CN106636409A - Universal fluorescent probe and detection method and application thereof - Google Patents
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Abstract
The invention relates to the field of real-time fluorescent PCRs (Polymerase Chain Reactions), in particular a universal fluorescent probe and a detection method and an application thereof. The universal fluorescent probe comprises a positive chain probe, a negative chain probe and a specific downstream primer, wherein the positive chain probe comprises an oligonucleotide positive chain sequence, a connection sequence and a specific upstream primer sequence which are connected in sequence from end 5' to end 3'; the negative chain probe comprises an oligonucleotide negative chain sequence which is complementary with the oligonucleotide positive chain sequence, the end 5' and the end 3' of the oligonucleotide negative chain sequence are connected with a fluorescent group and a quenching group respectively; the specific upstream primer sequence and the specific downstream primer are complementary with a target sequence of a target gene. According to the detection method, fluorescent PCR amplification is performed by the universal fluorescent probe; the universal fluorescent probe can be applied to genetic typing and mutation gene detection; by adopting the universal fluorescent probe and the detection method and the application thereof, real-time detection is realized, PCR subsequent treatment is not required, the designing and synthesizing time is saved, cost is lowered, and high result stability and easiness in analysis are realized.
Description
Technical Field
The invention relates to the field of real-time fluorescence PCR, in particular to a universal fluorescent probe and a detection method and application thereof.
Background
In the prior art, the application of a fluorescein labeling method in PCR is mainly embodied in two aspects of a fluorescent primer or a fluorescent probe.
The method of fluorescent primer mainly includes directly marking primer or marking joint primer, and PCR product adopts genetic analyzer, such as ABI genetic analyzer 310, 3130xl, 3730, etc., and is usually used for detecting microsatellite and genotyping, for example, the invention patent with Chinese patent publication No. CN104178566, which discloses a multiple fluorescent PCR universal joint for microsatellite detection and detection method and application, and its primer design diagram and detection flow diagram are respectively shown in FIG. 1 and FIG. 2. The disadvantages of this technique include: a) real-time detection cannot be realized; b) the result of the map is difficult to analyze and is easy to misjudge; c) PCR products need post-treatment and are easy to pollute the detection environment.
The fluorescent probe can be divided into a hydrolysis probe and a hybridization probe, and the fluorescent signal detection is mainly carried out by a real-time fluorescent quantitative PCR instrument. Wherein, 1) hydrolysis probe: as shown in fig. 3, taking TaqMan probe as an example for illustration, the 5 'end and the 3' end of the hydrolysis probe are respectively labeled with a reporter fluorophore and a quencher fluorophore, and when the probe is intact, the fluorescence signal generated by the system exciting the donor is absorbed by the adjacent quencher fluorophore, so that the donor fluorescence signal cannot be detected at this time; when Taq DNA polymerase is amplified to the site of the probe combined template in the PCR process, the activity of 5' -3' exonuclease cuts off a reporter group at the 5' end of the probe, namely a free reporter group is far away from a quenching group, the transfer of energy is broken, a fluorescence signal generated by exciting the reporter group can be detected by a fluorescence detection system, and the signal accumulation is detected. The TaqMan probe technology needs to design a specific probe aiming at a target gene or a mutation site, different genes or mutation sites need to be redesigned and synthesized, and the problems of high cost and time consumption exist. 2) And (3) hybridizing the two specific probes on the template during renaturation, enabling the two specific probes to approach each other to generate a detection signal, and detecting a real-time fluorescent signal when the temperature-rising denaturation probe is far away from the template and no signal exists.
A Molecular Beacon (MB) is a stem-loop double-labeled oligonucleotide probe with a hairpin structure, nucleic acid sequences at two ends are complementarily paired, a fluorescent group is closely adjacent to a quenching group labeled at the other end, a stem-loop structure is formed when a template does not exist at a renaturation temperature, and the complementary paired stem-loop double strands are separated when the template is heated and denatured, as shown in FIG. 4, if a loop sequence exists in the template, the Molecular beacon is paired with the template. Upon pairing with the template, the molecular beacon will chain rather than hairpin, leaving the fluorophore and quencher separated. When the fluorophore is excited, the quenching is released and an excitation photon is emitted. The molecular beacon technology needs to design a specific probe aiming at a target gene or a mutation site, different genes or mutation sites need to be redesigned and synthesized, and the problems of high cost and time consumption exist; in addition, the detection needs to be carried out through a melting curve Tm peak value, and the detection sensitivity is limited.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a universal fluorescent probe which can realize real-time detection, does not need PCR subsequent treatment, saves design and synthesis time, reduces cost, has good result stability and is easy to analyze, and a detection method and application thereof.
