探针具有选自下列的特征：Preferably, the at least one mediator probe has characteristics selected from the group consisting of:
(i)所述媒介子探针在其靶特异性序列的下游或3'端还包含第一发夹形成序列，所述第一发夹形成序列与所述媒介子探针的媒介子序列或其部分互补，由此，所述媒介子探针能够通过所述媒介子序列和所述第一发夹形成序列形成发夹结构；(i) the mediator probe further comprises a first hairpin-forming sequence downstream or at the 3' end of its target-specific sequence, the first hairpin-forming sequence and the mediator sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the mediator sequence and the first hairpin-forming sequence;
(ii)所述媒介子探针在其媒介子序列的上游或5'端还包含第二发夹形成序列，所述第二发夹形成序列与所述媒介子探针的靶特异性序列或其部分互补，由此，所述媒介子探针能够通过所述第二发夹形成序列和所述靶特异性序列形成发夹结构；(ii) the mediator probe further comprises a second hairpin-forming sequence upstream or 5' of its mediator sequence, the second hairpin-forming sequence and the target-specific sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the second hairpin-forming sequence and the target-specific sequence;
(iii)所述媒介子探针在其媒介子序列的上游或5'端还包含第三发夹形成序列，且在其靶特异性序列的下游或3'端还包含第四发夹形成序列，且所述第三发夹形成序列或其部分与所述第四发夹形成序列或其部分互补，由此，所述媒介子探针能够通过所述第三发夹形成序列和所述第四发夹形成序列形成发夹结构；(iii) the mediator probe further comprises a third hairpin-forming sequence upstream or 5' end of its mediator sequence, and further comprises a fourth hairpin-forming sequence downstream or 3' end of its target-specific sequence , and the third hairpin-forming sequence or part thereof is complementary to the fourth hairpin-forming sequence or part thereof, whereby the mediator probe can pass through the third hairpin-forming sequence and the third hairpin-forming sequence Four hairpin forming sequences form a hairpin structure;
并且，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列和媒介子探针接触；and, contacting the sample with the provided upstream oligonucleotide sequence and the mediator probe under conditions that allow nucleic acid hybridization;
(2)在允许切割媒介子探针的条件下，将步骤(1)的产物与具有5'核酸酶活性的酶接触；(2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
(3)提供m种检测探针，并且在允许核酸杂交的条件下，将步骤(2)的产物与所述m种检测探针接触，其中，m为大于0的整数，(3) providing m kinds of detection probes, and contacting the product of step (2) with the m kinds of detection probes under conditions that allow nucleic acid hybridization, wherein m is an integer greater than 0,
并且，每一种检测探针各自独立地从3'至5'方向包含，与一种或多种媒介子序列或其部分互补的一种或多种捕获序列，以及模板序列(templating sequence)；并且，所述m种检测探针包含至少n种捕获序列，其分别与步骤(1)中提供的n种媒介子探针的媒介子序列或其部分互补；并且，And, each detection probe independently comprises, from the 3' to 5' direction, one or more capture sequences complementary to one or more mediator subsequences or portions thereof, and a templating sequence; And, the m detection probes comprise at least n capture sequences, which are respectively complementary to the mediator sequences of the n mediator probes provided in step (1) or parts thereof; and,
每一种检测探针各自独立地标记有报告基团和淬灭基团，其中，所述报告基团能够发出信号，并且，所述淬灭基团能够吸收或淬灭所述报告基团发出的信号；并且，每一种检测探针在与其互补序列杂交的情况下发出的信号不同于在未与其互补序列杂交的情况下发出的信号；并且，Each detection probe is independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the reporter group emitting and, each detection probe emits a different signal when hybridized to its complementary sequence than when not hybridized to its complementary sequence; and,
(4)在允许核酸聚合酶进行延伸反应的条件下，将步骤(3)的产物与核酸聚合酶接触；(4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
(5)对步骤(4)的产物进行熔解曲线分析；并根据熔解曲线分析的结果，确定所述n种靶核酸序列是否存在于所述样品中。(5) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
在某些实施方案中，m为小于n且大于0的整数。在此类实施方案中，所使用的 检测探针的数目少于媒介子探针的数目。因此，至少一种检测探针包含两种或更多种捕获序列。在某些实施方案中，m等于n。在此类实施方案中，所使用的检测探针的数目等于媒介子探针的数目。因此，检测探针可以是与媒介子探针一一对应的。In certain embodiments, m is an integer less than n and greater than zero. In such embodiments, the number of detection probes used is less than the number of mediator probes. Thus, at least one detection probe comprises two or more capture sequences. In certain embodiments, m equals n. In such embodiments, the number of detection probes used is equal to the number of mediator probes. Therefore, the detection probes can be in a one-to-one correspondence with the mediator probes.
在某些实施方案中，m为≥1、≥2、≥3、≥4、≥5、≥6、≥8、≥10的整数(例如，m为1、2、3、4、5或6)；优选地，当m≥2时，所述m种检测探针各自标记有不同的报告基团。In certain embodiments, m is an integer >1, >2, >3, >4, >5, >6, >8, >10 (eg, m is 1, 2, 3, 4, 5, or 6 ); preferably, when m≧2, each of the m detection probes is labeled with a different reporter group.
在某些实施方案中，步骤(1)提供了至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少8种、至少10种、至少15种、至少20种、至少25种、至少30种、至少35种、至少40种、至少45种媒介子探针；并且，步骤(3)提供了至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少8种、或至少10种检测探针。In certain embodiments, step (1) provides at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 mediator probes; and, step (3) provides at least 1, at least 2, at least 3, at least 4 , at least 5, at least 6, at least 8, or at least 10 detection probes.
在某些实施方案中，至少1种、至少2种，至少3种，至少4种，至少5种，至少6种，至少10种，至少20种，至少30种，至少40种，至少45种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)。In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 20, at least 30, at least 40, at least 45 The mediator probes are each independently capable of forming a hairpin structure, for example each independently having features (i), (ii) or (iii) as defined in claim 1 .
在某些实施方案中，每一种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)。In certain embodiments, each mediator probe is independently capable of forming a hairpin structure, eg, each independently has features (i), (ii) or (iii) as defined in claim 1 .
在某些实施方案中，所述第一、第二、第三或第四发夹形成序列的长度各自独立地为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt。In certain embodiments, the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
在某些实施方案中，所述m种检测探针包含相同的报告基团；并且，在步骤(5)中，对步骤(4)的产物进行熔解曲线分析，并根据所获得的熔解曲线中的熔解峰(熔点)来确定某一种靶核酸序列的存在。In certain embodiments, the m detection probes comprise the same reporter group; and, in step (5), melting curve analysis is performed on the product of step (4), and according to the obtained melting curve the melting peak (melting point) to determine the presence of a certain target nucleic acid sequence.
在某些实施方案中，所述m种检测探针所包含的报告基团彼此不同；并且，在步骤(5)中，在对步骤(4)的产物进行熔解曲线分析时，分别实时监测每一种报告基团的信号，由此获得各自与一种报告基团的信号对应的多条熔解曲线；随后，根据报告基团的信号种类以及熔解曲线中的熔解峰(熔点)来确定某一种靶核酸序列的存在。In certain embodiments, the reporter groups contained in the m detection probes are different from each other; and, in step (5), when the melting curve analysis is performed on the product of step (4), each real-time monitoring A signal of a reporter group, thereby obtaining a plurality of melting curves corresponding to the signal of a reporter group; the presence of a target nucleic acid sequence.
在某些实施方案中，其中，m＝1，并且n为≥1的整数(例如，n为1、2、3、4、 5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更大的整数)。In certain embodiments, wherein m=1, and n is an integer > 1 (eg, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or larger integers).
在某些实施方案中，所述方法包括下述步骤：In certain embodiments, the method includes the steps of:
(1)针对待检测的每一种靶核酸序列，提供一种上游寡核苷酸序列和一种媒介子探针；其中，所述上游寡核苷酸序列包含与所述靶核酸序列互补的序列；并且，所述媒介子探针从5'至3'方向包含媒介子序列和靶特异性序列，所述媒介子序列包含不与所述靶核酸序列互补的序列，并且，所述靶特异性序列包含与所述靶核酸序列互补的序列；并且，当与所述靶核酸序列杂交时，所述上游寡核苷酸序列位于所述靶特异性序列的上游；并且，所有媒介子探针所包含的媒介子序列彼此不同；(1) For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence; and, the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator sequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes The included mediator subsequences differ from each other;
其中，至少一种媒介子探针自身能够形成发夹结构；例如，所述至少一种媒介子探针具有权利要求1中定义的特征(i)，(ii)或(iii)；wherein the at least one mediator probe itself is capable of forming a hairpin structure; for example, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1;
并且，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列和媒介子探针接触；and, contacting the sample with the provided upstream oligonucleotide sequence and the mediator probe under conditions that allow nucleic acid hybridization;
(2)在允许切割媒介子探针的条件下，将步骤(1)的产物与具有5'核酸酶活性的酶接触；(2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
(3)在允许核酸杂交的条件下，将步骤(2)的产物与一种检测探针接触，所述检测探针从3'至5'方向包含，与每一种媒介子序列或其部分互补的捕获序列，以及模板序列(templating sequence)；并且，所述检测探针标记有报告基团和淬灭基团，其中，所述报告基团能够发出信号，并且，所述淬灭基团能够吸收或淬灭所述报告基团发出的信号；并且，所述检测探针在与其互补序列杂交的情况下发出的信号不同于在未与其互补序列杂交的情况下发出的信号；(3) contacting the product of step (2) with a detection probe comprising from the 3' to 5' direction, under conditions allowing nucleic acid hybridization, with each of the mediator subsequences or portions thereof a complementary capture sequence, and a templating sequence; and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of signaling, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and, the detection probe emits a signal when hybridized to its complementary sequence that is different from the signal emitted when it is not hybridized to its complementary sequence;
(4)在允许核酸聚合酶进行延伸反应的条件下，将步骤(3)的产物与核酸聚合酶接触；(4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
(5)对步骤(4)的产物进行熔解曲线分析；并根据熔解曲线分析的结果，确定所述n种靶核酸序列是否存在于所述样品中。(5) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
在本申请的方法中，样品可以是任何待检测的样品。在某些实施方案中，所述样品包含或是DNA，或RNA，或核酸的混合物。在某些实施方案中，所述靶核酸序列是DNA或RNA；和/或，所述靶核酸序列是单链的或双链的。在某些实施方案中，所述样品或靶核酸序列获自选自下列的来源：原核生物，真核生物，病毒或类病毒。In the methods of the present application, the sample can be any sample to be detected. In certain embodiments, the sample comprises or is a mixture of DNA, or RNA, or nucleic acids. In certain embodiments, the target nucleic acid sequence is DNA or RNA; and/or, the target nucleic acid sequence is single-stranded or double-stranded. In certain embodiments, the sample or target nucleic acid sequence is obtained from a source selected from prokaryotes, eukaryotes, viruses, or viroids.
在某些实施方案中，所述媒介子探针包含或者由天然存在的核苷酸，经修饰的核 苷酸，非天然的核苷酸，或其任何组合组成。在某些优选的实施方案中，媒介子探针包含或者由天然的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)组成。在某些优选的实施方案中，媒介子探针包含经修饰的核苷酸，例如经修饰的脱氧核糖核苷酸或核糖核苷酸，例如5-甲基胞嘧啶或5-羟甲基胞嘧啶。在某些优选的实施方案中，媒介子探针包含非天然的核苷酸，例如脱氧次黄嘌呤，肌苷，1-(2'-脱氧-β-D-呋喃核糖基)-3-硝基吡咯，5-硝基吲哚或锁核酸(LNA)。In certain embodiments, the mediator probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the mediator probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the mediator probes comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine pyrimidine. In certain preferred embodiments, the mediator probe comprises non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitro pyrrole, 5-nitroindole or locked nucleic acid (LNA).
在某些实施方案中，所述媒介子探针的长度为15-150nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt，140-150nt。In certain embodiments, the mediator probe is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80 -90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
在某些实施方案中，所述媒介子探针中的靶特异性序列的长度为10-140nt，例如10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt。In certain embodiments, the target-specific sequence in the mediator probe is 10-140nt in length, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
在某些实施方案中，所述媒介子探针中的媒介子序列的长度可以为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt。In certain embodiments, the length of the mediator sequence in the mediator probe can be 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt , 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
不受理论限制，本申请发明人认为，尽管检测探针已被设计为具有稳定的发夹结构，但媒介子探针与检测探针仍然可能发生非特异性结合，这可使得检测探针的发夹结构被打开，导致其所携带的荧光基团与淬灭基团发生分离，进而导致非特异性信号的产生。因此，通过对媒介子探针进行改造(即，在其靶特异性序列的下游或3'端添加第一发夹形成序列、或者在其媒介子序列的上游或5'端添加第二发夹形成序列、或者在其媒介子序列的上游或5'端添加第三发夹形成序列，且在其靶特异性序列的下游或3'端添加第四发夹形成序列)，使媒介子探针自身能够形成发夹结构(借助于媒介子序列和第一发夹形成序列之间的碱基互补配对、或者第二发夹形成序列和靶特异性序列之间的碱基互补配对、或者第三发夹形成序列和第四发夹形成序列之间的碱基互补配对)，可进一步降低甚至消除媒介子探针与检测探针的非特异性结合，从而进一步降低甚至消除非特异性信号的产生。因此，在本申请的方法中，第一、第二、第三或第四发夹形成序列可以为任意的长度，而不受限制，只要其能够使媒介子探针形成发夹结构即可。然而，易于理解，如果第一、第二、第三或第四发夹形成序列的长度太短，那么媒介子探针所形成的发夹结构的稳定性将会下降，这可能影响(例如增加)媒介子探针与检测探针的非特异性结合。反之，如果第一、第二、第三或第 四发夹形成序列的长度太长，那么媒介子探针所形成的发夹结构的稳定性将会增加，这可能影响(例如减少)媒介子探针与靶核酸序列的特异性结合。因此，本领域技术人员可以根据实际需要，确定第一、第二、第三或第四发夹形成序列的最佳长度。在某些实施方案中，所述第一、第二、第三或第四发夹形成序列的长度各自独立地为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt。Without being limited by theory, the inventors of the present application believe that although the detection probe has been designed to have a stable hairpin structure, non-specific binding of the mediator probe to the detection probe may still occur, which can make the hairpin of the detection probe possible. The clip structure is opened, resulting in the separation of the fluorophore and the quencher group it carries, which in turn leads to the generation of a non-specific signal. Therefore, by engineering the mediator probe (ie, adding a first hairpin-forming sequence downstream or 3' of its target-specific sequence, or adding a second hairpin upstream or 5' of its mediator sequence forming sequence, or adding a third hairpin forming sequence upstream or 5' end of its mediator sequence, and adding a fourth hairpin forming sequence downstream or 3' end of its target-specific sequence), making the mediator probe Capable of forming a hairpin structure by itself (by means of complementary base pairing between an intermediary subsequence and a first hairpin forming sequence, or base pairing between a second hairpin forming sequence and a target-specific sequence, or a third The complementary base pairing between the hairpin-forming sequence and the fourth hairpin-forming sequence) can further reduce or even eliminate the non-specific binding between the mediator probe and the detection probe, thereby further reducing or even eliminating the generation of non-specific signals. Therefore, in the method of the present application, the first, second, third or fourth hairpin-forming sequence can be of any length without limitation as long as it enables the mediator probe to form a hairpin structure. However, it is readily understood that if the length of the first, second, third or fourth hairpin-forming sequence is too short, the stability of the hairpin structure formed by the mediator probe will decrease, which may affect (eg increase the ) non-specific binding of the mediator probe to the detection probe. Conversely, if the length of the first, second, third or fourth hairpin-forming sequence is too long, then the stability of the hairpin structure formed by the mediator probe will increase, which may affect (eg reduce) the mediator. Specific binding of probes to target nucleic acid sequences. Therefore, those skilled in the art can determine the optimal length of the first, second, third or fourth hairpin forming sequence according to actual needs. In certain embodiments, the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
此外，还有意外发现：除了降低非特异性信号之外，具有发夹结构的媒介子探针可能带来额外的潜在益处：能够增强阳性信号的强度，提高检测的灵敏度。不受理论限制，本申请发明人认为，具有发夹结构的媒介子探针的使用，降低了媒介子探针与检测探针的非特异性结合，这使得有反应体系中有更多的检测探针能够与切割下来的媒介子片段结合，从而针对同一阳性样品，可产生更强的阳性信号。In addition, it was unexpectedly discovered that, in addition to reducing non-specific signals, mediator probes with hairpin structures may bring additional potential benefits: the ability to enhance the intensity of positive signals and improve the sensitivity of detection. Without being limited by theory, the inventors of the present application believe that the use of the mediator probe with a hairpin structure reduces the non-specific binding between the mediator probe and the detection probe, which allows more detection probes in the reaction system. The needle can bind to the cleaved mediator fragment, resulting in a stronger positive signal for the same positive sample.
在某些实施方案中，所述媒介子探针的靶特异性序列与第一发夹形成序列之间还含有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is also included between the target-specific sequence of the mediator probe and the first hairpin-forming sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所述媒介子探针的第二发夹形成序列与媒介子序列之间还含有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所述媒介子探针的第三发夹形成序列与媒介子序列之间还含有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is also included between the third hairpin-forming sequence of the mediator probe and the mediator sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所述媒介子探针的靶特异性序列与第四发夹形成序列之间还含有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is further included between the target-specific sequence of the mediator probe and the fourth hairpin-forming sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所述第一发夹形成序列与所述媒介子序列完全互补或部分互补。在某些实施方案中，所述媒介子探针能够通过所述媒介子序列和所述第一发夹形成序列形成发夹结构，其中，所述发夹结构的臂具有平末端(即，不具有悬突)，或者所述臂具有5’悬突(例如，其5’端具有至少1个，至少2个，或者更多个游离碱基)，或者所述臂具有3’悬突(例如，其3’端具有至少1个，至少2个，或者更多个游离碱基)。In certain embodiments, the first hairpin-forming sequence is fully or partially complementary to the mediator sequence. In certain embodiments, the mediator probe is capable of forming a hairpin structure through the mediator sequence and the first hairpin-forming sequence, wherein the arms of the hairpin structure have blunt ends (ie, do not with an overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang (eg, , its 3' end has at least 1, at least 2, or more free bases).
