CN113862339B - Nucleic acid combination, detection kit and method for amplifying target nucleic acid - Google Patents
Nucleic acid combination, detection kit and method for amplifying target nucleic acid Download PDFInfo
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Abstract
The present invention relates to a nucleic acid combination, a detection kit and a method for amplifying a target nucleic acid. The nucleic acid combination product comprises at least one primer pair, wherein the primer pair comprises an upstream primer and a downstream primer, the upstream primer is in a stem-loop structure, when annealing, a loop part and a nucleic acid fragment at the 3 'end of the upstream primer are hybridized with a negative strand of a template, and the nucleic acid fragment at the 3' end of the upstream primer is not hybridized with the negative strand; the downstream primer also has a stem-loop structure, and when annealing, the loop part and the nucleic acid fragment at the 3 'end of the downstream primer hybridize with the positive strand of the template, and the nucleic acid fragment at the 3' end of the downstream primer does not hybridize with the positive strand. The primer pair of the nucleic acid combination product can improve the nonspecific amplification of the multiplex PCR.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a nucleic acid combination product, a detection kit and a method for amplifying a target nucleic acid.
Background
Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments, and is the most efficient method for obtaining DNA sequences in biotechnology. PCR consists of three basic reaction steps of denaturation-annealing-extension: firstly, the denaturation of template DNA: heating the template DNA to 93 ℃ for a certain time, dissociating the double-stranded template DNA or the double-stranded DNA formed by PCR amplification to form a single strand so that the single strand can be combined with the primer to prepare for the next reaction; annealing (renaturation) of template DNA to the primer: heating and denaturing the template DNA into single strands, cooling to about 55 ℃, and pairing and combining the primers and the complementary sequences of the template DNA single strands; extension of the primer: the DNA template-primer combination is characterized in that under the action of DNA polymerase (such as Taq DNA polymerase) at 72 ℃, dNTP is used as a reaction raw material, a target sequence is used as a template, a new half-retained replication chain complementary with a template DNA chain is synthesized according to the base complementary pairing and half-retained replication principle, and more 'half-retained replication chains' can be obtained by repeating the three processes of cyclic denaturation, annealing and extension, and the new chain can become a template of the next cycle. The amplification of the target gene to be amplified can be amplified by millions of times within 2-3 hours after each cycle is completed for 2-4 minutes. With the advancement of technology, PCR has been updated and applied much more.
Multiplex PCR refers to a PCR reaction in which two or more pairs of primers are added to a single reaction system to simultaneously amplify multiple nucleic acid fragments. It effectively solves the requirements of multi-site, multi-gene targeted amplification. The primer dimer is a pair of primers or a double-stranded DNA fragment with small molecular weight formed by complementary combination of the 3 'end part bases of the primers and extension from the 3' end under the action of Taq enzyme. The generation of primer dimer can reduce or even eliminate the yield of target product, and also affect the multiplex PCR. When the PCR multiplicity is larger, primer dimer of PCR becomes more serious. When multiplex PCR is repeated up to several hundreds or thousands, partial base complementarity at the 3' end is often unavoidable in theory.
At present, a plurality of methods for solving primer dimer, such as adding dimethyl sulfoxide (DMSO) into a reaction system, adopting hot start polymerase, increasing annealing temperature and the like, are available, but the problem of primer complementation is not solved fundamentally, and the nonspecific amplification of multiplex PCR is still obvious.
Disclosure of Invention
Based on this, there is a need for a nucleic acid combination that can improve non-specific amplification of multiplex PCR.
In addition, a detection kit and a method for amplifying a target nucleic acid which are less prone to non-specific amplification are provided.
A nucleic acid combination comprising at least one set of primer pairs, said primer pairs comprising an upstream primer and a downstream primer; the upstream primer and the downstream primer are stem-loop structures comprising a loop part and a stem part, when annealing, at least part of the loop part and at least part of the nucleic acid fragment at the 3 ' end of the upstream primer are hybridized with one strand of the template, at least part of the loop part and at least part of the nucleic acid fragment at the 3 ' end of the downstream primer are hybridized with the other strand of the template, and the nucleic acid fragments at the 5 ' ends of the upstream primer and the downstream primer are not hybridized with the template.
The upstream primer and the downstream primer of the primer pair of the nucleic acid combination product both have a stem-loop structure under a non-denaturing condition, when annealing is performed, at least part of the loop part and at least part of the nucleic acid fragment at the 3 ' end of the primer hybridized with the template are combined with the template in a complementary pairing manner, the rest part of the primer is not hybridized with the template, and meanwhile, the other primer not hybridized with the template is combined with the nucleic acid fragment at the 5 ' end of the primer and the nucleic acid fragment at the 3 ' end in a complementary pairing manner to form the stem-loop structure again, so that primer dimer is not easily formed, and non-specific amplification is not easily generated.