The invention provides a general fluorescent probe in a first aspect, which comprises a plus strand probe, a minus strand probe and a specific downstream primer; wherein,
the positive strand probe comprises an oligonucleotide positive strand sequence (UAP), a connecting sequence and a specific upstream primer sequence (ASO), which are sequentially connected from a 5 'end to a 3' end;
the negative strand probe comprises an oligonucleotide negative strand sequence which is complementary with an oligonucleotide positive strand sequence, and the 5 'end and the 3' end of the oligonucleotide negative strand sequence are respectively connected with a fluorescent group (R) and a quenching group (Q);
the specific upstream primer sequence (ASO) and the specific downstream primer (LSO) are mutually complementary with a target point sequence of a target gene.
Further, the linker sequence includes a TA Box sequence, which is a sequence including, but not limited to, AA, TT, AAA, TTT, AT, TA, ATA, TAT, AAT, TAA, ATTT, TAAA, TATT, AATA, TTAT, TATA, ATAT, ATATA, TATATA, TTTAA, TATTT, TTATT, TTTAT, TAATT, TTAAT, AAATT, ATAAA, AATAA, or AAATA.
It should be noted that, in order to achieve universal detection of the universal fluorescent probe in a certain class of target organisms, the oligonucleotide plus strand sequence has no homology or less than 30% sequence similarity with the sequence of the target organism detected by the universal fluorescent probe.
It should be noted that homology refers to the relationship between branches from the same ancestor during evolution, and homology is used to describe the evolutionary relationship between species, and that homologous sequences refer to different sequences that have evolved divergently from a common ancestor. Similarity refers to the ratio of identical DNA base or amino acid residue sequence between the detected sequence and the target sequence. It is generally accepted that the higher the similarity (similarity) between sequences, the greater the likelihood of homology between sequences. When the degree of similarity is more than 50%, it is relatively easy to presume that the detection sequence and the target sequence may be homologous sequences; when the degree of similarity is less than 20%, it is difficult to determine or impossible to determine whether the homology is present between the two.
Regarding the design of the universal fluorescent probe, if the target organism is a eukaryote, the oligonucleotide plus strand sequence can be designed according to the yeast genome sequence.
Further, the oligonucleotide positive strand sequence adopts a sequence shown as SEQ ID NO: 1-6, and correspondingly, the oligonucleotide negative strand sequence adopts a nucleotide sequence shown as SEQ ID NO: 7-12. The sequence information is specifically as follows:
GCGTGTTGAGTGTGCGGCGTAGA(SEQ ID NO：1)
GCGAAAGCCTGACGGAGCGAG(SEQ ID NO：2)
AGATGTAGCGATAGCCTGAGCGAGCGA(SEQ ID NO：3)
GATGAGTGTGTTGCGGCGTAGATGAGTG(SEQ ID NO：4)
TAGCGATAGCCTGAGCGAGCGA(SEQ ID NO：5)
GCGATGCGTGACGGAGAGTGAG(SEQ ID NO：6)
R-TCTACGCCGCACACTCAACACGC-Q(SEQ ID NO：7)
R-CTCGCTCCGTCAGGCTTTCGC-Q(SEQ ID NO：8)
R-TCGCTCGCTCAGGCTATCGCTACATCT-Q(SEQ ID NO：9)
R-CACTCATCTACGCCGCAACACACTCATC-Q(SEQ ID NO：10)
R-TCGCTCGCTCAGGCTATCGCTA-Q(SEQ ID NO：11)
R-CTCACTCTCCGTCACGCATCGC-Q(SEQ ID NO：12)
wherein R represents a fluorescent group and Q represents a quenching group.
Further, the fluorescent group includes, but is not limited to, FAM, TET, Texas Red, VIC, or Cy5, and the quenching group includes, but is not limited to, BHQ1 or BHQ 2.