在某些实施方案中，所述臂由完全互补的第一发夹形成序列与媒介子序列构成。由此，所述臂是对称的或具有平末端。在某些实施方案中，所述臂由部分互补的第一发夹形成序列与媒介子序列构成。由此，所述臂是非对称的或具有5’或3’悬突。In certain embodiments, the arm consists of a fully complementary first hairpin-forming sequence and a mediator subsequence. Thus, the arms are symmetrical or have blunt ends. In certain embodiments, the arm consists of a partially complementary first hairpin-forming sequence and a mediator subsequence. Thus, the arms are asymmetric or have 5' or 3' overhangs.
在某些实施方案中，所述第一发夹形成序列与所述媒介子序列的长度相同。在此类实施方案中，所述臂可具有平末端。In certain embodiments, the first hairpin-forming sequence is the same length as the mediator subsequence. In such embodiments, the arms may have blunt ends.
在某些实施方案中，所述第一发夹形成序列与所述媒介子序列的长度不同。例如，所述第一发夹形成序列的长度与所述媒介子序列的长度可相差1-10nt，例如1，2，3，4，5，6，7，8，9，10nt。在此类实施方案中，所述臂可具有5’或3’悬突。在某些实施方案中，所述第一发夹形成序列的长度比所述媒介子序列的长度多1-10nt，例如1，2，3，4，5，6，7，8，9，10nt。在某些实施方案中，所述媒介子序列的长度比所述第一发夹形成序列的长度多1-10nt，例如1，1，2，3，4，5，6，7，8，9，10nt。In certain embodiments, the first hairpin-forming sequence is different in length from the mediator subsequence. For example, the length of the first hairpin forming sequence and the length of the mediator subsequence may differ by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt. In such embodiments, the arms may have 5' or 3' overhangs. In certain embodiments, the length of the first hairpin-forming sequence is 1-10 nt longer than the length of the mediator subsequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt . In certain embodiments, the length of the mediator subsequence is 1-10 nt longer than the length of the first hairpin forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10nt.
在某些实施方案中，所述第二发夹形成序列与所述靶特异性序列完全互补或部分互补。在某些实施方案中，所述媒介子探针能够通过所述第二发夹形成序列和所述靶特异性序列形成发夹结构，其中，所述发夹结构的臂具有平末端(即，不具有悬突)，或者所述臂具有5’悬突(例如，其5’端具有至少1个，至少2个，或者更多个游离碱基)，或者所述臂具有3’悬突(例如，其3’端具有至少1个，至少2个，或者更多个游离碱基)。In certain embodiments, the second hairpin-forming sequence is fully or partially complementary to the target-specific sequence. In certain embodiments, the mediator probe is capable of forming a hairpin structure via the second hairpin-forming sequence and the target-specific sequence, wherein the arms of the hairpin structure have blunt ends (ie, no overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang ( For example, it has at least 1, at least 2, or more free bases at its 3' end).
在某些实施方案中，所述臂由完全互补的第二发夹形成序列与靶特异性序列构成。由此，所述臂是对称的或具有平末端。在某些实施方案中，所述臂由部分互补的第二发夹形成序列与靶特异性序列构成。由此，所述臂是非对称的或具有5’或3’悬突。In certain embodiments, the arm consists of a fully complementary second hairpin-forming sequence and a target-specific sequence. Thus, the arms are symmetrical or have blunt ends. In certain embodiments, the arm consists of a partially complementary second hairpin-forming sequence and a target-specific sequence. Thus, the arms are asymmetric or have 5' or 3' overhangs.
在某些实施方案中，所述第二发夹形成序列与所述靶特异性序列的长度相同。在此类实施方案中，所述臂可具有平末端。In certain embodiments, the second hairpin-forming sequence is the same length as the target-specific sequence. In such embodiments, the arms may have blunt ends.
在某些实施方案中，所述第二发夹形成序列与所述靶特异性序列的长度不同。例如，所述第二发夹形成序列的长度与所述靶特异性序列的长度可相差1-10nt，例如1，2，3，4，5，6，7，8，9，10nt。在此类实施方案中，所述臂可具有5’或3’悬突。在某些实施方案中，所述第二发夹形成序列的长度比所述靶特异性序列的长度多1-10nt，例如1，2，3，4，5，6，7，8，9，10nt。在某些实施方案中，所述靶特异性序列的长度比所述第二发夹形成序列的长度多1-10nt，例如1，1，2，3， 4，5，6，7，8，9，10nt。In certain embodiments, the second hairpin-forming sequence is not the same length as the target-specific sequence. For example, the length of the second hairpin forming sequence may differ from the length of the target-specific sequence by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt. In such embodiments, the arms may have 5' or 3' overhangs. In certain embodiments, the length of the second hairpin-forming sequence is 1-10 nt longer than the length of the target-specific sequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt. In certain embodiments, the length of the target-specific sequence is 1-10 nt longer than the length of the second hairpin forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
在某些实施方案中，所述第三发夹形成序列与所述第四发夹形成序列完全互补或部分互补。在某些实施方案中，所述媒介子探针能够通过所述第三发夹形成序列和所述第四发夹形成序列形成发夹结构，其中，所述发夹结构的臂具有平末端(即，不具有悬突)，或者所述臂具有5’悬突(例如，其5’端具有至少1个，至少2个，或者更多个游离碱基)，或者所述臂具有3’悬突(例如，其3’端具有至少1个，至少2个，或者更多个游离碱基)。In certain embodiments, the third hairpin-forming sequence is fully or partially complementary to the fourth hairpin-forming sequence. In certain embodiments, the mediator probe is capable of forming a hairpin structure via the third hairpin-forming sequence and the fourth hairpin-forming sequence, wherein the arms of the hairpin structure have blunt ends ( That is, no overhang), or the arm has a 5' overhang (eg, it has at least 1, at least 2, or more free bases at its 5' end), or the arm has a 3' overhang Overhang (eg, having at least 1, at least 2, or more free bases at its 3' end).
在某些实施方案中，所述臂由完全互补的第三发夹形成序列与第四发夹形成序列构成。由此，所述臂是对称的或具有平末端。在某些实施方案中，所述臂由部分互补的第三发夹形成序列与第四发夹形成序列构成。由此，所述臂是非对称的或具有5’或3’悬突。In certain embodiments, the arms are comprised of fully complementary third and fourth hairpin-forming sequences. Thus, the arms are symmetrical or have blunt ends. In certain embodiments, the arms are comprised of partially complementary third and fourth hairpin-forming sequences. Thus, the arms are asymmetric or have 5' or 3' overhangs.
在某些实施方案中，所述第三发夹形成序列与所述第四发夹形成序列的长度相同。在此类实施方案中，所述臂可具有平末端。In certain embodiments, the third hairpin-forming sequence is the same length as the fourth hairpin-forming sequence. In such embodiments, the arms may have blunt ends.
在某些实施方案中，所述第三发夹形成序列与所述第四发夹形成序列的长度不同。例如，所述第三发夹形成序列的长度与所述第四发夹形成序列的长度可相差1-10nt，例如1，2，3，4，5，6，7，8，9，10nt。在此类实施方案中，所述臂可具有5’或3’悬突。在某些实施方案中，所述第三发夹形成序列的长度比所述第四发夹形成序列的长度多1-10nt，例如1，2，3，4，5，6，7，8，9，10nt。在某些实施方案中，所述第四发夹形成序列的长度比所述第三发夹形成序列的长度多1-10nt，例如1，1，2，3，4，5，6，7，8，9，10nt。In certain embodiments, the third hairpin-forming sequence is different in length from the fourth hairpin-forming sequence. For example, the length of the third hairpin forming sequence may differ from the length of the fourth hairpin forming sequence by 1-10 nt, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nt. In such embodiments, the arms may have 5' or 3' overhangs. In certain embodiments, the length of the third hairpin-forming sequence is 1-10 nt longer than the length of the fourth hairpin-forming sequence, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt. In certain embodiments, the length of the fourth hairpin-forming sequence is 1-10 nt longer than the length of the third hairpin-forming sequence, eg, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10nt.
在某些实施方案中，所述媒介子探针具有3'-OH末端，或者其3'-末端是封闭的。在某些优选的实施方案中，媒介子探针的3'-末端是封闭的，以抑制其延伸。可通过各种方法来封闭核酸(例如媒介子探针)的3'-末端。例如，可通过对媒介子探针的最后一个核苷酸的3'-OH进行修饰，以封闭媒介子探针的3'-末端。在某些实施方案中，可通过在媒介子探针的最后一个核苷酸的3'-OH上添加化学部分(例如，生物素或烷基)，从而封闭媒介子探针的3'-末端。在某些实施方案中，可通过将媒介子探针的最后一个核苷酸的3'-OH去除，或者将所述最后一个核苷酸替换为双脱氧核苷酸，从而封闭媒介子探针的3'-末端。In certain embodiments, the mediator probe has a 3'-OH terminus, or its 3'-terminus is blocked. In certain preferred embodiments, the 3'-terminus of the mediator probe is blocked to inhibit its extension. The 3'-terminus of nucleic acids (eg, mediator probes) can be blocked by various methods. For example, the 3'-terminus of the mediator probe can be blocked by modifying the 3'-OH of the last nucleotide of the mediator probe. In certain embodiments, the 3'-terminus of the mediator probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the mediator probe . In certain embodiments, the mediator probe can be blocked by removing the 3'-OH of the last nucleotide of the mediator probe, or by replacing the last nucleotide with a dideoxynucleotide 3'-end.
在某些实施方案中，所述上游寡核苷酸序列包含或者由天然存在的核苷酸，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成。在某些优选的实施方案中，上 游寡核苷酸序列包含或者由天然的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)组成。在某些优选的实施方案中，上游寡核苷酸序列包含经修饰的核苷酸，例如经修饰的脱氧核糖核苷酸或核糖核苷酸，例如5-甲基胞嘧啶或5-羟甲基胞嘧啶。在某些优选的实施方案中，上游寡核苷酸序列包含非天然的核苷酸，例如脱氧次黄嘌呤，肌苷，1-(2'-脱氧-β-D-呋喃核糖基)-3-硝基吡咯，5-硝基吲哚或锁核酸(LNA)。In certain embodiments, the upstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the upstream oligonucleotide sequence comprises or consists of natural nucleotides such as deoxyribonucleotides or ribonucleotides. In certain preferred embodiments, the upstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethyl base cytosine. In certain preferred embodiments, the upstream oligonucleotide sequence comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
在本申请的方法中，上游寡核苷酸序列不受其长度的限制，只要其能够与靶核酸序列特异性杂交。在某些实施方案中，所述上游寡核苷酸序列的长度为15-150nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt，140-150nt。In the method of the present application, the upstream oligonucleotide sequence is not limited by its length, as long as it can specifically hybridize to the target nucleic acid sequence. In certain embodiments, the upstream oligonucleotide sequence is 15-150nt in length, eg, 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
在某些实施方案中，所述上游寡核苷酸序列在与靶核酸序列杂交后，位于媒介子探针的上游远端，或位于媒介子探针的上游邻近，或与媒介子探针的靶特异性序列具有部分重叠的序列。In certain embodiments, the upstream oligonucleotide sequence, after hybridization to the target nucleic acid sequence, is located at the upstream distal end of the mediator probe, or is located upstream adjacent to the mediator probe, or is adjacent to the mediator probe. Target-specific sequences have partially overlapping sequences.
在某些实施方案中，所述上游寡核苷酸序列为特异于靶核酸序列的引物或者特异于靶核酸序列的探针。In certain embodiments, the upstream oligonucleotide sequence is a primer specific for a target nucleic acid sequence or a probe specific for a target nucleic acid sequence.
在某些实施方案中，在步骤(2)中，所述具有5'核酸酶活性的酶切割与靶核酸序列杂交的媒介子探针，并释放出包含完整媒介子序列或者媒介子序列的一部分(5'-末端部分)的媒介子片段。In certain embodiments, in step (2), the enzyme with 5' nuclease activity cleaves the mediator probe hybridized to the target nucleic acid sequence, and releases the entire mediator sequence or a portion of the mediator sequence (5'-end portion) of the mediator fragment.
在某些实施方案中，所述具有5'核酸酶活性的酶为具有5'核酸酶活性(例如5'外切核酸酶活性)的核酸聚合酶(例如，DNA聚合酶，特别是热稳定的DNA聚合酶)。In certain embodiments, the enzyme having 5' nuclease activity is a nucleic acid polymerase (eg, a DNA polymerase, particularly a thermostable) having 5' nuclease activity (eg, 5' exonuclease activity) DNA polymerase).
在某些实施方案中，所述DNA聚合酶获自选自下列的细菌：Thermus aquaticus(Taq),Thermus thermophiles(Tth),Thermus filiformis,Thermis flavus,Thermococcus literalis,Thermus antranildanii,Thermus caldophllus,Thermus chliarophilus,Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima,Thermotoga neapolitana,Thermosiphoafricanus,Pyrococcus woesei,Pyrococcus horikoshii,Pyrococcus  abyssi,Pyrodictium occultum,Aquifexpyrophilus和Aquifex aeolieus。在某些优选地实施方案中，所述DNA聚合酶为Taq聚合酶。In certain embodiments, the DNA polymerase is obtained from a bacterium selected from the group consisting of: Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima,Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeolieus. In certain preferred embodiments, the DNA polymerase is Taq polymerase.
在某些实施方案中，在步骤(2)中，所述具有5'核酸酶活性的核酸聚合酶催化上游寡核苷酸序列的延伸，并诱导媒介子探针的切割。In certain embodiments, in step (2), the nucleic acid polymerase having 5' nuclease activity catalyzes the extension of the upstream oligonucleotide sequence and induces cleavage of the mediator probe.
在某些实施方案中，在步骤(2)中，使用核酸聚合酶以靶核酸序列为模板催化上游寡核苷酸序列的延伸，并且随后，所述具有5'核酸酶活性的酶结合至上游寡核苷酸序列的延伸产物，并催化媒介子探针的切割。In certain embodiments, in step (2), a nucleic acid polymerase is used to catalyze the extension of the upstream oligonucleotide sequence using the target nucleic acid sequence as a template, and then the enzyme having 5' nuclease activity is bound to the upstream An extension product of an oligonucleotide sequence and catalyzes cleavage of the mediator probe.
在某些优选的实施方案中，在步骤(1)和/或(2)中，还将样品与特异于靶核酸序列的下游寡核苷酸序列(或下游引物)接触。在某些实施方案中，核酸聚合酶和下游寡核苷酸序列(或下游引物)的使用是特别有利的。特别地，核酸聚合酶能够以靶核酸序列为模板，以上游寡核苷酸序列和下游寡核苷酸序列为引物，产生额外的靶核酸序列，从而可提高本发明方法的灵敏度。In certain preferred embodiments, in steps (1) and/or (2), the sample is also contacted with a downstream oligonucleotide sequence (or downstream primer) specific for the target nucleic acid sequence. In certain embodiments, the use of nucleic acid polymerases and downstream oligonucleotide sequences (or downstream primers) is particularly advantageous. In particular, the nucleic acid polymerase can use the target nucleic acid sequence as a template and the upstream oligonucleotide sequence and the downstream oligonucleotide sequence as primers to generate additional target nucleic acid sequences, thereby improving the sensitivity of the method of the present invention.
因此，在某些优选的实施方案中，在步骤(1)中，除了上文所定义的所述上游寡核苷酸序列和媒介子探针之外，针对待检测的每一种靶核酸序列，还提供一种下游寡核苷酸序列；其中，所述下游寡核苷酸序列包含与所述靶核酸序列互补的序列；并且，当与所述靶核酸序列杂交时，所述下游寡核苷酸序列位于所述靶特异性序列的下游；然后，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列、媒介子探针和下游寡核苷酸序列接触。Therefore, in certain preferred embodiments, in step (1), in addition to the upstream oligonucleotide sequences and mediator probes as defined above, for each target nucleic acid sequence to be detected , also provides a downstream oligonucleotide sequence; wherein, the downstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized with the target nucleic acid sequence, the downstream oligonucleotide sequence The nucleotide sequence is located downstream of the target-specific sequence; the sample is then contacted with the provided upstream oligonucleotide sequence, mediator probe, and downstream oligonucleotide sequence under conditions that allow nucleic acid hybridization .
在某些实施方案中，在步骤(2)中，在允许核酸扩增的条件下，将步骤(1)的产物与具有5'核酸酶活性的核酸聚合酶接触。In certain embodiments, in step (2), the product of step (1) is contacted with a nucleic acid polymerase having 5' nuclease activity under conditions that allow nucleic acid amplification.
在某些实施方案中所述下游寡核苷酸序列包含或者由天然存在的核苷酸，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成。在某些优选的实施方案中，下游寡核苷酸序列包含或者由天然的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)组成。在某些优选的实施方案中，下游寡核苷酸序列包含经修饰的核苷酸，例如经修饰的脱氧核糖核苷酸或核糖核苷酸，例如5-甲基胞嘧啶或5-羟甲基胞嘧啶。在某些优选的实施方案中，下游寡核苷酸序列包含非天然的核苷酸，例如脱氧次黄嘌呤，肌苷，1-(2'-脱氧-β-D-呋喃核糖基)-3-硝基吡咯，5-硝基吲哚或锁核酸(LNA)。In certain embodiments the downstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the downstream oligonucleotide sequence comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the downstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethyl base cytosine. In certain preferred embodiments, the downstream oligonucleotide sequence comprises non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3 - Nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).