In one embodiment, the upstream primer and/or the downstream primer further comprises a modification between the 5 'nucleic acid fragment of the corresponding primer and the loop portion or between the 3' nucleic acid fragment of the corresponding primer and the loop portion, wherein upon extension, the modification prevents the polymerase from continuing to move forward.
In one embodiment, the modification is selected from at least one of a spacer and NTP.
In one embodiment, the nucleic acid fragment 3' of the upstream primer comprises an upstream extension and an upstream specific segment, the upstream specific segment is located between the upstream loop portion and the upstream extension, the upstream extension can be hybridized with one strand of the template, and the upstream specific segment cannot be bound with the template in a complementary pairing manner;
and/or, the nucleic acid segment at the 3' end of the downstream primer comprises a downstream extension segment and a downstream specific segment, the downstream specific segment is positioned between the downstream loop part and the downstream extension segment, the downstream extension segment can be hybridized with the other strand of the template, and the downstream specific segment cannot be bound with the template in a complementary pairing manner.
In one embodiment, the length of the upstream extension section is 3 nt-20 nt, and the length of the upstream specific section is 3 nt-50 nt; the length of the downstream extension section is 3 nt-20 nt, and the length of the downstream specific section is 3 nt-50 nt.
In one embodiment, the forward primer and/or the reverse primer further comprises a tag segment between the nucleic acid fragment at the 5 'end of the respective primer and the loop portion or between the nucleic acid fragment at the 3' end of the respective primer and the loop portion.
In one embodiment, the lengths of the loop part of the upstream primer and the loop part of the downstream primer are respectively 10 nt-100 nt independently.
In one embodiment, the nucleic acid combination product further comprises at least one fluorescent probe, wherein the 5 'end of the fluorescent probe is connected with a fluorescent group, and the 3' end of the fluorescent probe is connected with a quenching group corresponding to the fluorescent group.
In one embodiment, the nucleic acid combination may further comprise at least one set of universal primer pairs that bind to the complementary pair of templates.
In one embodiment, the nucleic acid combination product comprises at least two of the following primer pairs:
the primer pair of HPV16 having sequences shown in seq id No.1 and seq id No.2, the primer pair of HPV18 having sequences shown in seq id No.3 and seq id No.4, the primer pair of HPV31 having sequences shown in seq id No.5 and seq id No.6, the primer pair of HPV33 having sequences shown in seq id No.7 and seq id No.8, the primer pair of HPV35 having sequences shown in seq id No.9 and seq id No.10, the primer pair of HPV39 having sequences shown in seq id No.11 and seq id No.12, the primer pair of HPV45 having sequences shown in seq id No.13 and seq id No.14, the primer pair of HPV51 having sequences shown in seq id No.15 and seq id No.16, the primer pair of HPV52 having sequences shown in seq id No.17 and seq id No.18, the primer pair of HPV53 having sequences shown in seq id No.19 and seq id No.20, the primer pair of HPV 3621 having sequences shown in seq id No.21 and seq id No.22, the primer pair of HPV 3626 having sequences shown in seq id No.26 and seq id No.26, and seq id No. 26.
In one embodiment, the nucleic acid combination product comprises at least two of the following primer pairs:
the primer pair SEPT9 having the sequences shown in SEQ ID No.62 and SEQ ID No.63, the primer pair VWC2 having the sequences shown in SEQ ID No.64 and SEQ ID No.65, the primer pair ADD2 having the sequences shown in SEQ ID No.66 and SEQ ID No.67, the primer pair INA having the sequences shown in SEQ ID No.68 and SEQ ID No.69, the primer pair POU4F1 having the sequences shown in SEQ ID No.70 and SEQ ID No.71, the primer pair HOXB4 having the sequences shown in SEQ ID No.72 and SEQ ID No.73, the primer pair SHOX2 having the sequences shown in SEQ ID No.74 and SEQ ID No.75, the primer pair PTGER4 having the sequences shown in SEQ ID No.76 and SEQ ID No.77, the primer pair THP having the sequences shown in SEQ ID No.78 and SEQ ID No.79, the primer pair THP 9 having the sequences shown in SEQ ID No.80 and SEQ ID No.81, the primer pair SENT No.25 having the sequences shown in SEQ ID No.92, the sequences shown in SEQ ID No.32, the primer pair SENT No.82 and SEQ ID No.82, the primer pair SENT No.94 having the sequences shown in SEQ ID No.95, the sequences shown in SEQ ID No.7 and SEQ ID No.94, the primer pair FLX 6394, the primer pair