The second aspect of the present invention provides a detection method using the above general fluorescent probe, comprising the following steps:
s1, extracting genome DNA of the sample;
s2, mixing the positive strand probe, the negative strand probe and the specific downstream primer in the universal fluorescent probe according to a certain proportion;
s3, preparing a PCR reaction system, and performing real-time fluorescence PCR amplification;
and S4, analyzing the result according to the fluorescence signal intensity detected in real time.
It should be noted that, for a low-abundance target sample or a low-recovery nucleic acid sample, after step S1, a step of pre-amplifying the genomic DNA of the sample by using an upstream primer (FP) and a downstream primer (RP) is further included, and then a plus strand probe, a minus strand probe and a specific downstream primer in the universal fluorescent probe are mixed according to a certain ratio and then subjected to PCR amplification; for a high-abundance target sample or a nucleic acid sample with high recovery rate, a positive strand probe, a negative strand probe and a specific downstream primer in the general fluorescent probe can be directly mixed according to a certain proportion, and then PCR amplification is carried out.
Further, the upstream primer and the downstream primer, and the universal fluorescent probe are arranged in multiple groups to detect multiple target genes simultaneously, or the universal fluorescent probe is arranged in multiple groups to detect multiple target genes, or multiple genotypes of a single target gene.
The third aspect of the invention provides an application of the general fluorescent probe in genotyping and detecting mutant genes.
By the scheme, the invention at least has the following advantages:
1) the general fluorescent probe has specific sequence and high universality;
2) the universal fluorescent probe can be used for SNP locus detection;
3) TA Box is introduced into the UAP sequence and the ASO sequence, so that the fluorescence quenching effect caused by the fluorescent group and the adjacent template base is eliminated, and the detection is prevented from being influenced;
4) the UAP sequence is flexibly changed, and the influence of a secondary mechanism of the primer on a detection result is eliminated;
5) the one-step detection method can obtain a real-time detection result without subsequent treatment, and is quick, accurate and pollution-free.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a diagram of a prior art multiplex fluorescent PCR universal adaptor primer design;
FIG. 2 is a flow chart of a prior art method for detecting a universal adaptor for multiplex fluorescent PCR for microsatellite detection;
FIG. 3 is a diagram of the working principle of a prior art TaqMan probe;
FIG. 4 is a diagram of the operation of a molecular beacon in the prior art;
FIG. 5 is a diagram showing a design of a universal fluorescent probe according to the first embodiment of the present invention;
FIG. 6 shows the principle of PCR amplification using a universal fluorescent probe according to the first embodiment of the present invention;
FIG. 7 is a graph showing the results of detection of mutant gene loci in alleles using a BIO-RAD fluorescence quantitative PCR instrument and a universal fluorescence probe;
FIG. 8 is a graph showing the results of detection of mutant gene loci in alleles using an ABI7500 fluorescent quantitative PCR instrument and a universal fluorescent probe;
FIG. 9 is a graph showing the result of genotyping in the second example of the present invention;
FIG. 10 is a graph showing the result of genotyping in the third example of the present invention;
FIG. 11 is a graph showing the result of genotyping in the fourth example of the present invention;
FIG. 12 is a graph showing the result of genotyping in the fifth example of the present invention;
FIG. 13 is a graph showing the result of genotyping in the sixth example of the present invention.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example one
A preferred embodiment of the present invention provides a set of universal fluorescent probes, as shown in FIG. 5, comprising a plus strand probe, a minus strand probe, and a specific downstream primer; wherein, the positive strand probe comprises an oligonucleotide positive strand sequence (UAP), a connecting sequence (TA Box) and a specific upstream primer sequence (ASO) which are sequentially connected from a 5 'end to a 3' end; the negative strand probe comprises an oligonucleotide negative strand sequence (UP) which is complementary with the oligonucleotide positive strand sequence, the 5 'end and the 3' end of the oligonucleotide negative strand sequence are respectively connected with a fluorescent group (R) and a quenching group (Q), the fluorescent group can be selected from FAM, TET, Texas Red, VIC or Cy5, and the quenching group can be selected from BHQ1 or BHQ 2; the specific upstream primer sequence (ASO) and the specific downstream primer (LSO) are mutually complementary with the target point sequence of the target gene.
The detection method adopting the universal fluorescent probe comprises the following steps:
s1, extracting genome DNA of the sample;
s2, mixing the positive strand probe, the negative strand probe and the specific downstream primer in the universal fluorescent probe according to a certain proportion;
s3, preparing a PCR reaction system, and performing real-time fluorescence PCR amplification;
and S4, analyzing the result according to the fluorescence signal intensity detected in real time.