在本申请的方法中，下游寡核苷酸序列不受其长度的限制，只要其能够与靶核酸序列特异性杂交。在某些实施方案中，所述下游寡核苷酸序列的长度为15-150nt， 例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt，140-150nt。In the method of the present application, the downstream oligonucleotide sequence is not limited by its length, as long as it can specifically hybridize to the target nucleic acid sequence. In certain embodiments, the downstream oligonucleotide sequence is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
在某些实施方案中，步骤(1)中提供的所有上游寡核苷酸序列和下游寡核苷酸序列在5'端具有一段相同的寡核苷酸序列。In certain embodiments, all upstream and downstream oligonucleotide sequences provided in step (1) have an identical stretch of oligonucleotide sequence at the 5' end.
在某些实施方案中，在步骤(1)中，除了所述上游寡核苷酸序列、媒介子探针和下游寡核苷酸序列之外，还提供一种通用引物，所述通用引物具有与所述相同的寡核苷酸序列互补的序列；然后，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列、媒介子探针、下游寡核苷酸序列和通用引物接触。In certain embodiments, in step (1), in addition to the upstream oligonucleotide sequence, the mediator probe and the downstream oligonucleotide sequence, a universal primer is provided, the universal primer has a sequence complementary to the same oligonucleotide sequence; the sample is then combined with the provided upstream oligonucleotide sequence, mediator probe, downstream oligonucleotide sequence under conditions that allow nucleic acid hybridization Contact with universal primers.
在某些优选的实施方案中，所述相同的寡核苷酸序列的长度为8-50nt，例如8-15nt，15-20nt，20-30nt，30-40nt，或40-50nt。相应地，所述通用引物的长度可以为8-50nt，例如8-15nt，15-20nt，20-30nt，30-40nt，或40-50nt。In certain preferred embodiments, the identical oligonucleotide sequences are 8-50 nt in length, eg, 8-15 nt, 15-20 nt, 20-30 nt, 30-40 nt, or 40-50 nt. Accordingly, the length of the universal primer may be 8-50nt, eg, 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
在本发明的某些实施方案中，通用引物可以包含或者由天然存在的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成。在某些优选的实施方案中，通用引物包含或者由天然的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)组成。在某些优选的实施方案中，通用引物包含经修饰的核苷酸，例如经修饰的脱氧核糖核苷酸或核糖核苷酸，例如5-甲基胞嘧啶或5-羟甲基胞嘧啶。在某些优选的实施方案中，通用引物包含非天然的核苷酸，例如脱氧次黄嘌呤，肌苷，1-(2'-脱氧-β-D-呋喃核糖基)-3-硝基吡咯，5-硝基吲哚或锁核酸(LNA)。In certain embodiments of the invention, universal primers may comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the universal primers comprise or consist of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the universal primers comprise modified nucleotides, eg, modified deoxyribonucleotides or ribonucleotides, eg, 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the universal primers comprise non-natural nucleotides such as deoxyhypoxanthine, inosine, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole , 5-nitroindole or locked nucleic acid (LNA).
在本申请的方法中，通用引物不受其长度的限制，只要其能够与上游和下游寡核苷酸序列中包含的所述相同的寡核苷酸序列特异性杂交。例如，通用引物的长度可以为8-50nt，例如8-15nt，15-20nt，20-30nt，30-40nt，或40-50nt。In the method of the present application, the universal primer is not limited by its length, as long as it can specifically hybridize to the same oligonucleotide sequence contained in the upstream and downstream oligonucleotide sequences. For example, a universal primer can be 8-50nt in length, such as 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
在某些实施方案中，所述检测探针包含多种捕获序列；并且，所述多种捕获序列以相邻的方式、以间隔有连接序列的方式，或者以重叠的方式排列。In certain embodiments, the detection probe comprises a plurality of capture sequences; and, the plurality of capture sequences are arranged adjacently, separated by linker sequences, or overlapping.
在某些实施方案中，所述检测探针包含或者由天然存在的核苷酸，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成。在某些优选的实施方案中，检测探针包含或者由天然的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)组成。在某些优选的实施方案中，检测探针包含经修饰的核苷酸，例如经修饰的脱氧核糖核苷酸或核糖 核苷酸，例如5-甲基胞嘧啶或5-羟甲基胞嘧啶。在某些优选的实施方案中，检测探针包含非天然的核苷酸，例如脱氧次黄嘌呤，肌苷，1-(2'-脱氧-β-D-呋喃核糖基)-3-硝基吡咯，5-硝基吲哚或锁核酸(LNA)。In certain embodiments, the detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the detection probe comprises or consists of natural nucleotides (eg, deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the detection probes comprise modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine . In certain preferred embodiments, the detection probe comprises non-natural nucleotides such as deoxyhyosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitro Pyrrole, 5-nitroindole or locked nucleic acid (LNA).
在某些实施方案中，所述检测探针的长度为15-1000nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-200nt，200-300nt，300-400nt，400-500nt，500-600nt，600-700nt，700-800nt，800-900nt，900-1000nt。In certain embodiments, the detection probe is 15-1000nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80- 90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000nt.
检测探针中的捕获序列可以是任何长度，只要其能够与媒介子片段特异性杂交。在某些实施方案中，所述检测探针中的捕获序列的长度为10-500nt，例如10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-150nt，150-200nt，200-250nt，250-300nt，300-350nt，350-400nt，400-450nt，450-500nt。The capture sequence in the detection probe can be of any length as long as it can specifically hybridize to the mediator fragment. In certain embodiments, the capture sequence in the detection probe is 10-500nt in length, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-nt 80nt, 80-90nt, 90-100nt, 100-150nt, 150-200nt, 200-250nt, 250-300nt, 300-350nt, 350-400nt, 400-450nt, 450-500nt.
检测探针中的模板序列可以是任何长度，只要其能够用作延伸媒介子片段的模板。在某些实施方案中，所述检测探针中的模板序列的长度为1-900nt，例如1-5nt，5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-200nt，200-300nt，300-400nt，400-500nt，500-600nt，600-700nt，700-800nt，800-900nt。The template sequence in the detection probe can be of any length as long as it can be used as a template for extending the mediator subfragment. In certain embodiments, the length of the template sequence in the detection probe is 1-900nt, such as 1-5nt, 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-nt 60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt.
在某些实施方案中，所述检测探针具有3'-OH末端，或者其3'-末端是封闭的，以抑制其延伸。可通过各种方法来封闭核酸(例如检测探针)的3'-末端。例如，可通过对检测探针的最后一个核苷酸的3'-OH进行修饰，以封闭检测探针的3'-末端。在某些实施方案中，可通过在检测探针的最后一个核苷酸的3'-OH上添加化学部分(例如，生物素或烷基)，从而封闭检测探针的3'-末端。在某些实施方案中，可通过将检测探针的最后一个核苷酸的3'-OH去除，或者将所述最后一个核苷酸替换为双脱氧核苷酸，从而封闭检测探针的3'-末端。In certain embodiments, the detection probe has a 3'-OH terminus, or its 3'-terminus is blocked to inhibit its extension. The 3'-terminus of nucleic acids (eg, detection probes) can be blocked by various methods. For example, the 3'-terminus of the detection probe can be blocked by modifying the 3'-OH of the last nucleotide of the detection probe. In certain embodiments, the 3'-terminus of the detection probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the detection probe. In certain embodiments, the 3'-OH of the detection probe can be blocked by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide. '-end.
在某些实施方案中，所述检测探针为自淬灭探针；例如，所述检测探针在其5'末端或上游标记有报告基团且在其3'末端或下游标记有淬灭基团，或者在其3'末端或下游标记报告基团且在5'末端或上游标记淬灭基团。在某些实施方案中，所述报告基团和淬灭基团相距10-80nt或更长的距离。In certain embodiments, the detection probe is a self-quenching probe; eg, the detection probe is labeled with a reporter group at its 5' end or upstream and labeled with a quencher at its 3' end or downstream group, or a reporter group is labeled at its 3' end or downstream and a quencher group is labeled at its 5' end or upstream. In certain embodiments, the reporter group and the quencher group are separated by a distance of 10-80 nt or more.
在某些实施方案中，所述检测探针中的报告基团为荧光基团(例如ALEX-350,FAM,VIC,TET,CAL
Figure PCTCN2020140053-appb-000001
Gold 540,JOE,HEX,CAL Fluor Orange 560,TAMRA, CAL Fluor Red 590,ROX,CAL Fluor Red 610,TEXAS RED,CAL Fluor Red 635,Quasar 670,CY3,CY5,CY5.5,Quasar 705)；并且，淬灭基团为能够吸收/淬灭所述荧光的分子或基团(例如DABCYL、BHQ(例如BHQ-1或者BHQ-2)、ECLIPSE、和/或TAMRA)。 In certain embodiments, the reporter group in the detection probe is a fluorophore (eg ALEX-350, FAM, VIC, TET, CAL
Figure PCTCN2020140053-appb-000001
Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and , a quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).
在某些实施方案中，所述检测探针具有修饰或不具有修饰。例如，所述检测探针具有抵抗核酸酶活性(例如5'核酸酶活性，例如5'至3'核酸外切酶活性)的抗性；例如，所述检测探针的主链包含抵抗核酸酶活性的修饰，例如硫代磷酸酯键，烷基磷酸三酯键，芳基磷酸三酯键，烷基膦酸酯键，芳基膦酸酯键，氢化磷酸酯键，烷基氨基磷酸酯键，芳基氨基磷酸酯键，2'-O-氨基丙基修饰，2'-O-烷基修饰，2'-O-烯丙基修饰，2'-O-丁基修饰，和1-(4'-硫代-PD-呋喃核糖基)修饰。In certain embodiments, the detection probes are modified or not. For example, the detection probe is resistant to nuclease activity (eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity); eg, the backbone of the detection probe comprises nuclease resistant Reactive modifications such as phosphorothioate bonds, alkyl phosphotriester bonds, aryl phosphotriester bonds, alkyl phosphonate bonds, aryl phosphonate bonds, hydrophosphate bonds, alkyl phosphoramidate bonds , aryl phosphoramidate linkages, 2'-O-aminopropyl modifications, 2'-O-alkyl modifications, 2'-O-allyl modifications, 2'-O-butyl modifications, and 1-( 4'-thio-PD-ribofuranosyl) modification.
在某些实施方案中，所述检测探针是线性的，或者具有发夹结构。在某些优选的实施方案中，所述检测探针是线性的。在某些优选的实施方案中，所述检测探针具有发夹结构。发夹结构可以是天然的，也可以是人工引入的。In certain embodiments, the detection probe is linear, or has a hairpin structure. In certain preferred embodiments, the detection probe is linear. In certain preferred embodiments, the detection probe has a hairpin structure. Hairpin structures can be natural or artificially introduced.
在某些实施方案中，在步骤(4)中，在允许核酸聚合酶进行延伸反应的条件下，所述核酸聚合酶以检测探针为模板，对杂交至检测探针的媒介子片段进行延伸，并由此形成双链体。In certain embodiments, in step (4), under conditions that allow the nucleic acid polymerase to carry out the extension reaction, the nucleic acid polymerase uses the detection probe as a template to extend the mediator fragment hybridized to the detection probe , and thus form a duplex.
在某些实施方案中，步骤(2)中，所使用的具有5'核酸酶活性的酶为具有5'核酸酶活性的核酸聚合酶，并且与步骤(4)中所使用的核酸聚合酶相同。In certain embodiments, in step (2), the enzyme with 5' nuclease activity used is a nucleic acid polymerase with 5' nuclease activity, and is the same as the nucleic acid polymerase used in step (4) .
在某些实施方案中，其中，步骤(1)-(5)通过包含下述步骤(I)-(VII)的方案来进行：In certain embodiments, wherein, steps (1)-(5) are carried out by a scheme comprising the following steps (I)-(VII):
(I)提供m种检测探针，并且针对待检测的每一种靶核酸序列，提供一种上游寡核苷酸序列、一种媒介子探针和一种下游寡核苷酸序列；并且，任选地，提供一种通用引物；其中，所述下游寡核苷酸序列如上所定义，并且所述通用引物如上所定义；(1) providing m detection probes, and for each target nucleic acid sequence to be detected, providing an upstream oligonucleotide sequence, a mediator probe and a downstream oligonucleotide sequence; and, Optionally, a universal primer is provided; wherein the downstream oligonucleotide sequence is as defined above, and the universal primer is as defined above;
(II)将待检测的样品与上游寡核苷酸序列，媒介子探针和下游寡核苷酸序列，以及具有5'核酸酶活性的模板依赖性核酸聚合酶(例如，DNA聚合酶，特别是热稳定的DNA聚合酶)混合；并且任选地，添加通用引物；(II) combining the sample to be detected with upstream oligonucleotide sequences, mediator probes and downstream oligonucleotide sequences, and a template-dependent nucleic acid polymerase with 5' nuclease activity (eg, DNA polymerase, particularly is a thermostable DNA polymerase) mix; and optionally, adding a universal primer;
(III)在允许核酸变性的条件下，温育前一步骤的产物；(III) incubating the product of the previous step under conditions that allow nucleic acid denaturation;
(IV)在允许核酸退火或杂交的条件下，温育前一步骤的产物；(IV) incubating the product of the previous step under conditions that allow nucleic acid annealing or hybridization;
(V)在允许核酸延伸的条件下，温育前一步骤的产物；(v) incubating the product of the previous step under conditions that allow nucleic acid extension;
(VI)任选地，重复步骤(III)-(V)一次或多次；和(VI) optionally, repeating steps (III)-(V) one or more times; and
(VII)对前一步骤的产物进行熔解曲线分析。(VII) Perform melting curve analysis on the product of the previous step.
在某些实施方案中，在所述方法的步骤(II)中，将所述样品与所述上游寡核苷酸序列，媒介子探针和下游寡核苷酸序列，以及核酸聚合酶混合，并进行PCR反应，然后，在PCR反应结束后，将检测探针加入到步骤(VI)的产物中，并进行熔解曲线分析；或者，在步骤(II)中，将所述样品与所述上游寡核苷酸序列，媒介子探针，下游寡核苷酸序列和所述检测探针，以及核酸聚合酶混合，并进行PCR反应，然后，在PCR反应结束后，进行熔解曲线分析。In certain embodiments, in step (II) of the method, the sample is mixed with the upstream oligonucleotide sequences, the mediator probe and downstream oligonucleotide sequences, and a nucleic acid polymerase, And carry out the PCR reaction, then, after the PCR reaction is completed, add the detection probe to the product of step (VI), and carry out melting curve analysis; or, in step (II), the sample and the upstream The oligonucleotide sequence, the mediator probe, the downstream oligonucleotide sequence, the detection probe, and the nucleic acid polymerase are mixed and subjected to a PCR reaction, and then, after the PCR reaction is completed, a melting curve analysis is performed.
在某些实施方案中，在所述方法的步骤(II)中，将所述样品与所述上游寡核苷酸序列，媒介子探针和下游寡核苷酸序列，核酸聚合酶，以及通用引物混合，并进行PCR反应，然后，在PCR反应结束后，将检测探针加入到步骤(VI)的产物中，并进行熔解曲线分析；或者，在步骤(II)中，将所述样品与所述上游寡核苷酸序列，媒介子探针，下游寡核苷酸序列和所述检测探针，核酸聚合酶，以及通用引物混合，并进行PCR反应，然后，在PCR反应结束后，进行熔解曲线分析。In certain embodiments, in step (II) of the method, the sample is combined with the upstream oligonucleotide sequences, the mediator probe and downstream oligonucleotide sequences, a nucleic acid polymerase, and a universal The primers are mixed, and a PCR reaction is performed, and then, after the PCR reaction is completed, the detection probe is added to the product of step (VI), and a melting curve analysis is performed; or, in step (II), the sample is mixed with. The upstream oligonucleotide sequence, the mediator probe, the downstream oligonucleotide sequence and the detection probe, nucleic acid polymerase, and universal primer are mixed, and a PCR reaction is performed, and then, after the PCR reaction is completed, a PCR reaction is performed. Melting curve analysis.
在某些实施方案中，在步骤(III)中，在80-105℃的温度下温育步骤(II)的产物，从而使核酸变性。In certain embodiments, in step (III), the product of step (II) is incubated at a temperature of 80-105°C, thereby denaturing the nucleic acid.
在某些实施方案中，在步骤(III)中，温育步骤(II)的产物10-20s，20-40s，40-60s，1-2min，或2-5min。In certain embodiments, in step (III), the product of step (II) is incubated for 10-20s, 20-40s, 40-60s, 1-2 min, or 2-5 min.
在某些实施方案中，在步骤(IV)中，在35-40℃，40-45℃，45-50℃，50-55℃，55-60℃，60-65℃，或65-70℃的温度下温育步骤(III)的产物，从而允许核酸退火或杂交。In certain embodiments, in step (IV), at 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, or 65-70°C The product of step (III) is incubated at a temperature to allow nucleic acid annealing or hybridization.
在某些实施方案中，在步骤(IV)中，温育步骤(III)的产物10-20s，20-40s，40-60s，1-2min，或2-5min。In certain embodiments, in step (IV), the product of step (III) is incubated for 10-20s, 20-40s, 40-60s, 1-2 min, or 2-5 min.
在某些实施方案中，在步骤(V)中，在35-40℃，40-45℃，45-50℃，50-55℃，55-60℃，60-65℃，65-70℃，70-75℃，75-80℃，80-85℃的温度下温育步骤(IV)的产物，从而允许核酸延伸。In certain embodiments, in step (V), at 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, 65-70°C, The product of step (IV) is incubated at temperatures of 70-75°C, 75-80°C, 80-85°C, thereby allowing nucleic acid extension.