SEID No.94, the primer pair SEED No.94 and SEQ ID No.94, the primer pair SED No.94, the primer pair SEED No.95, the primer pair SED No.94 and SEQ ID No.15 and SEQ ID No.94, the primer pair SED No.94 and SEQ ID No.94, the primer pair SED No.15 and SEQ ID No.94, the primer pair INA SEQ ID No.94, the primer pair SED No.94, the primer pair INA SEQ ID No.94, the primer pair SED No.94, the primer pair INA primer pair SED No.94 and SEQ ID No.94, the primer pair SED No.15 and SEQ ID No.94, the primer pair INA primer pair SED No.15 and SEQ ID No.94, the primer pair SED No.94, the primer pair INA SEQ ID No.94, the primer pair NANO. 94, the primer pair INA SEQ ID No.94 and SEQ ID No.94, the primer pair NANO. 94, the primer pair INA SEQ ID No.15 and SEQ ID No.94, the primer pair NANO. 94, the primer pair INA SEQ ID No.94 and SEQ ID No.94, the primer pair INA primer pair NANO. 94, the primer pair NANO. 15 and SEQ ID No.94, the primer pair INA primer pair NANO. 94, the primer pair NANO. 94 shown in SEQ ID No.94, the primer pair INA SEQ ID No.94, the primer pair NANO. 94 shown in SEQ ID No.94, the primer pair NANO. 15 shown in SEQ ID No.94, the primer pair NANO. 94 shown in SEQ ID No.94, the, A BOLL primer pair with sequences shown as SEQ ID No.100 and SEQ ID No.101, and an ACTB primer pair with sequences shown as SEQ ID No.102 and SEQ ID No. 103.
In one embodiment, the nucleic acid composition product further comprises an internal reference probe with a sequence shown as SEQ ID No.104, a detection probe with a sequence shown as SEQ ID No.105, and a universal primer pair with sequences shown as SEQ ID No.106 and SEQ ID No.107, wherein the internal reference probe and the detection probe are both connected with a fluorescent group, and the fluorescent group on the internal reference probe is different from the fluorescent group on the detection probe.
In one embodiment, the nucleic acid combination product comprises at least two of the following primer pairs:
the primer pair of EGFR18 with sequences shown as SEQ ID No.108 and SEQ ID No.109, the primer pair of EGFR19 with sequences shown as SEQ ID No.110 and SEQ ID No.111, the primer pair of EGFR20 with sequences shown as SEQ ID No.112 and SEQ ID No.113, the primer pair of EGFR21 with sequences shown as SEQ ID No.114 and SEQ ID No.115, the primer pair of KRAS2 with sequences shown as SEQ ID No.116 and SEQ ID No.117, the primer pair of KRAS3 with sequences shown as SEQ ID No.118 and SEQ ID No.119, the primer pair of KRAS4 with sequences shown as SEQ ID No.120 and SEQ ID No.121, the primer pair of BRAF with sequences shown as SEQ ID No.122 and SEQ ID No.123, the primer pair of PIK3CA with sequences shown as SEQ ID No.124 and SEQ ID No.125, and the primer pair of PIK3CA with sequences shown as SEQ ID No.126 and SEQ ID No. 127.
In one embodiment, the nucleic acid combination product further comprises a universal primer pair with sequences shown as SEQ ID No.128 and SEQ ID No. 129.
A detection kit comprises the nucleic acid combination product.
A method of amplifying a target nucleic acid, which method does not aim at the diagnosis and treatment of a disease, comprising the steps of:
mixing the template, the nucleic acid combination product, polymerase and a buffer agent to prepare PCR reaction liquid; and carrying out PCR reaction on the PCR reaction solution.
Drawings
FIG. 1 is a schematic diagram illustrating the operation of a nucleic acid composition according to an embodiment;
FIG. 2 is a graph showing the results of electrophoresis in example 1;
FIG. 3 is an amplification curve of the primer pair modified in example 2;
FIG. 4 is an amplification curve of the conventional primer pair in example 2;
FIGS. 5 to 10 are graphs showing the results of amplification curves in example 3;
FIG. 11 is a graph showing the results of electrophoresis in example 4.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. The term "and/or" includes any and all combinations of one or more of the associated listed items. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this context, "positive strand" and "negative strand" refer to the two single strands of nucleic acid of a DNA template that are complementary paired with the positive strand during PCR amplification, wherein: a nucleic acid single strand having a base sequence identical to mRNA corresponding to the DNA template (only the difference between T and U) is defined as a plus strand; a single nucleic acid strand having a sequence complementary to mRNA corresponding to the DNA template is designated as a negative strand. The term "upstream loop" as used herein means the same as "loop of upstream primer" and refers to the loop of stem-loop structure of upstream primer; the term "downstream loop" is used as the same meaning as "loop of downstream primer" and refers to the loop in the stem-loop structure of the downstream primer.