The principle of PCR amplification of a universal fluorescent probe is shown in FIG. 6:
the first step is as follows: amplifying a positive strand probe UAP-TA Box-ASO and a specific downstream primer LSO respectively by taking genome DNA as a template to generate a complementary strand;
the second step is that: taking the amplification product of the first step as a template, extending the specific downstream primer LSO to the UP position of the negative strand probe, and hydrolyzing a fluorescent group at the 5' end of the negative strand probe to generate a free fluorescent group;
the third step: and (4) performing fluorescence detection and analyzing the detection result.
For a low-abundance target sample or a nucleic acid sample with low recovery rate, the method further comprises a step of pre-amplifying the genomic DNA of the sample by using an upstream primer (FP) and a downstream primer (RP) after the step S1, then mixing a positive strand probe, a negative strand probe and a specific downstream primer in the universal fluorescent probe according to a certain proportion, and then carrying out PCR amplification; for a high-abundance target sample or a nucleic acid sample with high recovery rate, a positive strand probe, a negative strand probe and a specific downstream primer in the general fluorescent probe can be directly mixed according to a certain proportion, and then PCR amplification is carried out.
Further, the upstream primer and the downstream primer, and the universal fluorescent probe are arranged in multiple groups to detect multiple target genes simultaneously, or the universal fluorescent probe is arranged in multiple groups to detect multiple target genes, or multiple genotypes of a single target gene.
For example, the universal fluorescent probes are arranged into a plurality of groups to detect a plurality of genotypes of a single target gene, and for further explanation, fluorescent groups and quenching groups are marked at the 5 'end and the 3' end of the UP sequence and can be randomly combined with the UP sequence, but the fluorescent groups and the quenching groups of different universal fluorescent probes used at the same time cannot be repeated, and a one-to-one correspondence relationship needs to be established. The existing fluorescent groups (FAM, TET, Texas Red, VIC, Cy5 and the like) and quenching groups (BHQ1, BHQ2 and the like) are selected according to the detection channel and the detection type of an instrument, so that the spectrum overlapping is avoided.
Taking the detection of mutant gene loci in alleles as an example, the specific experimental procedures are as follows:
s1, designing a universal fluorescent probe, which comprises a positive strand probe (UAP-TA Box-ASO), a negative strand probe (UP), a specific upstream primer (FP) and a specific downstream primer (RP/LSO);
s2, a specific upstream primer (FP) and a specific downstream primer (RP) are used for configuring a reaction system to carry out DNA template pre-amplification;
s3, mixing a positive strand probe (UAP-TA Box-ASO), a negative strand probe (UP) and a specific downstream primer (LSO) according to a certain proportion to form a mixed primer, configuring a reaction system, and carrying out real-time fluorescent quantitative PCR detection on a pre-amplification product;
the reaction system is 10 mu L and comprises 2X TaKaRa TaqTMHS Perfect Mix 5ul, mixed primer 1.2 uL, fluorescent label joint 0.5 uL, deionized water 1 u L, DNA 20 ng.
And S4, analyzing results to generate a detection report, detecting the genotype by detecting the intensity of a fluorescence signal based on the conventional fluorescence quantitative PCR instrument, wherein the typing of different instruments is shown in FIGS. 7 and 8, which are a BIO-RAD fluorescence quantitative PCR instrument and an ABI7500 fluorescence quantitative PCR instrument respectively, so that the mutant gene loci in the alleles can be accurately detected.
Wherein, the sequences of the oligonucleotide plus strand sequence (UAP) and the oligonucleotide minus strand sequence (UP) can be designed as shown in Table 1 respectively.
TABLE 1 UAP and UP sequence Listing
The TA Box sequence combination is shown in table 2.
TABLE 2 TA Box sequence design
Example two
The invention provides a method for genotyping rs2294008 locus by using a universal fluorescent probe in a real-time fluorescent PCR instrument, which comprises the following steps:
s1, synthesizing a positive strand probe (allele-specific primer 1 and allele-specific primer 2), a negative strand probe (allele-specific primer 1 and allele-specific primer 2) complementary to the positive strand probe, and a specific downstream primer (LSO);
TABLE 3 rs2294008 genotype test primer design Table
In the table, the underlined part is the oligonucleotide plus strand sequence (UAP), the Box is the linker sequence (TA Box), and the unlabeled part is the specific forward primer sequence (ASO), corresponding to the wild type and mutant for detection of the rs2294008 site.