在某些实施方案中，在步骤(V)中，温育步骤(IV)的产物10-20s，20-40s，40-60s，1-2min，2-5min，5-10min，10-20min或20-30min。In certain embodiments, in step (V), the product of step (IV) is incubated for 10-20s, 20-40s, 40-60s, 1-2min, 2-5min, 5-10min, 10-20min or 20-30min.
在某些实施方案中，在相同或不同的温度下进行步骤(IV)和(V)。In certain embodiments, steps (IV) and (V) are performed at the same or different temperatures.
在某些实施方案中，重复步骤(III)-(V)至少一次，例如至少2次，至少5次，至少10次，至少20次，至少30次，至少40次，或至少50次。在某些实施方案中，当重复步骤(III)-(V)一次或多次时，每一个循环的步骤(III)-(V)所使用的条件各自独立地是相同的或不同的。In certain embodiments, steps (III)-(V) are repeated at least once, eg, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. In certain embodiments, when steps (III)-(V) are repeated one or more times, the conditions used in each cycle of steps (III)-(V) are each independently the same or different.
在某些实施方案中，在步骤(VII)中，对步骤(VI)的产物进行逐渐的升温或降温并实时监测每一种检测探针上的报告基团发出的信号，从而获得每一种报告基团的信号强度随着温度变化而变化的曲线。In certain embodiments, in step (VII), the product of step (VI) is gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining each A plot of the signal intensity of the reporter group as a function of temperature.
在某些实施方案中，对所获得的曲线进行求导，从而获得步骤(VI)的产物的熔解曲线。In certain embodiments, the obtained curve is derived to obtain a melting curve for the product of step (VI).
在某些实施方案中，根据熔解曲线中的熔解峰(熔点)，确定对应于所述熔解峰(熔点)的媒介子片段的存在；随后，通过媒介子片段中的媒介子序列与靶核酸序列的对应关系，确定与所述媒介子片段对应的靶核酸序列的存在。In certain embodiments, the presence of a mediator subfragment corresponding to the melting peak (melting point) is determined based on the melting peak (melting point) in the melting curve; then, the mediator subsequence in the mediator subfragment and the target nucleic acid sequence are determined The corresponding relationship is determined to determine the existence of the target nucleic acid sequence corresponding to the mediator fragment.
在另一个方面，本申请提供了一种探针组(probe set)，其包含一种检测探针，以及一种或多种(例如至少两种)的媒介子探针，其中，In another aspect, the application provides a probe set comprising a detection probe, and one or more (eg, at least two) mediator probes, wherein,
所述媒介子探针各自独立地从5'至3'方向包含媒介子序列和靶特异性序列，所述靶特异性序列包含与一种靶核酸序列互补的序列，所述媒介子序列包含不与所述靶核酸序列互补的序列，并且，所有媒介子探针所包含的媒介子序列彼此不同；The mediator probes each independently comprise, in the 5' to 3' direction, a mediator sequence comprising a sequence complementary to a target nucleic acid sequence and a target-specific sequence comprising a non-target nucleic acid sequence. a sequence complementary to the target nucleic acid sequence, and the mediator sequences contained in all mediator probes are different from each other;
其中，至少一种媒介子探针自身能够形成发夹结构；优选地，所述至少一种媒介子探针具有权利要求1中定义的特征(i)，(ii)或(iii)；和wherein the at least one mediator probe itself is capable of forming a hairpin structure; preferably, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1; and
所述检测探针从3'至5'方向包含，与每一种媒介子序列或其部分互补的捕获序列，以及模板序列(templating sequence)；并且，所述检测探针标记有报告基团和淬灭基团，其中，所述报告基团能够发出信号，并且，所述淬灭基团能够吸收或淬灭所述报告基团发出的信号；并且，所述检测探针在与其互补序列杂交的情况下发出的信号不同于在未与其互补序列杂交的情况下发出的信号。The detection probe comprises, from 3' to 5', a capture sequence complementary to each mediator subsequence or a portion thereof, and a templating sequence; and the detection probe is labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the detection probe is hybridized to its complementary sequence The signal emitted is different from the signal emitted in the absence of hybridization to its complementary sequence.
在某些实施方案中，所述探针组包含至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少8种、至少9种、至少10种、至少12种、至少15种、或至少20种媒介子探针。In certain embodiments, the probe set comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 mediator probes.
在某些实施方案中，至少1种、至少2种、至少3种、至少4种、至少5种、至 少6种、至少7种、至少8种、至少9种、至少10种、至少12种、至少15种、或至少20种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)。In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12 , at least 15, or at least 20 mediator probes each independently capable of forming a hairpin structure, eg each independently having features (i), (ii) or (iii) as defined in claim 1 .
在某些实施方案中，每一种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)。In certain embodiments, each mediator probe is independently capable of forming a hairpin structure, eg, each independently has features (i), (ii) or (iii) as defined in claim 1 .
在某些实施方案中，所述媒介子探针的长度为15-150nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt，140-150nt。In certain embodiments, the mediator probe is 15-150nt in length, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80 -90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.
在某些实施方案中，所述媒介子探针中的靶特异性序列的长度为10-140nt，例如10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt。In certain embodiments, the target-specific sequence in the mediator probe is 10-140nt in length, eg, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
在某些实施方案中，所述媒介子探针中的媒介子序列的长度可以为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt。In certain embodiments, the length of the mediator sequence in the mediator probe can be 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt , 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
在某些实施方案中，所述第一、第二、第三或第四发夹形成序列的长度各自独立地为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt。In certain embodiments, the first, second, third or fourth hairpin forming sequences are each independently 5-140nt in length, eg, 5-10nt, 10-20nt, 20-30nt, 30-40nt , 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt.
在某些实施方案中，所述媒介子探针具有3'-OH末端，或者其3'-末端是封闭的。在某些优选的实施方案中，媒介子探针的3'-末端是封闭的，以抑制其延伸。可通过各种方法来封闭核酸(例如媒介子探针)的3'-末端。例如，可通过对媒介子探针的最后一个核苷酸的3'-OH进行修饰，以封闭媒介子探针的3'-末端。在某些实施方案中，可通过在媒介子探针的最后一个核苷酸的3'-OH上添加化学部分(例如，生物素或烷基)，从而封闭媒介子探针的3'-末端。在某些实施方案中，可通过将媒介子探针的最后一个核苷酸的3'-OH去除，或者将所述最后一个核苷酸替换为双脱氧核苷酸，从而封闭媒介子探针的3'-末端。In certain embodiments, the mediator probe has a 3'-OH terminus, or its 3'-terminus is blocked. In certain preferred embodiments, the 3'-terminus of the mediator probe is blocked to inhibit its extension. The 3'-terminus of nucleic acids (eg, mediator probes) can be blocked by various methods. For example, the 3'-terminus of the mediator probe can be blocked by modifying the 3'-OH of the last nucleotide of the mediator probe. In certain embodiments, the 3'-terminus of the mediator probe can be blocked by adding a chemical moiety (eg, biotin or an alkyl group) to the 3'-OH of the last nucleotide of the mediator probe . In certain embodiments, the mediator probe can be blocked by removing the 3'-OH of the last nucleotide of the mediator probe, or by replacing the last nucleotide with a dideoxynucleotide 3'-end.
在某些实施方案中，所述媒介子探针的靶特异性序列与第一发夹形成序列之间还含有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is also included between the target-specific sequence of the mediator probe and the first hairpin forming sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所述媒介子探针的第二发夹形成序列与媒介子序列之间还含 有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所述媒介子探针的第三发夹形成序列与媒介子序列之间还含有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is also included between the third hairpin-forming sequence of the mediator probe and the mediator sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所述媒介子探针的靶特异性序列与第四发夹形成序列之间还含有连接体。在某些实施方案中，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)。In certain embodiments, a linker is further included between the target-specific sequence of the mediator probe and the fourth hairpin-forming sequence. In certain embodiments, the linker comprises 1 or more nucleotides (eg, 1, 2, 3, 4, 5, 8, 10, or more nucleosides acid).
在某些实施方案中，所有媒介子探针所包含的靶特异性序列彼此不同。In certain embodiments, all mediator probes comprise target-specific sequences that differ from each other.
在某些实施方案中，所有媒介子探针各自靶向不同的靶核酸序列。In certain embodiments, all mediator probes each target a different target nucleic acid sequence.
易于理解的是，此类探针组可用于实施上文所详细描述的本发明方法。因此，上文针对媒介子探针和检测探针所详细描述的各种技术特征同样可应用于探针组中的媒介子探针和检测探针。因此，在某些优选的实施方案中，所述探针组包含如上文所定义的媒介子探针。在某些优选的实施方案中，所述探针组包含如上文所定义的检测探针。It will be readily understood that such probe sets can be used to implement the methods of the invention described in detail above. Therefore, the various technical features described in detail above for the mediator probe and the detection probe are equally applicable to the mediator probe and the detection probe in the probe set. Thus, in certain preferred embodiments, the probe set comprises a mediator probe as defined above. In certain preferred embodiments, the probe set comprises detection probes as defined above.
在另一个方面，本申请提供了一种试剂盒，其包含一种或多种如前所定义的探针组。In another aspect, the application provides a kit comprising one or more probe sets as previously defined.
在某些实施方案中，所述试剂盒包含至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少8种、至少9种、至少10种探针组。In certain embodiments, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 probe sets.
在某些实施方案中，所述试剂盒中的所有媒介子序列各自靶向不同的靶核酸序列。In certain embodiments, all mediator sequences in the kit each target a different target nucleic acid sequence.
在某些实施方案中，所述试剂盒中的所有媒介子探针所包含的媒介子序列彼此不同。In certain embodiments, all the mediator probes in the kit contain mediator sequences that are different from each other.
在某些实施方案中，所述试剂盒中的所有媒介子探针所包含的靶特异性序列彼此不同。In certain embodiments, all mediator probes in the kit comprise target-specific sequences that differ from each other.
在某些实施方案中，所述试剂盒中的所有检测探针各自独立地标记有相同或不同的报告基团。In certain embodiments, all detection probes in the kit are each independently labeled with the same or different reporter groups.
易于理解的是，此类试剂盒可用于实施上文所详细描述的本发明方法。因此，上文针对媒介子探针和检测探针所详细描述的各种技术特征同样可应用于试剂盒中的媒介子探针和检测探针。并且，此类试剂盒还可包含实施本发明方法所需的其他 试剂。It will be readily understood that such kits can be used to practice the methods of the invention described in detail above. Therefore, the various technical features described in detail above for the mediator probe and the detection probe can also be applied to the mediator probe and the detection probe in the kit. Furthermore, such kits may also contain other reagents required for carrying out the methods of the present invention.
例如，在某些优选的实施方案中，所述试剂盒还可包含如上文所定义的上游寡核苷酸序列、下游寡核苷酸序列、通用引物、具有5'核酸酶活性的酶、核酸聚合酶、或其任何组合。在某些优选的实施方案中，所述试剂盒还可包含，用于进行核酸杂交的试剂、用于进行媒介子探针切割的试剂、用于进行核酸延伸的试剂、用于进行核酸扩增的试剂、或其任何组合。此类试剂可由本领域技术人员常规地确定，并且包括但不限于，酶(例如核酸聚合酶)的工作缓冲液、dNTPs、水、包含离子(例如Mg 2+)的溶液、单链DNA结合蛋白(Single Strand DNA-Binding Protein，SSB)、或其任何组合。 For example, in certain preferred embodiments, the kit may further comprise upstream oligonucleotide sequences, downstream oligonucleotide sequences, universal primers, enzymes with 5' nuclease activity, nucleic acids as defined above A polymerase, or any combination thereof. In certain preferred embodiments, the kit may further comprise, reagents for nucleic acid hybridization, reagents for mediator probe cleavage, reagents for nucleic acid extension, nucleic acid amplification reagents, or any combination thereof. Such reagents can be routinely determined by one of skill in the art, and include, but are not limited to, working buffers for enzymes (eg, nucleic acid polymerases), dNTPs, water, solutions containing ions (eg, Mg 2+ ), single-stranded DNA binding proteins (Single Strand DNA-Binding Protein, SSB), or any combination thereof.
本领域技术人员基于本申请所详细描述的原理，可对本发明技术方案的各种技术特征进行修饰、替换或组合，而不背离本发明的精神和范围。所有此类技术方案以及其变形都涵盖在本申请的权利要求书或其等同物的范围内。Based on the principles described in detail in this application, those skilled in the art can modify, replace or combine various technical features of the technical solutions of the present invention without departing from the spirit and scope of the present invention. All such technical solutions and modifications thereof are included within the scope of the claims of the present application or their equivalents.
术语定义Definition of Terms
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the operation steps such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA used in this paper are conventional steps widely used in the corresponding fields. . Meanwhile, for better understanding of the present invention, definitions and explanations of related terms are provided below.
如本文中所使用的，术语“媒介子探针”是指，从5'至3'方向含有媒介子序列(mediator sequence)和靶向序列(targeting sequence；即，靶特异性序列)的单链核酸分子，其任选地还可在靶向序列的下游或3’端包含第一发夹形成序列，或者在媒介子序列的上游或5'端包含第二发夹形成序列，或者在媒介子序列的上游或5'端包含第三发夹形成序列，且在靶特异性序列的下游或3'端包含第四发夹形成序列。当媒介子探针含有第一、第二、第三或第四发夹形成序列时，媒介子探针能够通过所述媒介子序列和所述第一发夹形成序列、或者所述第二发夹形成序列和所述靶特异性序列、或者所述第三发夹形成序列和所述第四发夹形成序列形成发夹结构。在本申请中，媒介子序列不含与靶核酸序列互补的序列，靶特异性序列包含与靶核酸序列互补的序列。因此，在允许核酸杂交、退火或扩增的条件下，媒介子探针通过靶特异性序列与靶核酸序列杂交或退火(即，形成双链结构)，并且媒介子探针 中的媒介子序列不与所述靶核酸序列杂交。As used herein, the term "mediator probe" refers to a single-stranded chain containing a mediator sequence and a targeting sequence (ie, a target-specific sequence) from the 5' to 3' direction A nucleic acid molecule, which optionally may also comprise a first hairpin-forming sequence downstream or 3' of the targeting sequence, or a second hairpin-forming sequence upstream or 5' of the mediator sequence, or at the mediator A third hairpin-forming sequence is included upstream or 5' of the sequence, and a fourth hairpin-forming sequence is included downstream or 3' of the target-specific sequence. When the mediator probe contains a first, second, third or fourth hairpin-forming sequence, the mediator probe can pass through the mediator sequence and the first hairpin-forming sequence, or the second hairpin-forming sequence The clip-forming sequence and the target-specific sequence, or the third hairpin-forming sequence and the fourth hairpin-forming sequence, form a hairpin structure. In the present application, the mediator subsequence does not contain a sequence complementary to the target nucleic acid sequence, and the target specific sequence comprises a sequence complementary to the target nucleic acid sequence. Thus, the mediator probe hybridizes or anneals (ie, forms a double-stranded structure) to the target nucleic acid sequence through the target-specific sequence under conditions that allow nucleic acid hybridization, annealing, or amplification, and the mediator sequence in the mediator probe Does not hybridize to the target nucleic acid sequence.
如本文中所使用的，术语“靶向序列”和“靶特异性序列”是指，在允许核酸杂交、退火或扩增的条件下，能够与靶核酸序列选择性/特异性杂交或退火的序列，其包含与靶核酸序列互补的序列。在本申请中，术语“靶向序列”和“靶特异性序列”具有相同的含义，并且可互换使用。易于理解的是，靶向序列或靶特异性序列对于靶核酸序列是特异性的。换言之，在允许核酸杂交、退火或扩增的条件下，靶向序列或靶特异性序列仅与特定的靶核酸序列杂交或退火，而不与其他的核酸序列杂交或退火。As used herein, the terms "targeting sequence" and "target-specific sequence" refer to those capable of selectively/specifically hybridizing or annealing to a target nucleic acid sequence under conditions that permit hybridization, annealing, or amplification of the nucleic acid. A sequence comprising a sequence complementary to a target nucleic acid sequence. In this application, the terms "targeting sequence" and "target-specific sequence" have the same meaning and are used interchangeably. It is readily understood that a targeting sequence or target-specific sequence is specific for a target nucleic acid sequence. In other words, a targeting sequence or target-specific sequence only hybridizes or anneals to a specific target nucleic acid sequence, and not to other nucleic acid sequences, under conditions that allow nucleic acid hybridization, annealing, or amplification.
如本文中所使用的，术语“媒介子序列”是指，媒介子探针中不与靶核酸序列互补的一段寡核苷酸序列，其位于靶特异性序列的上游(5'端)。在本申请中，针对每一种靶核酸序列，设计或提供一条独特的媒介子探针，其具有独特的媒介子序列(换言之，所使用的所有媒介子探针中的媒介子序列彼此不同)；由此，每一种靶核酸序列与一条独特的媒介子探针(独特的媒介子序列)相对应。因此，通过检测所述独特的媒介子序列，可以检测与之相对应的靶核酸序列。As used herein, the term "mediator sequence" refers to a stretch of oligonucleotide sequence in the mediator probe that is not complementary to the target nucleic acid sequence and is located upstream (5' end) of the target-specific sequence. In this application, for each target nucleic acid sequence, a unique mediator probe is designed or provided, which has a unique mediator sequence (in other words, the mediator sequences in all mediator probes used are different from each other) Thus, each target nucleic acid sequence corresponds to a unique mediator probe (unique mediator sequence). Thus, by detecting the unique mediator sequence, the corresponding target nucleic acid sequence can be detected.
如本文中所使用的，术语“上游寡核苷酸序列”是指，包含与靶核酸序列互补的序列的一段寡核苷酸序列，其在允许核酸杂交(或退火)或扩增的条件下，能够与靶核酸序列杂交(或退火)，并且，当与靶核酸序列杂交时，其位于媒介子探针的上游。As used herein, the term "upstream oligonucleotide sequence" refers to an oligonucleotide sequence comprising a sequence complementary to a target nucleic acid sequence under conditions that allow nucleic acid hybridization (or annealing) or amplification , is capable of hybridizing (or annealing) to the target nucleic acid sequence and, when hybridized to the target nucleic acid sequence, is located upstream of the mediator probe.