One embodiment of the present application provides a nucleic acid combination comprising at least one primer pair comprising an upstream primer and a downstream primer; the upstream primer and the downstream primer are both stem-loop structures comprising a loop portion and a stem portion, and when annealed, at least a portion of the loop portion and at least a portion of the 3 'end of the nucleic acid fragment of the upstream primer hybridizes to one strand of the template (e.g., the negative strand), at least a portion of the loop portion and at least a portion of the 3' end of the nucleic acid fragment of the downstream primer hybridizes to the other strand of the template (e.g., the positive strand), and neither the 5 'end of the nucleic acid fragment of the upstream primer nor the 5' end of the downstream primer hybridizes to the template.
Referring to fig. 1, the primer pair in the above-mentioned nucleic acid combination product is obtained by designing the upstream primer and the downstream primer into a stem-loop structure, where the upstream primer and the downstream primer are both: under non-denaturing (e.g., at room temperature) conditions, the 5 '-end nucleic acid fragment and the 3' -end nucleic acid fragment are complementarily paired and bound to form a stem portion, and the nucleic acid fragment located between the 5 '-end nucleic acid fragment and the 3' -end nucleic acid fragment forms a loop portion. Upon annealing, the loop portion and the 3 'nucleic acid fragment of the forward primer bind (hybridize) to one strand of the template in complementary pairing, but the 5' nucleic acid fragment of the forward primer does not hybridize to the template; simultaneously, the loop part and the nucleic acid segment at the 3 'end of the downstream primer are hybridized with the other part of the template, but the nucleic acid segment at the 5' end of the downstream primer is not hybridized with the template; in this case, the primer in which the 5 '-end nucleic acid fragment and the 3' -end nucleic acid fragment of the primer are complementarily paired and bound to form the stem-loop structure again without the other primer bound to the template is blocked at the 5 '-end and the 3' -end, and thus a primer dimer is not easily formed. Proved by verification, the primer designed according to the method has good specificity and is not easy to cause nonspecific amplification.
Specifically, the upstream primer is in a stem-loop structure, the upstream primer comprises an upstream stem part and an upstream loop part connected with the upstream stem part, a nucleic acid fragment at the 5 'end of the upstream primer is combined with a nucleic acid fragment at the 3' end of the upstream primer in a complementary pairing manner to form the upstream stem part, the upstream loop part is positioned between the nucleic acid fragment at the 5 'end of the upstream primer and the nucleic acid fragment at the 3' end of the upstream primer, when annealing is carried out, at least part of the upstream loop part and at least part of the nucleic acid fragment at the 3 'end of the upstream primer are hybridized with one strand of the template, and the nucleic acid fragment at the 5' end of the upstream primer cannot be complementarily paired with the strand; the downstream primer is also in a stem-loop structure, the downstream primer comprises a downstream stem part and a downstream loop part connected with the downstream stem part, a nucleic acid fragment at the 5 'end of the downstream primer is combined with a nucleic acid fragment at the 3' end of the downstream primer in a complementary pairing mode to form the downstream stem part, the downstream loop part is positioned between the nucleic acid fragment at the 5 'end of the downstream primer and the nucleic acid fragment at the 3' end of the downstream primer, when annealing is carried out, at least part of the downstream loop part and at least part of the nucleic acid fragment at the 3 'end of the downstream primer are hybridized with the other strand of the template, and the nucleic acid fragment at the 5' end of the downstream primer cannot be subjected to complementary pairing with the strand.
Specifically, the nucleic acid fragment at the 5 'end of the upstream primer is used for complementary pairing and combination with the nucleic acid fragment at the 3' end of the upstream primer under a non-denaturing condition (such as normal temperature); the nucleic acid fragment at the 3' end of the forward primer is used to hybridize to one of the strands (e.g., the negative strand) of the template upon annealing, thereby allowing the polymerase to extend using that strand as the template. The nucleic acid fragment at the 5 'end of the downstream primer is used for complementary pairing and combination with the nucleic acid fragment at the 3' end of the downstream primer under a non-denaturing condition (such as normal temperature); the nucleic acid fragment at the 3' end of the downstream primer is used to hybridize to the other strand of the template (e.g., the plus strand) upon annealing, thereby allowing the polymerase to extend using the strand as a template. The following description will be given by taking an example in which the forward primer binds to the minus strand of the template and the reverse primer binds to the plus strand of the template.