S2, selecting a human saliva sample, and extracting the genome DNA of the sample;
s3, selecting a newly synthesized upstream primer (FP) and downstream primer (RP) of the rs2294008 locus to pre-amplify the DNA template;
s4, mixing a positive strand probe (UAP-TA Box-ASO), a negative strand probe (UP) and a specific downstream primer (LSO) according to a certain proportion (molar ratio) by adopting the universal fluorescent probe in the table 3, and uniformly mixing on a vortex mixer to form a mixed primer;
s5, configuring the mixed primers into a PCR reaction system, and carrying out real-time fluorescence PCR amplification on the pre-amplification product;
s6, analyzing the result according to the fluorescence signal intensity detected in real time; genotyping was performed based on the results of fluorescence detection, and the results are shown in FIG. 9, which shows that the universal fluorescent probe can well separate the mutant type and the wild type of the target gene to be detected.
EXAMPLE III
The invention provides a method for genotyping an rs6010620 locus by using a universal fluorescent probe in a real-time fluorescent PCR (polymerase chain reaction) instrument, which comprises the following specific steps of:
s1, synthesizing a positive strand probe (allele-specific primer 1 and allele-specific primer 2), a negative strand probe (allele-specific primer 1 and allele-specific primer 2) complementary to the positive strand probe, and a specific downstream primer (LSO);
TABLE 4 rs6010620 genotyping detection primer design Table
In the table, the underlined part is the oligonucleotide plus strand sequence (UAP), the Box is the linker sequence (TA Box), and the unlabeled part is the specific forward primer sequence (ASO), corresponding to the wild type and mutant for detection of the rs6010620 site.
S2, selecting a human saliva sample, and extracting the genome DNA of the sample;
s3, selecting a newly synthesized upstream primer (FP) and downstream primer (RP) of the rs6010620 locus to pre-amplify the DNA template;
s4, mixing a positive strand probe (UAP-TA Box-ASO), a negative strand probe (UP) and a specific downstream primer (LSO) according to a certain proportion (molar ratio) by adopting the universal fluorescent probe in the table 4, and uniformly mixing on a vortex mixer to form a mixed primer;
s5, configuring the mixed primers into a PCR reaction system, and carrying out real-time fluorescence PCR amplification on the pre-amplification product;
s6, analyzing the result according to the fluorescence signal intensity detected in real time; genotyping was performed based on the results of fluorescence detection, and the results are shown in FIG. 10, which shows that the universal fluorescent probe can well separate the mutant type and the wild type of the target gene to be detected.
Example four
The invention provides a method for genotyping an rs339331 locus by using a universal fluorescent probe in a real-time fluorescent PCR (polymerase chain reaction) instrument, which comprises the following specific steps of:
s1, synthesizing a positive strand probe (allele-specific primer 1 and allele-specific primer 2), a negative strand probe (allele-specific primer 1 and allele-specific primer 2) complementary to the positive strand probe, and a specific downstream primer (LSO);
TABLE 5rs339331 Gene typing detection primer design Table
In the table, the underlined part is the oligonucleotide plus strand sequence (UAP), the Box is the linker sequence (TA Box), and the unlabeled part is the specific forward primer sequence (ASO), corresponding to the wild type and mutant detected at the rs339331 site.
S2, selecting a human saliva sample, and extracting the genome DNA of the sample;
s3, selecting a newly synthesized upstream primer (FP) and downstream primer (RP) of the locus rs339331 to pre-amplify the DNA template;
s4, mixing a positive strand probe (UAP-TA Box-ASO), a negative strand probe (UP) and a specific downstream primer (LSO) according to a certain proportion (molar ratio) by adopting the universal fluorescent probe in the table 5, and uniformly mixing on a vortex mixer to form a mixed primer;
s5, configuring the mixed primers into a PCR reaction system, and carrying out real-time fluorescence PCR amplification on the pre-amplification product;
s6, analyzing the result according to the fluorescence signal intensity detected in real time; genotyping was performed based on the results of fluorescence detection, and the results are shown in FIG. 11, which shows that the universal fluorescent probe can well separate the mutant type and the wild type of the target gene to be detected.