如本文中所使用的，术语“互补”意指，两条核酸序列能够根据碱基配对原则(Waston-Crick原则)在彼此之间形成氢键，并由此形成双链体。在本申请中，术语“互补”包括“实质上互补”和“完全互补”。如本文中所使用的，术语“完全互补”意指，一条核酸序列中的每一个碱基都能够与另一条核酸链中的碱基配对，而不存在错配或缺口。如本文中所使用的，术语“实质上互补”意指，一条核酸序列中的大部分碱基都能够与另一条核酸链中的碱基配对，其允许存在错配或缺口(例如，一个或数个核苷酸的错配或缺口)。通常，在允许核酸杂交、退火或扩增的条件下，“互补”(例如实质上互补或完全互补)的两条核酸序列将选择性地/特异性地发生杂交或退火，并形成双链体。相应地，术语“不互补”意指，两条核酸序列在允许核酸杂交、退火或扩增的条件下不能发生杂交或退火，无法形成双链体。如本文中所使用的，术语“不能完全互补”意指，一条核酸序列中的碱基不能够与另一条核酸链中的碱基完全配对，至少存在一个错配或缺口。As used herein, the term "complementary" means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the principles of base pairing (Waston-Crick principle), and thereby forming duplexes. In this application, the term "complementary" includes "substantially complementary" and "completely complementary". As used herein, the term "completely complementary" means that every base in one nucleic acid sequence is capable of pairing with bases in another nucleic acid strand without mismatches or gaps. As used herein, the term "substantially complementary" means that a majority of bases in one nucleic acid sequence are capable of pairing with bases in the other nucleic acid strand, which allows for mismatches or gaps (eg, one or mismatches or gaps of several nucleotides). Typically, two nucleic acid sequences that are "complementary" (eg, substantially complementary or fully complementary) will selectively/specifically hybridize or anneal under conditions that allow nucleic acid hybridization, annealing, or amplification, and form a duplex . Accordingly, the term "non-complementary" means that two nucleic acid sequences cannot hybridize or anneal under conditions that permit hybridization, annealing, or amplification of the nucleic acids to form a duplex. As used herein, the term "not perfectly complementary" means that bases in one nucleic acid sequence cannot perfectly pair with bases in another nucleic acid strand, at least one mismatch or gap exists.
如本文中所使用的，术语“杂交”和“退火”意指，互补的单链核酸分子形成双链核酸的过程。在本申请中，“杂交”和“退火”具有相同的含义，并且可互换使用。通常，完全互补或实质上互补的两条核酸序列可发生杂交或退火。两条核酸序列发生杂交或退火所需要的互补性取决于所使用的杂交条件，特别是温度。As used herein, the terms "hybridization" and "annealing" mean the process by which complementary single-stranded nucleic acid molecules form a double-stranded nucleic acid. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Typically, two nucleic acid sequences that are completely complementary or substantially complementary can hybridize or anneal. The complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, in particular the temperature.
如本文中所使用的，术语“PCR反应”具有本领域技术人员通常理解的含义，其是指使用核酸聚合酶和引物来扩增靶核酸的反应(聚合酶链式反应)。如本文中所使用的，术语“多重扩增”是指，在同一个反应体系中对多个靶核酸进行扩增。如本文中所使用的，术语“不对称扩增”是指，对靶核酸进行扩增所获得的扩增产物中，两条互补的核酸链的量不相同，一条核酸链的量大于另一条核酸链。As used herein, the term "PCR reaction" has the meaning commonly understood by those skilled in the art, which refers to a reaction (polymerase chain reaction) that amplifies a target nucleic acid using a nucleic acid polymerase and primers. As used herein, the term "multiplex amplification" refers to the amplification of multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" means that in the amplification product obtained by amplifying a target nucleic acid, the amount of two complementary nucleic acid strands is different, and the amount of one nucleic acid strand is greater than that of the other. nucleic acid strands.
如本文中所使用的，术语“检测探针”标记有报告基团和淬灭基团。简言之，在环境温度下，检测探针能够通过碱基配对作用与其互补序列形成双链体。在此情况下，检测探针上的报告基团(例如荧光基团)和淬灭基团彼此分离，淬灭基团无法吸收报告基团发出的信号(例如荧光信号)，此时，能够检测到最强的信号(例如荧光信号)。随着温度的升高，双链体的两条链开始解离(即，检测探针逐渐从其互补序列上解离)，并且解离下的检测探针呈单链自由卷曲状态。在此情况下，解离下的检测探针上的报告基团(例如荧光基团)和淬灭基团互相靠近，由此报告基团(例如荧光基团)发出的信号(例如荧光信号)被淬灭基团所吸收。因此，随着温度的升高，所检测到信号(例如荧光信号)逐渐变弱。当双链体的两条链完全解离时，所有的检测探针均呈单链自由卷曲状态。在此情况下，所有的检测探针上的报告基团(例如荧光基团)发出的信号(例如荧光信号)都被淬灭基团所吸收。因此，基本上无法检测到报告基团(例如荧光基团)发出的信号(例如荧光信号)。As used herein, the term "detection probe" is labeled with a reporter group and a quencher group. Briefly, at ambient temperature, detection probes are capable of forming duplexes with their complementary sequences through base pairing. In this case, the reporter group (such as a fluorophore) and the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. to the strongest signal (e.g. fluorescence signal). As the temperature increases, the two strands of the duplex begin to dissociate (ie, the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe is in a single-stranded free coil state. In this case, the reporter group (eg, fluorophore) and the quencher group on the dissociated detection probe are in close proximity to each other, whereby the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescent signal) gradually becomes weaker. When the two strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, all the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the detection probe is absorbed by the quencher group. Thus, the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) is substantially undetectable.
如本文中所使用的，术语“熔解曲线分析”具有本领域技术人员通常理解的含义，其是指，通过测定双链核酸分子的熔解曲线来分析双链核酸分子存在或其身份(identity)的方法，其通常用于评估双链核酸分子在加热过程中的解离特征。用于进行熔解曲线分析的方法是本领域技术人员熟知的(参见例如The Journal of Molecular Diagnostics2009,11(2):93-101)。在本申请中，术语“熔解曲线分析”和“熔解分析”具有相同的含义，并且可互换使用。As used herein, the term "melting curve analysis" has the meaning commonly understood by those skilled in the art and refers to the analysis of the presence or identity of a double-stranded nucleic acid molecule by determining the melting curve of the double-stranded nucleic acid molecule. method, which is commonly used to assess the dissociation characteristics of double-stranded nucleic acid molecules during heating. Methods for performing melting curve analysis are well known to those skilled in the art (see, eg, The Journal of Molecular Diagnostics 2009, 11(2):93-101). In this application, the terms "melting curve analysis" and "melting analysis" have the same meaning and are used interchangeably.
在本申请的某些优选实施方案中，可通过使用标记有报告基团和淬灭基团的检测探针来进行熔解曲线分析。简言之，在环境温度下，检测探针能够通过碱基配对作用与其互补序列形成双链体。在此情况下，检测探针上的报告基团(例如荧光基团)和淬灭基 团彼此分离，淬灭基团无法吸收报告基团发出的信号(例如荧光信号)，此时，能够检测到最强的信号(例如荧光信号)。随着温度的升高，双链体的两条链开始解离(即，检测探针逐渐从其互补序列上解离)，并且解离下的检测探针呈单链自由卷曲状态。在此情况下，解离下的检测探针上的报告基团(例如荧光基团)和淬灭基团互相靠近，由此报告基团(例如荧光基团)发出的信号(例如荧光信号)被淬灭基团所吸收。因此，随着温度的升高，所检测到信号(例如荧光信号)逐渐变弱。当双链体的两条链完全解离时，所有的检测探针均呈单链自由卷曲状态。在此情况下，所有的检测探针上的报告基团(例如荧光基团)发出的信号(例如荧光信号)都被淬灭基团所吸收。因此，基本上无法检测到报告基团(例如荧光基团)发出的信号(例如荧光信号)。因此，对包含检测探针的双链体在升温或降温过程中发出的信号(例如荧光信号)进行检测，就能观察到检测探针与其互补序列的杂交和解离过程，形成信号强度随着温度变化而变化的曲线。进一步，对所获得的曲线进行求导分析，可获得以信号强度变化速率为纵坐标，温度为横坐标的曲线(即，该双链体的熔解曲线)。该熔解曲线中的峰即为熔解峰，其所对应的温度即为所述双链体的熔点(T m值)。通常而言，检测探针与互补序列的匹配程度越高(例如，错配的碱基越少，配对的碱基越多)，那么双链体的T m值就越高。因此，通过检测双链体的T m值，可确定双链体中与检测探针互补的序列的存在和身份。在本文中，术语“熔解峰”、“熔点”和“T m值”具有相同的含义，并且可互换使用。 In certain preferred embodiments of the present application, melting curve analysis can be performed by using detection probes labeled with reporter and quencher groups. Briefly, at ambient temperature, detection probes are capable of forming duplexes with their complementary sequences through base pairing. In this case, the reporter group (such as a fluorophore) and the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group. to the strongest signal (e.g. fluorescence signal). As the temperature increases, the two strands of the duplex begin to dissociate (ie, the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe assumes a single-stranded free coil state. In this case, the reporter group (eg, fluorophore) and the quencher group on the dissociated detection probe are in close proximity to each other, whereby the signal (eg, fluorescence signal) emitted by the reporter group (eg, fluorophore) absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescent signal) gradually becomes weaker. When the two strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coil state. In this case, all the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the detection probe is absorbed by the quencher group. Thus, the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) is substantially undetectable. Therefore, by detecting the signal (such as a fluorescent signal) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation process of the detection probe and its complementary sequence can be observed, and the signal intensity changes with temperature. changing curve. Further, by derivation analysis of the obtained curve, a curve with the change rate of signal intensity as the ordinate and the temperature as the abscissa (ie, the melting curve of the duplex) can be obtained. The peak in the melting curve is the melting peak, and the corresponding temperature is the melting point (T m value) of the duplex. In general, the more closely the detection probe matches the complementary sequence (eg, fewer bases are mismatched and more bases are paired), the higher the Tm value of the duplex will be. Thus, by detecting the Tm value of the duplex, the presence and identity of the sequence complementary to the detection probe in the duplex can be determined. Herein, the terms "melting peak", "melting point" and " Tm value" have the same meaning and are used interchangeably.
发明的有益效果Beneficial Effects of Invention
本申请的方法能够有效降低(甚至消除)使用媒介子探针和检测探针的多重实时PCR检测方法的非特异性信号，显著提高了所述检测方法的特异性。此外，本申请的方法还能够一定程度的提升阳性信号的强度，提高了检测的灵敏度。因此，本申请的方法是特别有利的，特别适合用于靶核酸序列的多重检测。The method of the present application can effectively reduce (or even eliminate) the non-specific signal of the multiplex real-time PCR detection method using the mediator probe and the detection probe, and significantly improve the specificity of the detection method. In addition, the method of the present application can also increase the intensity of the positive signal to a certain extent, thereby improving the detection sensitivity. Therefore, the methods of the present application are particularly advantageous, particularly suitable for multiplex detection of target nucleic acid sequences.
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, rather than limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.
附图说明Description of drawings
图1显示了本发明三种发夹媒介子探针的结构。其中，图1A显示了当媒介子探针含有媒介子序列、靶特异性序列和第一发夹形成序列时，在媒介子序列被剪切之前呈发夹结构。图1B显示了当媒介子探针含有媒介子序列、靶特异性序列和第二发夹形成序列时，在媒介子序列被剪切之前呈发夹结构。图1C显示了当媒介子探针含有媒介子序列、靶特异性序列、第三发夹形成序列和第四发夹形成序列时，在媒介子序列被剪切之前呈发夹结构。Figure 1 shows the structures of three hairpin mediator probes of the present invention. 1A shows that when the mediator probe contains the mediator sequence, the target-specific sequence and the first hairpin forming sequence, the mediator subsequence is in a hairpin structure before being cleaved. Figure IB shows that when the mediator probe contains a mediator sequence, a target-specific sequence and a second hairpin forming sequence, the mediator sequence is in a hairpin structure before the mediator sequence is cleaved. Figure 1C shows that the mediator probe assumes a hairpin structure before the mediator sequence is cleaved when the mediator probe contains a mediator sequence, a target-specific sequence, a third hairpin-forming sequence, and a fourth hairpin-forming sequence.
图2示例性的显示了本发明***的原理。图2A显示了当媒介子探针含有媒介子序列和靶特异性序列时，在媒介子序列被剪切之前呈线性结构，容易与检测探针非特异性结合。图2B显示了当媒介子探针含有媒介子序列、靶特异性序列和第一发夹形成序列时，在媒介子序列被剪切之前呈发夹结构，显著减少了与检测探针的非特异性结合。Figure 2 schematically shows the principle of the system of the present invention. Figure 2A shows that when the mediator probe contains a mediator sequence and a target-specific sequence, before the mediator sequence is cleaved, it has a linear structure, which is easy to non-specifically bind to the detection probe. Figure 2B shows that when the mediator probe contains a mediator sequence, a target-specific sequence and a first hairpin-forming sequence, the mediator sequence is in a hairpin structure before the mediator sequence is cleaved, significantly reducing non-specificity with the detection probe combine.
图3显示了实施例1的反应体系的检测结果，其中，图3A为使使用线性媒介子探针的Gp8wzx检测体系所产生的检测结果；图3B为使用发夹媒介子探针的Gp8wzx检测体系所产生的检测结果；图3C为使用线性媒介子探针的O52wzm检测体系所产生的检测结果；图3D为使用发夹媒介子探针的O52wzm检测体系所产生的检测结果。并且，图中的实线表示以质粒(4份样品)为模板的检测结果；虚线表示以TE缓冲液(10mM Tris-HCl,1mM EDTA,pH 8.5)为模板的检测结果(阴性对照)。Figure 3 shows the detection results of the reaction system of Example 1, wherein Figure 3A is the detection result produced by the Gp8wzx detection system using the linear mediator probe; Figure 3B is the Gp8wzx detection system using the hairpin mediator probe The detection results generated; Figure 3C shows the detection results generated by the O52wzm detection system using the linear mediator probes; Figure 3D shows the detection results generated by the O52wzm detection system using the hairpin mediator probes. In addition, the solid line in the figure represents the detection result using the plasmid (4 samples) as the template; the dotted line represents the detection result (negative control) using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template.
图4显示了Gp8wzx检测体系的检测结果，其中，图4A为使用线性媒介子探针的检测结果；图4B为使用互补碱基数为6的发夹媒介子探针的检测结果；图4C为使用互补碱基数为8的发夹媒介子探针的检测结果；图4D为使用互补碱基数为10的发夹媒介子探针的检测结果。并且，图中的实线表示以大肠杆菌Gp8wzx质粒为模板的检测结果；虚线表示以TE缓冲液(10mM Tris-HCl,1mM EDTA,pH 8.5)为模板的检测结果(阴性对照)。Figure 4 shows the detection results of the Gp8wzx detection system, wherein Figure 4A is the detection result using the linear mediator probe; Figure 4B is the detection result using the hairpin mediator probe with 6 complementary bases; Figure 4C is the detection result. The detection result using the hairpin mediator probe with 8 complementary bases; Fig. 4D is the detection result using the hairpin mediator probe with 10 complementary bases. And, the solid line in the figure represents the detection result using Escherichia coli Gp8wzx plasmid as the template; the dotted line represents the detection result (negative control) using TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template.
图5显示了实施例3的反应体系的检测结果，其中，图5A为使使用线性媒介子探针的检测体系；图5B为使用发夹媒介子探针的检测体系。并且，图中的实线表示以含有靶基因1-6的质粒为模板的检测结果；虚线表示以TE缓冲液(10mM Tris-HCl,1mM EDTA,pH 8.5)为模板的检测结果(阴性对照)。Figure 5 shows the detection results of the reaction system of Example 3, wherein Figure 5A is the detection system using the linear mediator probe; Figure 5B is the detection system using the hairpin mediator probe. In addition, the solid line in the figure represents the detection result using the plasmid containing the target gene 1-6 as the template; the dotted line represents the detection result using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template (negative control) .
图6显示了实施例4的反应体系的检测结果，其中，图6A为使使用线性媒介子探针的检测体系；图6B为使用发夹媒介子探针的检测体系。并且，图中的实线表示以含有靶基因7-12的质粒为模板的检测结果；虚线表示以TE缓冲液(10mM Tris-HCl,1mM  EDTA,pH 8.5)为模板的检测结果(阴性对照)。Figure 6 shows the detection results of the reaction system of Example 4, wherein Figure 6A is the detection system using the linear mediator probe; Figure 6B is the detection system using the hairpin mediator probe. In addition, the solid line in the figure represents the detection result using the plasmid containing the target gene 7-12 as the template; the dashed line represents the detection result using the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5) as the template (negative control) .
序列描述sequence description
本申请涉及的部分序列的信息如下：The information on the partial sequences involved in this application is as follows:
Figure PCTCN2020140053-appb-000002
Figure PCTCN2020140053-appb-000002
Figure PCTCN2020140053-appb-000003
Figure PCTCN2020140053-appb-000003
Figure PCTCN2020140053-appb-000004
Figure PCTCN2020140053-appb-000004
具体实施方式Detailed ways
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。The present invention will now be described with reference to the following examples, which are intended to illustrate, but not limit, the invention.