In some embodiments, upon annealing, the nucleic acid fragment 3' of the portion of the forward primer hybridizes to the negative strand of the template. Specifically, the nucleic acid fragment at the 3' -end of the upstream primer includes an upstream loop portion and an upstream specific segment, the upstream specific segment is located between the upstream loop portion and the upstream extension, the upstream extension can hybridize with the negative strand in the template, and the upstream specific segment and the negative strand cannot be complementarily paired and combined. The specificity of the upstream primer is improved by providing an upstream specific segment that does not hybridize to the minus strand, so that the binding of the 3' end of the upstream primer to the complementary pair of the minus strand is dependent on the upstream extension, so that the upstream extension requires a polymerase to initiate extension after complete complementary pairing with the minus strand. Optionally, the length of the upstream extension segment is 3 nt-20 nt, and the length of the upstream specific segment is 3 nt-50 nt. Furthermore, the length of the upstream extension section is 6 nt-15 nt, and the length of the upstream specific section is 15 nt-35 nt. In other embodiments, the nucleic acid segment at the 3' end of the forward primer hybridizes exclusively to the negative strand upon annealing.
In some embodiments, upon annealing, the nucleic acid fragment at the 3' end of the portion of the downstream primer hybridizes to the positive strand of the template. Specifically, the nucleic acid segment at the 3' end of the downstream primer comprises a downstream extension segment and a downstream specific segment, the downstream specific segment is positioned between the downstream loop part and the downstream extension segment, the downstream extension segment can be hybridized with the positive strand, and the downstream specific segment cannot be complementarily paired and combined with the positive strand. The downstream specific segment which cannot be hybridized with the positive strand is arranged, so that the complementary pairing and combination of the 3' end of the downstream primer and the positive strand depend on the downstream extension segment, the polymerase can start extension after the downstream extension segment is completely complementary paired with the positive strand, and the specificity of the downstream primer is improved. Optionally, the length of the downstream extension section is 3 nt-20 nt, and the length of the downstream specific section is 3 nt-50 nt. Furthermore, the length of the downstream extension section is 6 nt-15 nt, and the length of the downstream specific section is 15 nt-35 nt. In other embodiments, upon annealing, the nucleic acid segment at the 3' end of the downstream primer hybridizes fully to the negative strand.
Specifically, the upstream loop portion is used for binding to the minus strand of the template, and facilitates binding of the nucleic acid fragment at the 3' -end of the upstream primer to the minus strand. At least a portion of the upstream loop portion is complementary to the negative strand. In some embodiments, the upstream loop has a length of 10nt to 100 nt. Further, the length of the upstream ring part is 18nt to 60 nt. In one embodiment, a portion of the upstream loop portion can be complementarily paired with the negative strand. Specifically, the length of the nucleic acid fragment complementary-paired with the negative strand in the upstream loop portion is 10nt to 50 nt. Alternatively, in the upstream loop portion, the length of the nucleic acid fragment complementarily pairing to the negative strand is 15nt, 20nt, 30nt, 40nt, or 45 nt. In another embodiment, the upstream loop portions are all complementary-paired with the negative strand.
Specifically, the downstream loop portion is used for binding with the positive strand of the template, and facilitates the binding of the nucleic acid fragment at the 3' end of the downstream primer with the positive strand. At least a portion of the downstream loop portion is complementary to the negative strand. In some embodiments, the downstream loop portion has a length of 10nt to 100 nt. Further, the length of the downstream ring part is 18 nt-60 nt. In one embodiment, a portion of the downstream loop portion can be complementarily paired with the positive strand. Specifically, in the downstream loop portion, the length of the nucleic acid fragment complementarily paired with the positive strand is 10nt to 50 nt. Optionally, in the downstream loop portion, the nucleic acid fragment complementarily pairing with the positive strand has a length of 15nt, 20nt, 30nt, 40nt, or 45 nt. In another embodiment, the downstream loop portions are all capable of complementary pairing with the positive strand.
In some embodiments, the forward primer and/or the reverse primer further comprises a modification between the nucleic acid fragment at the 5 'end of the respective primer and the loop or between the nucleic acid fragment at the 3' end of the respective primer and the loop, which upon extension, prevents the polymerase from continuing forward movement.
In one embodiment, the upstream primer further comprises an upstream modification part, and the upstream modification part is positioned between the nucleic acid fragment at the 5' end of the upstream primer and the upstream loop part; upon extension, the upstream modification prevents the polymerase from continuing forward movement. By providing the upstream modifying portion between the nucleic acid fragment at the 5 '-end of the upstream primer and the upstream loop portion, when a single-stranded nucleic acid containing the upstream primer is used as a template, the movement of the DNA polymerase is stopped at the nucleic acid fragment at the 5' -end of the upstream primer on the template, and the extension is terminated, thereby avoiding the occurrence of non-specific amplification by extending the complementary strand of the nucleic acid fragment synthesized to the 5 '-end of the upstream primer (i.e., the 5' -end of the minus strand at this time). In this context, the term "polymerase" refers to DNA polymerase; the polymerase proceeds from the 5 'end of the template to the 3' end.