EXAMPLE five
The invention provides a method for genotyping an rs17728461 locus by using a universal fluorescent probe in a real-time fluorescent PCR (polymerase chain reaction) instrument, which comprises the following specific steps of:
s1, synthesizing a positive strand probe (allele-specific primer 1 and allele-specific primer 2), a negative strand probe (allele-specific primer 1 and allele-specific primer 2) complementary to the positive strand probe, and a specific downstream primer (LSO);
TABLE 6 primer design table for rs17728461 genotyping detection
In the table, the underlined part is the oligonucleotide plus strand sequence (UAP), the Box is the linker sequence (TA Box), and the unlabeled part is the specific forward primer sequence (ASO), corresponding to the wild type and mutant detected at rs 17728461.
S2, selecting a human saliva sample, and extracting the genome DNA of the sample;
s3, selecting a newly synthesized upstream primer (FP) and downstream primer (RP) of the locus rs17728461 to pre-amplify the DNA template;
s4, mixing a positive strand probe (UAP-TA Box-ASO), a negative strand probe (UP) and a specific downstream primer (LSO) according to a certain proportion (molar ratio) by adopting the universal fluorescent probe in the table 6, and uniformly mixing on a vortex mixer to form a mixed primer;
s5, configuring the mixed primers into a PCR reaction system, and carrying out real-time fluorescence PCR amplification on the pre-amplification product;
s6, analyzing the result according to the fluorescence signal intensity detected in real time; genotyping was performed based on the results of fluorescence detection, and the results are shown in FIG. 12, which shows that the universal fluorescent probe can well separate the mutant type and the wild type of the target gene to be detected.
EXAMPLE six
The invention provides a method for genotyping rs11903757 locus by using a universal fluorescent probe in a real-time fluorescent PCR instrument, which comprises the following steps:
s1, synthesizing a positive strand probe (allele-specific primer 1 and allele-specific primer 2), a negative strand probe (allele-specific primer 1 and allele-specific primer 2) complementary to the positive strand probe, and a specific downstream primer (LSO);
TABLE 7 rs11903757 genotyping detection primer design Table
In the table, the underlined part is the oligonucleotide plus strand sequence (UAP), the Box is the linker sequence (TA Box), and the unlabeled part is the specific upstream primer sequence (ASO), corresponding to the wild type and mutant for detection of the rs11903757 site.
S2, selecting a human saliva sample, and extracting the genome DNA of the sample;
s3, selecting a newly synthesized upstream primer (FP) and downstream primer (RP) of the rs11903757 locus to pre-amplify the DNA template;
s4, mixing a positive strand probe (UAP-TA Box-ASO), a negative strand probe (UP) and a specific downstream primer (LSO) according to a certain proportion (molar ratio) by adopting the universal fluorescent probe in the table 7, and uniformly mixing on a vortex mixer to form a mixed primer;
s5, configuring the mixed primers into a PCR reaction system, and carrying out real-time fluorescence PCR amplification on the pre-amplification product;
s6, analyzing the result according to the fluorescence signal intensity detected in real time; genotyping was performed based on the results of fluorescence detection, and the results are shown in FIG. 13, which shows that the universal fluorescent probe can well separate the mutant type and the wild type of the target gene to be detected.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Jianlu Biotechnology (Suzhou) Ltd
<120> general fluorescent probe and detection method and application thereof
<130>2016
<160>27
<170>PatentIn version 3.3
<210>1
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<213> Artificial sequence
<400>1
gcgtgttgag tgtgcggcgt aga 23
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gcgaaagcct gacggagcga g 21
<210>3
<211>27
<212>DNA
<213> Artificial sequence
<400>3
agatgtagcg atagcctgag cgagcga 27
<210>4
<211>28
<212>DNA
<213> Artificial sequence
<400>4
gatgagtgtg ttgcggcgta gatgagtg 28
<210>5
<211>22
<212>DNA
<213> Artificial sequence
<400>5
tagcgatagc ctgagcgagc ga 22
<210>6
<211>22
<212>DNA
<213> Artificial sequence
<400>6
gcgatgcgtg acggagagtg ag 22
<210>7
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tctacgccgc acactcaaca cgc 23
<210>8
<211>21
<212>DNA
<213> Artificial sequence
<400>8
ctcgctccgt caggctttcg c 21
<210>9
<211>27
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<213> Artificial sequence
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tcgctcgctc aggctatcgc tacatct 27
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cactcatcta cgccgcaaca cactcatc 28