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的生物化学、化学、分子生物学等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))。Unless otherwise indicated, the experiments and methods described in the Examples were performed essentially according to conventional methods well known in the art and described in various references. For example, for conventional techniques of biochemistry, chemistry, molecular biology, etc. used in the present invention, see Sambrook, Fritsch, and Maniatis, "Molecular Cloning: Experimental MOLECULAR CLONING: A LABORATORY MANUAL, 2nd editor (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (edited by F.M. Ausubel et al., (1987) )); "METHODS IN ENZYMOLOGY" series (academic publishing company): "PCR 2: A PRACTICAL APPROACH" (M.J. MacPherson), B.D. Hems ( B.D. Hames) and G.R. Taylor (edited by G.R. Taylor (1995)).
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。In addition, if the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market. Those skilled in the art appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
实施例1.发夹媒介子探针降低非特异熔解信号的可行性Example 1. Feasibility of hairpin mediator probes to reduce non-specific melting signals
为考察发夹媒介子探针是否可用于双探针实时PCR检测，本实施例分别使用线性媒介子探针和发夹媒介子探针进行对比，并且检测探针为同一个。In order to investigate whether the hairpin mediator probe can be used for dual-probe real-time PCR detection, in this example, a linear mediator probe and a hairpin mediator probe are used for comparison, and the detection probe is the same.
实验分别以大肠杆菌O抗原特异基因Gp8wzx和O52wzm为检测对象，每25μL PCR反应体系包括1×PCR buffer(致善生物科技有限公司,厦门)，7.0mM MgCl 2，0.2mM dNTPs，3U Taq01酶(致善生物科技有限公司,厦门)，引物和探针(用量见表1)，以及5μL模板(阳性对照的模板分别为大肠杆菌Gp8wzx质粒和O52wzm质粒(10000copies/ul)，将如SEQ ID NO:56和SEQ ID NO:57所示的核苷酸序列分别***puc57载体的多克隆位点中，分别获得大肠杆菌Gp8wzx质粒和O52wzm质粒；阴性对照的模板为TE缓冲液(10mM Tris-HCl,1mM EDTA,pH 8.5)。PCR反应程序为：50℃保温2min，95℃变性5min，然后95℃ 20s和60℃ 1min共40循环，60℃采集荧光，PCR完成后熔 解分析的程序如下：35℃杂交延伸20min，然后95℃变性2min，45℃保温2min，随后按0.04℃/s的升温速率从45℃升温至95℃，并采集CY5通道的荧光信号。实验仪器为SLAN 96S实时PCR仪(宏石医疗科技有限公司，上海)，引物和探针均由上海生物工程有限公司合成。 The experiment used Escherichia coli O antigen-specific genes Gp8wzx and O52wzm as detection objects, and each 25 μL PCR reaction system included 1×PCR buffer (Zhishan Biotechnology Co., Ltd., Xiamen), 7.0 mM MgCl 2 , 0.2 mM dNTPs, 3 U Taq01 enzyme ( Zhishan Biotechnology Co., Ltd., Xiamen), primers and probes (see Table 1 for dosage), and 5 μL of templates (the templates of positive control are E. coli Gp8wzx plasmid and O52wzm plasmid (10000 copies/ul), respectively, will be as SEQ ID NO: The nucleotide sequences shown in 56 and SEQ ID NO: 57 were inserted into the multi-cloning site of the puc57 vector to obtain the Escherichia coli Gp8wzx plasmid and the O52wzm plasmid respectively; the template of the negative control was TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.5). The PCR reaction program is: incubation at 50 °C for 2 min, denaturation at 95 °C for 5 min, then 40 cycles of 20 s at 95 °C and 1 min at 60 °C, fluorescence collection at 60 °C, and the procedure for melting analysis after PCR is as follows: Hybridization at 35 °C Extend for 20min, then denature at 95°C for 2min, keep at 45°C for 2min, then heat up from 45°C to 95°C at a heating rate of 0.04°C/s, and collect the fluorescence signal of the CY5 channel. The experimental instrument is a SLAN 96S real-time PCR instrument (Hongshi Medical Technology Co., Ltd., Shanghai), primers and probes were synthesized by Shanghai Bioengineering Co., Ltd.
反应A为Gp8wzx单重检测体系，使用线性媒介子探针；反应B为Gp8wzx单重检测体系，使用发夹媒介子探针。结果表明，反应A(图3A)的阴性对照(虚线)在52℃产生了明显的非特异熔解峰，而反应B(图3B)的阴性对照(虚线)无明显的非特异熔解峰。实验结果说明，发夹媒介子探针相比线性媒介子探针能够明显降低非特异熔解信号。反应C为O52wzm单重检测体系，使用线性媒介子探针；反应D为O52wzm单重检测体系，使用发夹媒介子探针。结果表明，反应C(图3C)的阴性对照(虚线)在阳性对照熔解峰对应熔点65℃处产生了非特熔解峰，即产生了假阳性信号，且45～50℃存在低温非特异信号，而反应D(图3D)阴性对照(虚线)无假阳性信号，45～50℃低温非特异信号也显著降低。并且，与反应C相比，阳性样本熔解峰信号提升。实验结果说明，发夹媒介子探针相比线性媒介子探针能够降低甚至几乎消除假阳性信号，并能够提升阳性样本熔解峰信号，有效减少非特异信号对检测体系结果判读的干扰。Reaction A is a Gp8wzx singleplex detection system, using a linear mediator probe; Reaction B is a Gp8wzx singleplex detection system, using a hairpin mediator probe. The results showed that the negative control (dotted line) of reaction A (Fig. 3A) produced an obvious non-specific melting peak at 52°C, while the negative control (dotted line) of reaction B (Fig. 3B) had no obvious non-specific melting peak. The experimental results show that the hairpin mediator probe can significantly reduce the non-specific melting signal compared with the linear mediator probe. Reaction C is an O52wzm singleplex detection system, using a linear mediator probe; Reaction D is an O52wzm singleplex detection system, using a hairpin mediator probe. The results showed that the negative control (dotted line) of reaction C (Fig. 3C) produced a non-specific melting peak at the melting point of the positive control at 65 °C, that is, a false positive signal was generated, and there was a low-temperature non-specific signal at 45-50 °C, while The negative control (dotted line) of reaction D (Fig. 3D) had no false positive signal, and the non-specific signal at low temperature of 45-50°C was also significantly reduced. Also, compared with reaction C, the melting peak signal of the positive sample was increased. The experimental results show that the hairpin mediator probe can reduce or even almost eliminate false positive signals compared with the linear mediator probe, and can improve the melting peak signal of positive samples, effectively reducing the interference of non-specific signals on the interpretation of the detection system results.
表1.实施例1中所用引物和探针Table 1. Primers and probes used in Example 1
Figure PCTCN2020140053-appb-000005
Figure PCTCN2020140053-appb-000005
注：斜体加下划线的碱基表示发夹媒介子探针相较于线性媒介子探针在3’端增加的序列。通用引物Tag在Gp8 wzx单重检测体系和O52 wzm单重检测体系均有使用。Note: Italicized underlined bases indicate the increased sequence at the 3' end of the hairpin mediator probe compared to the linear mediator probe. The universal primer Tag is used in both the Gp8 wzx singleplex detection system and the O52 wzm singleplex detection system.
实施例2.媒介子探针发夹结构互补碱基数考察Example 2. Investigation of the number of complementary bases in the hairpin structure of the mediator probe
本实施例采用实施例1中描述的Gp8 wzx单重检测体系，考察线性媒介子探针和发夹结构互补碱基数分别为6，8，10个碱基的媒介子探针用于熔解曲线分析的可行性。反应体系组成与PCR反应条件均与实施例1相同，本实施例所使用的线性媒介子探针也与实施例1相同，发夹媒介子探针如表2所示。In this example, the Gp8 wzx singleplex detection system described in Example 1 was used to investigate the use of the linear mediator probe and the mediator probe with hairpin structure complementary bases of 6, 8, and 10 bases, respectively, for melting curves. Feasibility of analysis. The composition of the reaction system and the PCR reaction conditions are the same as those in Example 1. The linear mediator probes used in this example are also the same as those in Example 1. Table 2 shows the hairpin mediator probes.
检测结果如图4所示，线性媒介子探针(图4A)的阴性对照(虚线)在50℃左右出现了明显的非特异性信号，而无论发夹媒介子探针的互补碱基数为多少，与线性媒介子探针相比，发夹媒介子探针的非特异性信号均显著降低(图4B-3D)。当发夹结构互补碱基数小于等于8个碱基的时候(图4B和图4C)，随着发夹结构的增强，阴性对照(虚线)非特异熔解峰逐渐减弱，而阳性标本扩增曲线及熔解峰不受影响；当发夹结构互补碱基数大于8个碱基的时候(图4D)，随着发夹结构的增强，阴性对照非特异熔解峰继续减弱，但是阳性标本扩增曲线及熔解峰也受到了一定的影响。The detection results are shown in Figure 4. The negative control (dotted line) of the linear mediator probe (Figure 4A) showed an obvious non-specific signal at around 50 °C, regardless of the number of complementary bases of the hairpin mediator probe. , the non-specific signals of the hairpin mediator probes were significantly reduced compared to the linear mediator probes (Figures 4B-3D). When the number of complementary bases of the hairpin structure is less than or equal to 8 bases (Figure 4B and Figure 4C), with the increase of the hairpin structure, the non-specific melting peak of the negative control (dotted line) gradually weakened, while the amplification curve of the positive sample and melting peaks are not affected; when the number of complementary bases in the hairpin structure is greater than 8 bases (Figure 4D), with the enhancement of the hairpin structure, the non-specific melting peak of the negative control continues to weaken, but the amplification curve of the positive sample and melting peaks were also affected to some extent.
表2.实施例2中所用发夹媒介子探针Table 2. Hairpin Mediator Probes Used in Example 2
Figure PCTCN2020140053-appb-000006
Figure PCTCN2020140053-appb-000006
注：斜体加下划线的碱基表示发夹媒介子探针相较于线性媒介子探针在3’端增加的序列。Note: Italicized underlined bases indicate the increased sequence at the 3' end of the hairpin mediator probe compared to the linear mediator probe.
实施例3.六重PCR熔解曲线分析检测体系Example 3. Six-fold PCR melting curve analysis detection system
本实施例采用六重PCR熔解曲线分析体系，检测并区分6个不同的靶序列。以5种靶基因(靶基因1-5)和1个阳性质控基因(靶基因6)为检测对象。将如SEQ ID NO:58至SEQ ID NO:63所示的核苷酸序列分别***puc57载体的多克隆位点中，分别获得含有靶基因1-6的质粒。本实施例共进行两个反应体系的检测，反应体系A为对照体系，使用6条线性媒介子探针和2条荧光探针。反应体系B为实验体系，使用4条线性媒介子探针、2条发夹媒介子探针和2条荧光探针，其中，4条线性媒介子探针分别对应的靶基因1、2、3和5，2条发夹媒介子探针对应阳性质控基因以及靶基因4。反应体系组成与PCR反应条件与实施例1相同，检测的模板为携带靶基因或质控基因的6个阳性质粒(浓度为1000copies/ul)。本实施例所使用的引物和探针具体如表3所示。In this example, a six-fold PCR melting curve analysis system was used to detect and distinguish 6 different target sequences. Five target genes (target genes 1-5) and one positive quality control gene (target gene 6) were used as detection objects. The nucleotide sequences shown in SEQ ID NO: 58 to SEQ ID NO: 63 were inserted into the multiple cloning sites of the puc57 vector, respectively, to obtain plasmids containing target genes 1-6. In this example, a total of two reaction systems were tested, and reaction system A was a control system, using 6 linear mediator probes and 2 fluorescent probes. Reaction system B is an experimental system, using 4 linear mediator probes, 2 hairpin mediator probes and 2 fluorescent probes, wherein the 4 linear mediator probes correspond to target genes 1, 2, and 3 respectively And 5, 2 hairpin mediator probes correspond to positive quality control gene and target gene 4. The composition of the reaction system and the PCR reaction conditions were the same as those in Example 1, and the detected templates were 6 positive plasmids (concentration: 1000 copies/ul) carrying target genes or quality control genes. The primers and probes used in this example are specifically shown in Table 3.
实验结果如图5所示，反应体系A(图5A)和反应体系B(图5B)对于每一个靶基因都检测出了相同T m值的熔解峰，但是，反应体系B(具有发夹媒介子探针)的熔解峰信号整体上高于反应体系A(全部为线性媒介子探针)。并且，相比于反应体系A，反应体系B在45～50℃的非特异熔解信号明显降低。 The experimental results are shown in Fig. 5. Reaction system A (Fig. 5A) and reaction system B (Fig. 5B) detected melting peaks with the same Tm value for each target gene, however, reaction system B (with a hairpin medium) The melting peak signal of the sub-probe) is generally higher than that of the reaction system A (all of which are linear mediator sub-probes). Moreover, compared with the reaction system A, the non-specific melting signal of the reaction system B at 45-50 °C was significantly reduced.
表3.实施例3中所用的引物和探针Table 3. Primers and probes used in Example 3
Figure PCTCN2020140053-appb-000007
Figure PCTCN2020140053-appb-000007
注：斜体加下划线的碱基表示发夹媒介子探针相较于线性媒介子探针在3’端增加的序列。Note: Italicized underlined bases indicate the increased sequence at the 3' end of the hairpin mediator probe compared to the linear mediator probe.
实施例4.六重PCR熔解曲线分析检测体系Example 4. Six-fold PCR melting curve analysis detection system
本实施例采用六重PCR熔解曲线分析体系，检测并区分6个不同的微生物的靶基因。以靶基因7-12为检测对象。将如SEQ ID NO:64至SEQ ID NO:69所示的核苷酸序列分别***puc57载体的多克隆位点中，分别获得含有靶基因7-12的质粒。In this example, a six-fold PCR melting curve analysis system is used to detect and distinguish the target genes of 6 different microorganisms. The target gene 7-12 was used as the detection object. The nucleotide sequences shown in SEQ ID NO: 64 to SEQ ID NO: 69 were inserted into the multiple cloning sites of the puc57 vector, respectively, to obtain plasmids containing target genes 7-12.
本实施例共进行两个反应体系的检测，反应体系A为对照体系，使用6条线性媒介 子探针和2条荧光探针，反应体系B为实验体系，使用5条线性媒介子探针、1条发夹媒介子探针和2条荧光探针。反应体系组成与PCR反应条件与实施例1相同，熔解曲线分析体系使用ROX荧光通道。检测的模板为携带靶基因的6个阳性质粒(浓度为1000copies/ul)。本实施例所使用的引物和探针具体如表4所示。In this example, a total of two reaction systems were tested. Reaction system A was a control system, using 6 linear mediator probes and 2 fluorescent probes, and reaction system B was an experimental system, using 5 linear mediator probes, 1 hairpin mediator probe and 2 fluorescent probes. The composition of the reaction system and the PCR reaction conditions were the same as those in Example 1, and the melting curve analysis system used the ROX fluorescence channel. The detected templates were 6 positive plasmids (concentration of 1000 copies/ul) carrying the target gene. The primers and probes used in this example are specifically shown in Table 4.
实验结果如图6所示，反应体系A(图6A)和反应体系B(图6B)对于每一个靶基因都检测出了相同T m值的熔解峰，但是，反应体系B(具有发夹媒介子探针)的熔解峰信号整体上高于反应体系A(全部为线性媒介子探针)。并且，相比于反应体系A，反应体系B在45～50℃的非特异熔解信号完全消除。 The experimental results are shown in Fig. 6. Reaction system A (Fig. 6A) and reaction system B (Fig. 6B) detected melting peaks with the same Tm value for each target gene, however, reaction system B (with a hairpin medium) The melting peak signal of the sub-probe) is generally higher than that of the reaction system A (all of which are linear mediator sub-probes). Moreover, compared with the reaction system A, the non-specific melting signal of the reaction system B at 45-50°C was completely eliminated.
表4.实施例4中所用的引物和探针Table 4. Primers and probes used in Example 4
Figure PCTCN2020140053-appb-000008
Figure PCTCN2020140053-appb-000008
注：斜体加下划线的碱基表示发夹媒介子探针相较于线性媒介子探针在3’端增加的序列。前面带有“+”的碱基为锁核酸(Locked Nucleic Acid，LNA)修饰的碱基。Note: Italicized underlined bases indicate the increased sequence at the 3' end of the hairpin mediator probe compared to the linear mediator probe. The base with "+" in front is the base modified by Locked Nucleic Acid (LNA).
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据 已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention . The full division of the invention is given by the appended claims and any equivalents thereof.