Optionally, the upstream modification is selected from at least one of Spacer and NTP. In one embodiment, the Spacer is at least one of Spacer C3, Spacer C6, Spacer C9, and Spacer C18, wherein Spacer C3 is-CH2CH2CH2-, Spacer C6 means 6-CH2CH2CH2CH2CH2CH2-, Spacer C9 means 3 consecutive glycols (-CH)2CH2O-) of a linker chain, Spacer C18, refers to 6 consecutive glycols (-CH)2CH2O-). It is understood that the specific composition of the upstream modification part is not limited to the above, and may be other substances capable of preventing the polymerase from continuing to move toward the 5' end of the upstream primer.
In one embodiment, the downstream primer further comprises a downstream modification portion located between the nucleic acid fragment at the 5' end of the downstream primer and the downstream loop portion, wherein the downstream modification portion prevents the polymerase from moving forward when extended. By providing the downstream modifying part between the nucleic acid fragment at the 5 ' end of the downstream primer and the downstream loop part, when the nucleic acid single strand containing the downstream primer is used as a template, the DNA polymerase stops moving when moving to the nucleic acid fragment at the 5 ' end of the downstream primer, the extension is terminated, and the non-specific amplification caused by the extension of the complementary strand of the nucleic acid fragment synthesized to the 5 ' end of the downstream primer is avoided.
Optionally, the downstream modification is selected from at least one of a Spacer and NTP. In one embodiment, the Spacer is at least one of Spacer C3, Spacer C6, Spacer C9, and Spacer C18, wherein Spacer C3 is-CH2CH2CH2-, Spacer C6 means-CH2CH2CH2CH2CH2CH2-, Spacer C9 means 3 consecutive glycols (-CH)2CH2O-) of a linker chain, Spacer C18, refers to 6 consecutive glycols (-CH)2CH2O-). It is understood that the specific composition of the downstream modification part is not limited to the above, and other substances capable of preventing the polymerase from continuing to move to the 5' -end of the downstream primer may be used.
In some embodiments, the forward primer and/or the reverse primer further comprises a tag segment between the nucleic acid fragment at the 5 'end of the respective primer and the loop portion or between the nucleic acid fragment at the 3' end of the respective primer and the loop portion.
In one embodiment, the upstream primer further comprises an upstream tag segment located between the nucleic acid fragment at the 5' end of the upstream primer and the upstream loop portion, wherein the upstream tag segment is not capable of complementary pairing with the minus strand. The upstream tag segment is used for introducing a universal primer sequence, a molecular tag and the like into a newly synthesized chain, so that a sequencing library can be conveniently constructed subsequently.
In one embodiment, the downstream primer further comprises a downstream tag segment located between the nucleic acid segment at the 5' end of the downstream primer and the downstream loop portion, wherein the downstream tag segment is not capable of complementary pairing with the positive strand. The downstream tag segment is used for introducing a universal primer sequence, a molecular tag and the like into a newly synthesized chain, so that a sequencing library can be conveniently constructed subsequently.
In some embodiments, the upstream primer is attached to a fluorophore. In an alternative specific example, the 5 'end of the upstream primer is connected with a fluorescent group, and the 3' end of the upstream primer is connected with a quenching group corresponding to the fluorescent group. By attaching a fluorophore to the upstream primer, the degree of amplification can be characterized by the intensity of fluorescence. It is understood that the fluorescent group and the quencher group are not particularly limited.
In some embodiments, the downstream primer is attached to a fluorophore. In an alternative specific example, the 5 'end of the downstream primer is connected with a fluorescent group, and the 3' end of the downstream primer is connected with a quenching group corresponding to the fluorescent group. By attaching a fluorophore to the downstream primer, the degree of amplification can be characterized by the fluorescence intensity. It is understood that the fluorescent group and the quencher group are not particularly limited.
In some embodiments, at least one fluorescent probe is included in the nucleic acid combination product. In an alternative embodiment, the fluorescent probe has a fluorophore attached to its 5 'end and a quencher corresponding to the fluorophore attached to its 3' end. The probe is degraded by enzyme digestion in the amplification process to emit fluorescence, and the amplification degree can be represented by the fluorescence intensity. It is understood that the fluorescent group and the quencher group are not particularly limited.
In one embodiment, at least one set of universal primer pairs is included in the nucleic acid combination product. The universal primer pair is complementary to the template pair for sequencing.