<210>11
<211>22
<212>DNA
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tcgctcgctc aggctatcgc ta 22
<210>12
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ctcactctcc gtcacgcatc gc 22
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gcgtgttgag tgtgcggcgt agaatataca gcccaccagt gaccac 46
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<213> Artificial sequence
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<210>18
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<213> Artificial sequence
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<400>25
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<213> Artificial sequence
<400>26
gatgagtgtg ttgcggcgta gatgagtgtt ttggacttgt aactggtgtt tgcg 54
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gtacactttg catgaactct ctctccc 27
Claims (10)
1. A universal fluorescent probe characterized by: comprises a positive strand probe, a negative strand probe and a specific downstream primer; wherein,
the positive strand probe comprises an oligonucleotide positive strand sequence, a connecting sequence and a specific upstream primer sequence which are sequentially connected from a 5 'end to a 3' end;
the negative strand probe comprises an oligonucleotide negative strand sequence which is complementary with the oligonucleotide positive strand sequence, and the 5 'end and the 3' end of the oligonucleotide negative strand sequence are respectively connected with a fluorescent group and a quenching group;
the specific upstream primer sequence and the specific downstream primer are mutually complementary with the target point sequence of the target gene.
2. The universal fluorescent probe of claim 1, wherein: the linker sequence includes a TA Box sequence, which is a sequence including, but not limited to, AA, TT, AAA, TTT, AT, TA, ATA, TAT, AAT, TAA, ATTT, TAAA, TATT, AATA, TTAT, TATA, ATAT, ATATA, TATATA, TTTAA, TATTT, TTATT, TTTAT, TAATT, TTAAT, AAATT, ATAAA, AATAA, or AAATA.
3. The universal fluorescent probe of claim 1, wherein: compared with the sequence of a target organism detected by the universal fluorescent probe, the oligonucleotide positive strand sequence has no homology or the sequence similarity is less than 30 percent.
4. The universal fluorescent probe of claim 3, wherein: the target organism is a eukaryote, and the oligonucleotide positive strand sequence is designed according to a yeast genome sequence.
5. The universal fluorescent probe of claim 1, wherein: the oligonucleotide positive strand sequence adopts a sequence shown as SEQ ID NO: 1-6, wherein the oligonucleotide negative strand sequence adopts a nucleotide sequence shown as SEQ ID NO: 7-12.
6. The universal fluorescent probe of claim 1, wherein: the fluorescent group includes, but is not limited to, FAM, TET, Texas Red, VIC or Cy5, and the quenching group includes, but is not limited to, BHQ1 or BHQ 2.
7. A detection method using the universal fluorescent probe according to any one of claims 1 to 6, characterized in that: the method comprises the following steps:
s1, extracting genome DNA of the sample;
s2, mixing the positive strand probe, the negative strand probe and the specific downstream primer in the universal fluorescent probe according to a certain proportion;
s3, preparing a PCR reaction system, and performing real-time fluorescence PCR amplification;
and S4, analyzing the result according to the fluorescence signal intensity detected in real time.
8. The detection method according to claim 7, characterized in that: for a low-abundance target sample or a nucleic acid sample with low recovery rate, the method also comprises a step of pre-amplifying the genomic DNA of the sample by adopting an upstream primer and a downstream primer after the step S1, then mixing a positive strand probe, a negative strand probe and a specific downstream primer in the universal fluorescent probe according to a certain proportion, and then carrying out PCR amplification; for a high-abundance target sample or a nucleic acid sample with high recovery rate, a positive strand probe, a negative strand probe and a specific downstream primer in the general fluorescent probe are directly mixed according to a certain proportion, and then PCR amplification is carried out.
9. The detection method according to claim 7, characterized in that: the upstream primer, the downstream primer and the universal fluorescent probe are arranged into multiple groups to detect multiple target genes simultaneously, or the universal fluorescent probe is arranged into multiple groups to detect multiple target genes or multiple genotypes of a single target gene.
10. Use of a universal fluorescent probe according to any of claims 1 to 6 for genotyping and detecting mutant genes.
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