Claims (12)

  1. 一种检测n种靶核酸序列在样品中的存在的方法，其中，n为≥1的整数(例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40或更大的整数)，并且，所述方法包括以下步骤：A method of detecting the presence of n target nucleic acid sequences in a sample, wherein n is an integer ≥ 1 (eg, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or greater integers), and the method includes the steps of:
    (1)针对待检测的每一种靶核酸序列，提供一种上游寡核苷酸序列和一种媒介子探针；其中，所述上游寡核苷酸序列包含与所述靶核酸序列互补的序列；(1) For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence;
    并且，所述媒介子探针从5'至3'方向包含媒介子序列和靶特异性序列，所述媒介子序列包含不与所述靶核酸序列互补的序列，并且，所述靶特异性序列包含与所述靶核酸序列互补的序列；并且，当与所述靶核酸序列杂交时，所述上游寡核苷酸序列位于所述靶特异性序列的上游；并且，所有媒介子探针所包含的媒介子序列彼此不同；Also, the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator subsequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes comprise The mediator subsequences of are different from each other;
    其中，至少一种媒介子探针自身能够形成发夹结构；Wherein, at least one mediator probe itself can form a hairpin structure;
    优选地，所述至少一种媒介子探针具有选自下列的特征：Preferably, the at least one mediator probe has characteristics selected from the group consisting of:
    (i)所述媒介子探针在其靶特异性序列的下游或3'端还包含第一发夹形成序列，所述第一发夹形成序列与所述媒介子探针的媒介子序列或其部分互补，由此，所述媒介子探针能够通过所述媒介子序列和所述第一发夹形成序列形成发夹结构；(i) the mediator probe further comprises a first hairpin-forming sequence downstream or at the 3' end of its target-specific sequence, the first hairpin-forming sequence and the mediator sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the mediator sequence and the first hairpin-forming sequence;
    (ii)所述媒介子探针在其媒介子序列的上游或5'端还包含第二发夹形成序列，所述第二发夹形成序列与所述媒介子探针的靶特异性序列或其部分互补，由此，所述媒介子探针能够通过所述第二发夹形成序列和所述靶特异性序列形成发夹结构；(ii) the mediator probe further comprises a second hairpin-forming sequence upstream or 5' of its mediator sequence, the second hairpin-forming sequence and the target-specific sequence of the mediator probe or It is partially complementary, whereby the mediator probe is capable of forming a hairpin structure through the second hairpin-forming sequence and the target-specific sequence;
    (iii)所述媒介子探针在其媒介子序列的上游或5'端还包含第三发夹形成序列，且在其靶特异性序列的下游或3'端还包含第四发夹形成序列，且所述第三发夹形成序列或其部分与所述第四发夹形成序列或其部分互补，由此，所述媒介子探针能够通过所述第三发夹形成序列和所述第四发夹形成序列形成发夹结构；(iii) the mediator probe further comprises a third hairpin-forming sequence upstream or 5' end of its mediator sequence, and further comprises a fourth hairpin-forming sequence downstream or 3' end of its target-specific sequence , and the third hairpin-forming sequence or part thereof is complementary to the fourth hairpin-forming sequence or part thereof, whereby the mediator probe can pass through the third hairpin-forming sequence and the third hairpin-forming sequence Four hairpin forming sequences form a hairpin structure;
    并且，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列和媒介子探针接触；and, contacting the sample with the provided upstream oligonucleotide sequence and the mediator probe under conditions that allow nucleic acid hybridization;
    (2)在允许切割媒介子探针的条件下，将步骤(1)的产物与具有5'核酸酶活性的酶接触；(2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
    (3)提供m种检测探针，并且在允许核酸杂交的条件下，将步骤(2)的产物与所述m种检测探针接触，其中，m为大于0的整数，(3) providing m kinds of detection probes, and contacting the product of step (2) with the m kinds of detection probes under conditions that allow nucleic acid hybridization, wherein m is an integer greater than 0,
    并且，每一种检测探针各自独立地从3'至5'方向包含，与一种或多种媒介子序列或其部分互补的一种或多种捕获序列，以及模板序列(templating sequence)；并且，所述m种检测探针包含至少n种捕获序列，其分别与步骤(1)中提供的n种媒介子探针的媒介子序列或其部分互补；并且，And, each detection probe independently comprises, from the 3' to 5' direction, one or more capture sequences complementary to one or more mediator subsequences or portions thereof, and a templating sequence; And, the m detection probes comprise at least n capture sequences, which are respectively complementary to the mediator sequences of the n mediator probes provided in step (1) or parts thereof; and,
    每一种检测探针各自独立地标记有报告基团和淬灭基团，其中，所述报告基团能够发出信号，并且，所述淬灭基团能够吸收或淬灭所述报告基团发出的信号；并且，每一种检测探针在与其互补序列杂交的情况下发出的信号不同于在未与其互补序列杂交的情况下发出的信号；并且，Each detection probe is independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the reporter group emitting and, each detection probe emits a different signal when hybridized to its complementary sequence than when not hybridized to its complementary sequence; and,
    (4)在允许核酸聚合酶进行延伸反应的条件下，将步骤(3)的产物与核酸聚合酶接触；(4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
    (5)对步骤(4)的产物进行熔解曲线分析；并根据熔解曲线分析的结果，确定所述n种靶核酸序列是否存在于所述样品中。(5) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
  2. 权利要求1的方法，所述方法具有选自下列的一个或多个技术特征：The method of claim 1, which has one or more technical features selected from the group consisting of:
    (a)m为小于等于n且大于0的整数；(a) m is an integer less than or equal to n and greater than 0;
    (b)m为≥1、≥2、≥3、≥4、≥5、≥6、≥8、≥10的整数(例如，m为1、2、3、4、5或6)；优选地，当m≥2时，所述m种检测探针各自标记有不同的报告基团；(b) m is an integer ≥1, ≥2, ≥3, ≥4, ≥5, ≥6, ≥8, ≥10 (eg, m is 1, 2, 3, 4, 5, or 6); preferably , when m≥2, the m detection probes are each labeled with different reporter groups;
    (c)步骤(1)提供了至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少8种、至少10种、至少15种、至少20种、至少25种、至少30种、至少35种、至少40种、至少45种媒介子探针；并且，步骤(3)提供了至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少8种、或至少10种检测探针；(c) step (1) provides at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 mediator probes; and, step (3) provides at least 1, at least 2, at least 3, at least 4, at least 5 , at least 6, at least 8, or at least 10 detection probes;
    (d)至少1种，至少2种，至少3种，至少4种，至少5种，至少6种，至少10种，至少20种，至少30种，至少40种，至少45种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)；(d) at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 20, at least 30, at least 40, at least 45 mediator probes each independently capable of forming a hairpin structure, for example each independently having features (i), (ii) or (iii) as defined in claim 1;
    (e)每一种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)；(e) each mediator probe is independently capable of forming a hairpin structure, for example each independently having features (i), (ii) or (iii) as defined in claim 1;
    (f)所述第一、第二、第三或第四发夹形成序列的长度各自独立地为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt， 70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt；(f) the length of the first, second, third or fourth hairpin forming sequence is each independently 5-140nt, eg 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt , 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt;
    (g)所述m种检测探针包含相同的报告基团；并且，在步骤(5)中，对步骤(4)的产物进行熔解曲线分析，并根据所获得的熔解曲线中的熔解峰(熔点)来确定某一种靶核酸序列的存在；(g) the m kinds of detection probes comprise the same reporter group; and, in step (5), the product of step (4) is subjected to melting curve analysis, and according to the melting peak ( melting point) to determine the presence of a certain target nucleic acid sequence;
    (h)所述m种检测探针所包含的报告基团彼此不同；并且，在步骤(5)中，在对步骤(4)的产物进行熔解曲线分析时，分别实时监测每一种报告基团的信号，由此获得各自与一种报告基团的信号对应的多条熔解曲线；随后，根据报告基团的信号种类以及熔解曲线中的熔解峰(熔点)来确定某一种靶核酸序列的存在；(h) the reporter groups contained in the m kinds of detection probes are different from each other; and, in step (5), when melting curve analysis is performed on the product of step (4), each reporter group is monitored in real time respectively. Then, a certain target nucleic acid sequence is determined according to the signal type of the reporter group and the melting peak (melting point) in the melting curve. The presence;
    (i)所述媒介子探针的靶特异性序列与第一发夹形成序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；(i) a linker is further included between the target-specific sequence of the mediator probe and the first hairpin-forming sequence; preferably, the linker comprises one or more nucleotides (eg, one, 2, 3, 4, 5, 8, 10, or more nucleotides);
    (j)所述媒介子探针的第二发夹形成序列与媒介子序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；(j) A linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence; preferably, the linker comprises 1 or more nucleotides (eg, 1, 2 , 3, 4, 5, 8, 10, or more nucleotides);
    (k)所述媒介子探针的第三发夹形成序列与媒介子序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；(k) A linker is also included between the third hairpin-forming sequence of the mediator probe and the mediator sequence; preferably, the linker comprises 1 or more nucleotides (eg, 1, 2 , 3, 4, 5, 8, 10, or more nucleotides);
    (l)所述媒介子探针的靶特异性序列与第四发夹形成序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；(1) a linker is also included between the target-specific sequence of the mediator probe and the fourth hairpin-forming sequence; preferably, the linker comprises 1 or more nucleotides (for example, 1, 2, 3, 4, 5, 8, 10, or more nucleotides);
    (m)所述第一发夹形成序列与所述媒介子序列完全互补或部分互补；(m) the first hairpin-forming sequence is fully or partially complementary to the mediator sequence;
    (n)所述第二发夹形成序列与所述靶特异性序列完全互补或部分互补；(n) the second hairpin-forming sequence is fully or partially complementary to the target-specific sequence;
    (o)所述第三发夹形成序列与所述第四发夹形成序列完全互补或部分互补；(o) the third hairpin-forming sequence is fully or partially complementary to the fourth hairpin-forming sequence;
    (q)所述媒介子探针能够通过所述媒介子序列和所述第一发夹形成序列、或者所述第二发夹形成序列和所述靶特异性序列、或者所述第三发夹形成序列和所述第四发夹形成序列形成发夹结构，其中，所述发夹结构的臂具有平末端，或者所述臂具有5’悬突(例如，其5’端具有至少1个，至少2个，或者更多个游离碱基)，或者所述臂具有3’悬突(例如，其3’端具有至少1个，至少2个，或者更多个游离碱基)。(q) The mediator probe can pass through the mediator sequence and the first hairpin-forming sequence, or the second hairpin-forming sequence and the target-specific sequence, or the third hairpin The forming sequence and the fourth hairpin forming sequence form a hairpin structure, wherein the arms of the hairpin structure have blunt ends, or the arms have 5' overhangs (eg, the 5' ends have at least one, at least 2, or more free bases), or the arm has a 3' overhang (eg, its 3' end has at least 1, at least 2, or more free bases).
  3. 权利要求1或2的方法，其中，m＝1，并且n为≥1的整数(例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更大的整数)；The method of claim 1 or 2, wherein m=1, and n is an integer > 1 (eg, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater);
    优选地，所述方法包括下述步骤：Preferably, the method comprises the steps of:
    (1)针对待检测的每一种靶核酸序列，提供一种上游寡核苷酸序列和一种媒介子探针；其中，所述上游寡核苷酸序列包含与所述靶核酸序列互补的序列；并且，所述媒介子探针从5'至3'方向包含媒介子序列和靶特异性序列，所述媒介子序列包含不与所述靶核酸序列互补的序列，并且，所述靶特异性序列包含与所述靶核酸序列互补的序列；并且，当与所述靶核酸序列杂交时，所述上游寡核苷酸序列位于所述靶特异性序列的上游；并且，所有媒介子探针所包含的媒介子序列彼此不同；(1) For each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence and a mediator probe are provided; wherein, the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence sequence; and, the mediator probe comprises a mediator sequence and a target-specific sequence from the 5' to 3' direction, the mediator sequence comprises a sequence that is not complementary to the target nucleic acid sequence, and the target-specific sequence and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence; and, all mediator probes The included mediator subsequences differ from each other;
    其中，至少一种媒介子探针自身能够形成发夹结构；例如，所述至少一种媒介子探针具有权利要求1中定义的特征(i)，(ii)或(iii)；并且，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列和媒介子探针接触；wherein the at least one mediator probe itself is capable of forming a hairpin structure; for example, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1; and, in contacting the sample with the provided upstream oligonucleotide sequence and the mediator probe under conditions that allow nucleic acid hybridization;
    (2)在允许切割媒介子探针的条件下，将步骤(1)的产物与具有5'核酸酶活性的酶接触；(2) contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;
    (3)在允许核酸杂交的条件下，将步骤(2)的产物与一种检测探针接触，所述检测探针从3'至5'方向包含，与每一种媒介子序列或其部分互补的捕获序列，以及模板序列(templating sequence)；并且，所述检测探针标记有报告基团和淬灭基团，其中，所述报告基团能够发出信号，并且，所述淬灭基团能够吸收或淬灭所述报告基团发出的信号；并且，所述检测探针在与其互补序列杂交的情况下发出的信号不同于在未与其互补序列杂交的情况下发出的信号；(3) contacting the product of step (2) with a detection probe comprising from the 3' to 5' direction, under conditions allowing nucleic acid hybridization, with each of the mediator subsequences or portions thereof a complementary capture sequence, and a templating sequence; and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of signaling, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and, the detection probe emits a signal when hybridized to its complementary sequence that is different from the signal emitted when it is not hybridized to its complementary sequence;
    (4)在允许核酸聚合酶进行延伸反应的条件下，将步骤(3)的产物与核酸聚合酶接触；(4) contacting the product of step (3) with the nucleic acid polymerase under conditions that allow the nucleic acid polymerase to carry out the extension reaction;
    (5)对步骤(4)的产物进行熔解曲线分析；并根据熔解曲线分析的结果，确定所述n种靶核酸序列是否存在于所述样品中。(5) Perform melting curve analysis on the product of step (4); and determine whether the n target nucleic acid sequences exist in the sample according to the result of the melting curve analysis.
  4. 权利要求1-3任一项的方法，其中，所述方法具有选自下列的一个或多个技术特征：The method of any one of claims 1-3, wherein the method has one or more technical features selected from the following:
    (a)所述样品包含或是DNA，或RNA，或核酸的混合物；(a) the sample comprises or is a mixture of DNA, or RNA, or nucleic acids;
    (b)所述靶核酸序列是DNA或RNA；和/或，所述靶核酸序列是单链的或双链的；(b) the target nucleic acid sequence is DNA or RNA; and/or, the target nucleic acid sequence is single-stranded or double-stranded;
    (c)所述样品或靶核酸序列获自选自下列的来源：原核生物，真核生物，病毒或类病毒。(c) The sample or target nucleic acid sequence is obtained from a source selected from the group consisting of prokaryotes, eukaryotes, viruses or viroids.
    (d)所述媒介子探针包含或者由天然存在的核苷酸，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；(d) the mediator probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof;
    (e)所述媒介子探针的长度为15-150nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt，140-150nt；(e) The length of the mediator probe is 15-150nt, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90 -100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt;
    (f)所述媒介子探针中的靶特异性序列的长度为10-140nt，例如10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt；(f) The length of the target-specific sequence in the mediator probe is 10-140nt, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt;
    (g)所述媒介子探针中的媒介子序列的长度可以为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt；(g) The length of the mediator sequence in the mediator probe can be 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt , 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt;
    (h)所述媒介子探针具有3'-OH末端，或者其3'-末端是封闭的；(h) the mediator probe has a 3'-OH terminus, or its 3'-terminus is blocked;
    (i)所述上游寡核苷酸序列包含或者由天然存在的核苷酸，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；(i) the upstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof;
    (j)所述上游寡核苷酸序列的长度为15-150nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt，140-150nt；(j) The length of the upstream oligonucleotide sequence is 15-150nt, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt , 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt;
    (k)所述上游寡核苷酸序列在与靶核酸序列杂交后，位于媒介子探针的上游远端，或位于媒介子探针的上游邻近，或与媒介子探针的靶特异性序列具有部分重叠的序列；和(k) The upstream oligonucleotide sequence is located at the upstream distal end of the mediator probe after hybridization with the target nucleic acid sequence, or is located in the upstream vicinity of the mediator probe, or is located at the target-specific sequence of the mediator probe have partially overlapping sequences; and
    (l)所述上游寡核苷酸序列为特异于靶核酸序列的引物或者特异于靶核酸序列的探针。(1) The upstream oligonucleotide sequence is a primer specific to the target nucleic acid sequence or a probe specific to the target nucleic acid sequence.
  5. 权利要求1-4任一项的方法，其中，所述方法具有选自下列的一个或多个技术特征：The method of any one of claims 1-4, wherein the method has one or more technical features selected from the following:
    (a)在步骤(2)中，所述具有5'核酸酶活性的酶切割与靶核酸序列杂交的媒介子探针，并释放出包含完整媒介子序列或者媒介子序列的一部分(5'-末端部分)的 媒介子片段；(a) In step (2), the enzyme with 5' nuclease activity cleaves the mediator probe hybridized with the target nucleic acid sequence, and releases a part (5'- end portion) of the mediator subfragment;
    (b)所述具有5'核酸酶活性的酶为具有5'核酸酶活性(例如5'外切核酸酶活性)的核酸聚合酶(例如，DNA聚合酶，特别是热稳定的DNA聚合酶)；(b) the enzyme having 5' nuclease activity is a nucleic acid polymerase (eg, DNA polymerase, especially a thermostable DNA polymerase) having 5' nuclease activity (eg, 5' exonuclease activity) ;
    优选地，所述DNA聚合酶获自选自下列的细菌：Thermus aquaticus(Taq),Thermus thermophiles(Tth),Thermus filiformis,Thermis flavus,Thermococcus literalis,Thermus antranildanii,Thermus caldophllus,Thermus chliarophilus,Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima,Thermotoga neapolitana,Thermosiphoafricanus,Pyrococcus woesei,Pyrococcus horikoshii,Pyrococcus abyssi,Pyrodictium occultum,Aquifexpyrophilus和Aquifex aeolieus；特别优选地，所述DNA聚合酶为Taq聚合酶；Preferably, the DNA polymerase is obtained from a bacterium selected from the group consisting of: Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae ,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi,Thermococcus gorgonarius,Thermotoga maritima,Thermotoga neapolitana,Thermosiphoafricanus,Pyrococcus woesei , Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeolieus; Particularly preferably, the DNA polymerase is Taq polymerase;
    优选地，在步骤(2)中，所述具有5'核酸酶活性的核酸聚合酶催化上游寡核苷酸序列的延伸，并诱导媒介子探针的切割；Preferably, in step (2), the nucleic acid polymerase with 5' nuclease activity catalyzes the extension of the upstream oligonucleotide sequence and induces the cleavage of the mediator probe;
    (c)在步骤(2)中，使用核酸聚合酶以靶核酸序列为模板催化上游寡核苷酸序列的延伸，并且随后，所述具有5'核酸酶活性的酶结合至上游寡核苷酸序列的延伸产物，并催化媒介子探针的切割。(c) in step (2), using a nucleic acid polymerase to catalyze the extension of the upstream oligonucleotide sequence with the target nucleic acid sequence as a template, and then, the enzyme having 5' nuclease activity is bound to the upstream oligonucleotide Sequence extension product and catalyzes cleavage of the mediator probe.