In one embodiment, at least one of the forward primer and the reverse primer is a stem loop primer. Further, at least one of the upstream primer and the downstream primer is a molecular beacon type primer.
In some embodiments, at least two sets of primer pairs are included in the nucleic acid combination product, with different primer pairs being used to amplify different nucleic acid fragments. Specifically, the structure of each primer pair is as described above, and each primer pair differs in that the target regions targeted by different primer pairs differ. In some embodiments, the different primer pairs are packaged in a mixture. In other embodiments, different primer pairs are packaged separately.
In one embodiment, the nucleic acid combination product is a product for detecting HPV virus. In particular, the nucleic acid combination product comprises at least two of the following primer pairs: HPV16 primer pairs with sequences shown as SEQ ID No.1 and SEQ ID No.2, HPV18 primer pairs with sequences shown as SEQ ID No.3 and SEQ ID No.4, HPV31 primer pairs with sequences shown as SEQ ID No.5 and SEQ ID No.6, HPV33 primer pairs with sequences shown as SEQ ID No.7 and SEQ ID No.8, HPV35 primer pairs with sequences shown as SEQ ID No.9 and SEQ ID No.10, HPV39 primer pairs with sequences shown as SEQ ID No.11 and SEQ ID No.12, HPV45 primer pairs with sequences shown as SEQ ID No.13 and SEQ ID No.14, HPV51 primer pairs with sequences shown as SEQ ID No.15 and SEQ ID No.16, HPV52 primer pairs with sequences shown as SEQ ID No.17 and SEQ ID No.18, HPV53 primer pairs with sequences shown as SEQ ID No.19 and SEQ ID No.20, and HPV 5824 primer pairs with sequences shown as SEQ ID No.21 and SEQ ID No. 56, and SEQ ID No.24, An HPV59 primer pair with sequences shown as SEQ ID No.25 and SEQ ID No.26 and an HPV66 primer pair with sequences shown as SEQ ID No.27 and SEQ ID No. 28.
In one embodiment, the nucleic acid combination product is a product for detecting tumor gene methylation. In particular, the nucleic acid combination product comprises at least two of the following primer pairs: SEPT9 primer pair with sequences shown as SEQ ID No.62 and SEQ ID No.63, VWC2 primer pair with sequences shown as SEQ ID No.64 and SEQ ID No.65, ADD2 primer pair with sequences shown as SEQ ID No.66 and SEQ ID No.67, INA primer pair with sequences shown as SEQ ID No.68 and SEQ ID No.69, POU4F1 primer pair with sequences shown as SEQ ID No.70 and SEQ ID No.71, HOXB4 primer pair with sequences shown as SEQ ID No.72 and SEQ ID No.73, SHOX2 primer pair with sequences shown as SEQ ID No.74 and SEQ ID No.75, PTGER4 primer pair with sequences shown as SEQ ID No.76 and SEQ ID No.77, THBD primer pair with sequences shown as SEQ ID No.78 and SEQ ID No.79, THPT 2 primer pair with sequences shown as SEQ ID No.80 and SEQ ID No.81, SOX 3582 and SOX 3584 primer pair with sequences shown as SEQ ID No.85 and SEQ ID No.83, GHSR primer pair with sequences shown as SEQ ID No.86 and SEQ ID No.87, JAM3 primer pair with sequences shown as SEQ ID No.88 and SEQ ID No.89, PRDM14 primer pair with sequences shown as SEQ ID No.90 and SEQ ID No.91, UCHL1 primer pair with sequences shown as SEQ ID No.92 and SEQ ID No.93, IRF4 primer pair with sequences shown as SEQ ID No.94 and SEQ ID No.95, ZNF583 primer pair with sequences shown as SEQ ID No.96 and SEQ ID No.97, FLT1 primer pair with sequences shown as SEQ ID No.98 and SEQ ID No.99, BOLL primer pair with sequences shown as SEQ ID No.100 and SEQ ID No.101, and ACTB primer pair with sequences shown as SEQ ID No.102 and SEQ ID No. 103.
Further, the nucleic acid composition product for detecting tumor gene methylation also comprises an internal reference probe with a sequence shown in SEQ ID No.104, a detection probe with a sequence shown in SEQ ID No.105 and a universal primer pair with sequences shown in SEQ ID No.106 and SEQ ID No. 107. The reference probe and the detection probe are both connected with fluorescent groups, and the fluorescent groups on the reference probe are different from those on the detection probe.