  6. 权利要求1-5任一项的方法，其中，在步骤(1)中，除了所述上游寡核苷酸序列和媒介子探针之外，针对待检测的每一种靶核酸序列，还提供一种下游寡核苷酸序列；其中，所述下游寡核苷酸序列包含与所述靶核酸序列互补的序列；并且，当与所述靶核酸序列杂交时，所述下游寡核苷酸序列位于所述靶特异性序列的下游；然后，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列、媒介子探针和下游寡核苷酸序列接触；The method of any one of claims 1-5, wherein, in step (1), in addition to the upstream oligonucleotide sequence and the mediator probe, for each target nucleic acid sequence to be detected, also provide A downstream oligonucleotide sequence; wherein the downstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the downstream oligonucleotide sequence downstream of the target-specific sequence; then, contacting the sample with the provided upstream oligonucleotide sequence, mediator probe, and downstream oligonucleotide sequence under conditions that allow nucleic acid hybridization;
    优选地，在步骤(2)中，在允许核酸扩增的条件下，将步骤(1)的产物与具有5'核酸酶活性的核酸聚合酶接触；Preferably, in step (2), the product of step (1) is contacted with a nucleic acid polymerase having 5' nuclease activity under conditions that allow nucleic acid amplification;
    优选地，所述下游寡核苷酸序列具有选自下列的一个或多个特征：Preferably, the downstream oligonucleotide sequence has one or more characteristics selected from the group consisting of:
    (a)所述下游寡核苷酸序列包含或者由天然存在的核苷酸，经修饰的核苷酸，非 天然的核苷酸，或其任何组合组成；和(a) the downstream oligonucleotide sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and
    (b)所述下游寡核苷酸序列的长度为15-150nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt，140-150nt；(b) the length of the downstream oligonucleotide sequence is 15-150nt, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt , 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt;
    优选地，步骤(1)中提供的所有上游寡核苷酸序列和下游寡核苷酸序列在5'端具有一段相同的寡核苷酸序列；Preferably, all upstream oligonucleotide sequences and downstream oligonucleotide sequences provided in step (1) have an identical oligonucleotide sequence at the 5' end;
    优选地，在步骤(1)中，除了所述上游寡核苷酸序列、媒介子探针和下游寡核苷酸序列之外，还提供一种通用引物，所述通用引物具有与所述相同的寡核苷酸序列互补的序列；然后，在允许核酸杂交的条件下，将所述样品与所提供的上游寡核苷酸序列、媒介子探针、下游寡核苷酸序列和通用引物接触；Preferably, in step (1), in addition to the upstream oligonucleotide sequence, the mediator probe and the downstream oligonucleotide sequence, a universal primer is also provided, and the universal primer has the same the sequence complementary to the oligonucleotide sequence of the ;
    优选地，所述通用引物具有选自下列的一个或多个特征：Preferably, the universal primer has one or more characteristics selected from the group consisting of:
    (a)所述通用引物包含或者由天然存在的核苷酸，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；和(a) the universal primer comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and
    (b)所述通用引物的长度为8-50nt，例如8-15nt，15-20nt，20-30nt，30-40nt，或40-50nt。(b) The length of the universal primer is 8-50nt, eg, 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.
  7. 权利要求1-6任一项的方法，其中，所述检测探针具有选自下列的一个或多个特征：The method of any one of claims 1-6, wherein the detection probe has one or more characteristics selected from the group consisting of:
    (a)所述检测探针包含多种捕获序列；并且，所述多种捕获序列以相邻的方式、以间隔有连接序列的方式，或者以重叠的方式排列；(a) the detection probe comprises a plurality of capture sequences; and, the plurality of capture sequences are arranged in an adjacent manner, in a manner of being spaced by a linking sequence, or in an overlapping manner;
    (b)所述检测探针包含或者由天然存在的核苷酸，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；(b) the detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof;
    (c)所述检测探针的长度为15-1000nt，例如15-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-200nt，200-300nt，300-400nt，400-500nt，500-600nt，600-700nt，700-800nt，800-900nt，900-1000nt；(c) The length of the detection probe is 15-1000nt, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90- 100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000nt;
    (d)所述检测探针中的捕获序列的长度为10-500nt，例如10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-150nt，150-200nt，200-250nt，250-300nt，300-350nt，350-400nt，400-450nt，450-500nt；(d) The length of the capture sequence in the detection probe is 10-500nt, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80- 90nt, 90-100nt, 100-150nt, 150-200nt, 200-250nt, 250-300nt, 300-350nt, 350-400nt, 400-450nt, 450-500nt;
    (e)所述检测探针中的模板序列的长度为1-900nt，例如1-5nt，5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-200nt，200-300nt，300-400nt，400-500nt，500-600nt，600-700nt，700-800nt，800-900nt；(e) The length of the template sequence in the detection probe is 1-900nt, such as 1-5nt, 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-nt 70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt;
    (f)所述检测探针具有3'-OH末端，或者其3'-末端是封闭的；(f) the detection probe has a 3'-OH terminus, or its 3'-terminus is blocked;
    (g)所述检测探针为自淬灭探针；例如，所述检测探针在其5'末端或上游标记有报告基团且在其3'末端或下游标记有淬灭基团，或者在其3'末端或下游标记报告基团且在5'末端或上游标记淬灭基团；优选地，所述报告基团和淬灭基团相距10-80nt或更长的距离；(g) the detection probe is a self-quenching probe; for example, the detection probe is labeled with a reporter group at its 5' end or upstream and labeled with a quencher group at its 3' end or downstream, or A reporter group is labeled at its 3' end or downstream and a quencher group is labeled at its 5' end or upstream; preferably, the reporter group and the quencher group are separated by a distance of 10-80 nt or more;
    (h)所述检测探针中的报告基团为荧光基团(例如ALEX-350,FAM,VIC,TET,CAL
    Figure PCTCN2020140053-appb-100001
    Gold 540,JOE,HEX,CAL Fluor Orange 560,TAMRA,CAL Fluor Red 590,ROX,CAL Fluor Red 610,TEXAS RED,CAL Fluor Red 635,Quasar 670,CY3,CY5,CY5.5,Quasar 705)；并且，淬灭基团为能够吸收/淬灭所述荧光的分子或基团(例如DABCYL、BHQ(例如BHQ-1或者BHQ-2)、ECLIPSE、和/或TAMRA)；     (h) The reporter group in the detection probe is a fluorescent group (such as ALEX-350, FAM, VIC, TET, CAL
    Figure PCTCN2020140053-appb-100001
    Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and , the quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg DABCYL, BHQ (eg BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA);
    (i)所述检测探针是线性的，或者具有发夹结构。(i) The detection probe is linear, or has a hairpin structure.
  8. 权利要求1-7任一项的方法，其中，在步骤(4)中，在允许核酸聚合酶进行延伸反应的条件下，所述核酸聚合酶以检测探针为模板，对杂交至检测探针的媒介子片段进行延伸，并由此形成双链体；The method of any one of claims 1-7, wherein, in step (4), under conditions that allow nucleic acid polymerase to carry out an extension reaction, the nucleic acid polymerase uses the detection probe as a template to hybridize to the detection probe. The mediator sub-segment is extended, and thus a duplex is formed;
    优选地，步骤(2)中所使用的具有5'核酸酶活性的酶为具有5'核酸酶活性的核酸聚合酶，并且与步骤(4)中所使用的核酸聚合酶相同。Preferably, the enzyme with 5' nuclease activity used in step (2) is a nucleic acid polymerase with 5' nuclease activity, and is the same as the nucleic acid polymerase used in step (4).
  9. 权利要求1-8任一项的方法，其中，步骤(1)-(4)通过包含下述步骤(I)-(VI)的方案来进行：The method of any one of claims 1-8, wherein steps (1)-(4) are carried out by a scheme comprising the following steps (I)-(VI):
    (I)提供m种检测探针，并且针对待检测的每一种靶核酸序列，提供一种上游寡核苷酸序列、一种媒介子探针和一种下游寡核苷酸序列；并且，任选地，提供一种通用引物；其中，所述下游寡核苷酸序列如权利要求6所定义，并且所述通用引物如权利要求6所定义；(1) providing m detection probes, and for each target nucleic acid sequence to be detected, providing an upstream oligonucleotide sequence, a mediator probe and a downstream oligonucleotide sequence; and, Optionally, a universal primer is provided; wherein the downstream oligonucleotide sequence is as defined in claim 6, and the universal primer is as defined in claim 6;
    (II)将待检测的样品与所提供的检测探针，上游寡核苷酸序列，媒介子探针和下游寡核苷酸序列，以及具有5'核酸酶活性的模板依赖性核酸聚合酶(例如，DNA聚 合酶，特别是热稳定的DNA聚合酶)混合；并且任选地，添加通用引物；(II) combining the sample to be detected with the provided detection probe, upstream oligonucleotide sequence, mediator probe and downstream oligonucleotide sequence, and a template-dependent nucleic acid polymerase with 5' nuclease activity ( For example, a DNA polymerase, especially a thermostable DNA polymerase) is mixed; and optionally, a universal primer is added;
    (III)在允许核酸变性的条件下，温育前一步骤的产物；(III) incubating the product of the previous step under conditions that allow nucleic acid denaturation;
    (IV)在允许核酸退火或杂交的条件下，温育前一步骤的产物；(IV) incubating the product of the previous step under conditions that allow nucleic acid annealing or hybridization;
    (V)在允许核酸延伸的条件下，温育前一步骤的产物；和(v) incubating the product of the previous step under conditions that allow nucleic acid extension; and
    (VI)任选地，重复步骤(III)-(V)一次或多次。(VI) Optionally, repeat steps (III)-(V) one or more times.
  10. 权利要求1-9任一项的方法，其中，在步骤(5)中，对步骤(4)的产物进行逐渐的升温或降温并实时监测每一种检测探针上的报告基团发出的信号，从而获得每一种报告基团的信号强度随着温度变化而变化的曲线；The method of any one of claims 1-9, wherein, in step (5), the product of step (4) is gradually heated or cooled and the signal sent by the reporter group on each detection probe is monitored in real time , so as to obtain the curve of the signal intensity of each reporter group as a function of temperature;
    优选地，对所获得的曲线进行求导，从而获得步骤(4)的产物的熔解曲线；Preferably, the obtained curve is derived to obtain the melting curve of the product of step (4);
    优选地，根据熔解曲线中的熔解峰(熔点)，确定对应于所述熔解峰(熔点)的媒介子片段的存在；随后，通过媒介子片段中的媒介子序列与靶核酸序列的对应关系，确定与所述媒介子片段对应的靶核酸序列的存在。Preferably, according to the melting peak (melting point) in the melting curve, the existence of the mediator sub-fragment corresponding to the melting peak (melting point) is determined; then, through the correspondence between the mediator subsequence in the mediator sub-fragment and the target nucleic acid sequence, The presence of a target nucleic acid sequence corresponding to the mediator fragment is determined.
  11. 一种探针组(probe set)，其包含一种检测探针，以及一种或多种(例如至少两种)的媒介子探针，其中，A probe set comprising a detection probe, and one or more (for example at least two) mediator probes, wherein,
    所述媒介子探针各自独立地从5'至3'方向包含媒介子序列和靶特异性序列，所述靶特异性序列包含与一种靶核酸序列互补的序列，所述媒介子序列包含不与所述靶核酸序列互补的序列，并且，所有媒介子探针所包含的媒介子序列彼此不同；The mediator probes each independently comprise, in the 5' to 3' direction, a mediator sequence comprising a sequence complementary to a target nucleic acid sequence and a target-specific sequence comprising a non-target nucleic acid sequence. a sequence complementary to the target nucleic acid sequence, and the mediator sequences contained in all mediator probes are different from each other;
    其中，至少一种媒介子探针自身能够形成发夹结构；优选地，所述至少一种媒介子探针具有权利要求1中定义的特征(i)，(ii)或(iii)；和wherein the at least one mediator probe itself is capable of forming a hairpin structure; preferably, the at least one mediator probe has features (i), (ii) or (iii) as defined in claim 1; and
    所述检测探针从3'至5'方向包含，与每一种媒介子序列或其部分互补的捕获序列，以及模板序列(templating sequence)；并且，所述检测探针标记有报告基团和淬灭基团，其中，所述报告基团能够发出信号，并且，所述淬灭基团能够吸收或淬灭所述报告基团发出的信号；并且，所述检测探针在与其互补序列杂交的情况下发出的信号不同于在未与其互补序列杂交的情况下发出的信号；The detection probe comprises, from 3' to 5', a capture sequence complementary to each mediator subsequence or a portion thereof, and a templating sequence; and the detection probe is labeled with a reporter group and a quenching group, wherein the reporter group can emit a signal, and the quenching group can absorb or quench the signal emitted by the reporter group; and the detection probe is hybridized to its complementary sequence The signal is different from the signal when it is not hybridized to its complementary sequence;
    优选地，所述探针组包含至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少8种、至少9种、至少10种、至少12种、至少15种、或至少20种媒介子探针；Preferably, the probe set comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 mediator probes;
    优选地，至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至 少7种、至少8种、至少9种、至少10种、至少12种、至少15种、或至少20种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)；Preferably, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15 , or at least 20 mediator probes each independently capable of forming a hairpin structure, for example each independently having features (i), (ii) or (iii) as defined in claim 1;
    优选地，每一种媒介子探针各自独立地能够形成发夹结构，例如各自独立地具有权利要求1中定义的特征(i)，(ii)或(iii)；Preferably, each mediator probe is independently capable of forming a hairpin structure, eg each independently has features (i), (ii) or (iii) as defined in claim 1;
    优选地，所述第一、第二、第三或第四发夹形成序列的长度各自独立地为5-140nt，例如5-10nt，10-20nt，20-30nt，30-40nt，40-50nt，50-60nt，60-70nt，70-80nt，80-90nt，90-100nt，100-110nt，110-120nt，120-130nt，130-140nt；Preferably, the length of the first, second, third or fourth hairpin forming sequence is each independently 5-140nt, such as 5-10nt, 10-20nt, 20-30nt, 30-40nt, 40-50nt , 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt;
    优选地，所述媒介子探针的靶特异性序列与第一发夹形成序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；Preferably, a linker is further included between the target-specific sequence of the mediator probe and the first hairpin-forming sequence; preferably, the linker comprises one or more nucleotides (eg, one, 2, 3, 4, 5, 8, 10, or more nucleotides);
    优选地，所述媒介子探针的第二发夹形成序列与媒介子序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；Preferably, a linker is further included between the second hairpin-forming sequence of the mediator probe and the mediator sequence; preferably, the linker comprises 1 or more nucleotides (for example, 1, 2 , 3, 4, 5, 8, 10, or more nucleotides);
    优选地，所述媒介子探针的第三发夹形成序列与媒介子序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；Preferably, a linker is further included between the third hairpin-forming sequence of the mediator probe and the mediator sequence; preferably, the linker comprises 1 or more nucleotides (for example, 1, 2 , 3, 4, 5, 8, 10, or more nucleotides);
    优选地，所述媒介子探针的靶特异性序列与第四发夹形成序列之间还含有连接体；优选地，所述连接体包含1个或多个核苷酸(例如，1个，2个，3个，4个，5个，8个，10个，或更多个核苷酸)；Preferably, a linker is further included between the target-specific sequence of the mediator probe and the fourth hairpin forming sequence; preferably, the linker comprises one or more nucleotides (eg, one, 2, 3, 4, 5, 8, 10, or more nucleotides);
    优选地，所有媒介子探针所包含的靶特异性序列彼此不同；Preferably, the target-specific sequences contained in all mediator probes are different from each other;
    优选地，所有媒介子探针各自靶向不同的靶核酸序列；Preferably, all mediator probes each target a different target nucleic acid sequence;
    优选地，所述媒介子探针如权利要求1、2和4所定义；和/或，所述检测探针如权利要求1和7所定义。Preferably, the mediator probe is as defined in claims 1, 2 and 4; and/or the detection probe is as defined in claims 1 and 7.
  12. 一种试剂盒，其包含一种或多种如权利要求11所定义的探针组；A kit comprising one or more probe sets as defined in claim 11;
    优选地，所述试剂盒包含至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少8种、至少9种、至少10种探针组；Preferably, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 probe sets ;
    优选地，所述试剂盒中的所有媒介子序列各自靶向不同的靶核酸序列；Preferably, all mediator sequences in the kit each target a different target nucleic acid sequence;
    优选地，所述试剂盒中的所有媒介子探针所包含的媒介子序列彼此不同；Preferably, the mediator sequences contained in all mediator probes in the kit are different from each other;
    优选地，所述试剂盒中的所有媒介子探针所包含的靶特异性序列彼此不同；Preferably, the target-specific sequences contained in all the mediator probes in the kit are different from each other;
    优选地，所述试剂盒中的所有检测探针各自独立地标记有相同或不同的报告基团；Preferably, all detection probes in the kit are independently labeled with the same or different reporter groups;
    优选地，所述试剂盒还包含：上游寡核苷酸序列(例如如权利要求1或4所定义的上游寡核苷酸序列)、下游寡核苷酸序列(例如如权利要求6所定义的下游寡核苷酸序列)、通用引物(例如如权利要求6所定义的通用引物)、具有5'核酸酶活性的酶、核酸聚合酶、或其任何组合；Preferably, the kit further comprises: an upstream oligonucleotide sequence (eg as defined in claim 1 or 4), a downstream oligonucleotide sequence (eg as defined in claim 6) downstream oligonucleotide sequences), universal primers (e.g., universal primers as defined in claim 6), enzymes with 5' nuclease activity, nucleic acid polymerases, or any combination thereof;
    优选地，所述试剂盒还包含：用于进行核酸杂交的试剂、用于进行媒介子探针切割的试剂、用于进行核酸延伸的试剂、用于进行核酸扩增的试剂、或其任何组合。Preferably, the kit further comprises: reagents for nucleic acid hybridization, reagents for mediator probe cleavage, reagents for nucleic acid extension, reagents for nucleic acid amplification, or any combination thereof .
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