In one embodiment, the nucleic acid combination product is a product for detecting a mutation in a tumor gene. In particular, the nucleic acid combination product comprises at least two of the following primer pairs: an EGFR18 primer pair with sequences shown as SEQ ID No.108 and SEQ ID No.109, an EGFR19 primer pair with sequences shown as SEQ ID No.110 and SEQ ID No.111, an EGFR20 primer pair with sequences shown as SEQ ID No.112 and SEQ ID No.113, an EGFR21 primer pair with sequences shown as SEQ ID No.114 and SEQ ID No.115, a KRAS2 primer pair with sequences shown as SEQ ID No.116 and SEQ ID No.117, a KRAS3 primer pair with sequences shown as SEQ ID No.118 and SEQ ID No.119, a KRAS4 primer pair with sequences shown as SEQ ID No.120 and SEQ ID No.121, a BRAF primer pair with sequences shown as SEQ ID No.122 and SEQ ID No.123, a PIK3CA primer pair with sequences shown as SEQ ID No.124 and SEQ ID No.125, and a PIK3CA primer pair with sequences shown as SEQ ID No.126 and SEQ ID No. 127.
Furthermore, the nucleic acid combination product for detecting the tumor gene mutation also comprises a universal primer pair with the sequences shown as SEQ ID No.128 and SEQ ID No. 129.
In some embodiments, the primer pairs in the nucleic acid combination product are in one set. In one embodiment, primer pairs are used to amplify a sequence on the human ACTB genome. In a specific example, the primer pair of the nucleic acid combination product is shown as SEQ ID No.29 and SEQ ID No. 30.
In addition, an embodiment of the present application further provides a detection kit, comprising the nucleic acid combination product of any one of the above embodiments.
Optionally, the detection kit further comprises at least one of a polymerase, a buffer and a universal primer for sequencing.
Specifically, the polymerase is used to extend at the 3' end of the primer during PCR. The buffer provides a buffer system for the PCR reaction. The universal primers are used for amplifying products to prepare a sequencing library. It is understood that the universal primer pair for sequencing contained in the nucleic acid composition may be omitted from the detection kit.
The detection kit comprises the nucleic acid combination product, the primer pair of the nucleic acid combination product solves the problem that the primers are easy to be complementarily paired to form a dimer from the structure of the primers, and when the nucleic acid combination product is used for multiplex PCR detection, the dimer and non-specific amplification are not easy to occur between the primers, so that the non-specific amplification of the multiplex PCR can be improved.
In addition, an embodiment of the present application also provides a method for amplifying a target nucleic acid, which is not intended for the diagnosis and treatment of diseases, comprising the steps of: mixing the template, the nucleic acid combination product of any one of the embodiments, polymerase and a buffer to prepare a PCR reaction solution; and carrying out PCR reaction on the PCR reaction solution.
Specifically, the method for amplifying a target nucleic acid can be used for amplification of a nucleic acid fragment before preparation of a library. In one embodiment, a method of amplifying a target nucleic acid is used to construct a sequencing library.
In one embodiment, the conditions of the PCR reaction are: pre-denaturation: 1 min-10 min at 90-98 ℃; denaturation: the temperature is 90-98 ℃ for 2-60 s; annealing: 10 s-120 min at 48-68 ℃; extension: the temperature is 68-75 ℃ for 10 s-10 min; the number of cycles: 2 to 60.
The method for amplifying the target nucleic acid has the advantages of good specificity and difficult occurrence of nonspecific amplification by adopting the nucleic acid combination product.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. In the following examples, the primers were all "spacers" as Spacer C9.
Example 1 amplification of human ACTB Gene
A section of human ACTB genome sequence (i.e., target nucleic acid) is amplified by respectively adopting a conventional PCR primer pair and an improved primer pair to verify the good amplification performance of the improved primer pair. The base sequence of the target nucleic acid is shown in SEQ ID No. 31. The specific verification steps comprise:
(1) designing a primer:
a: conventional primer pairs: f: 5'-TCCTTCCTGGGCATGGAG-3' (SEQ ID NO. 32); r: 5'-CATTGTGCTGGGTGCCAG-3' (SEQ ID NO. 33);
b: the modified primer pair, underlined is the sequence that hybridizes to the template: f: 5' -ACAGGACACATCCTT CCTGGGCATGGAGAACCATCCTGT -3’（SEQ ID No.29）；R：5’-CACTGCTCTCCATTGTGCTGGGTGCCAGACACCGCAGTG -3’（SEQ ID No.30）。
(2) Preparing a PCR reaction solution:
PCR reaction systems (total system volume 25. mu.L) of the conventional primer pair and the improved primer pair were prepared respectively according to Table 1:
TABLE 1
(3) And (3) PCR amplification: vortex mixing, instantaneous centrifugation, and PCR reaction in a PCR instrument according to the reaction program set in the table 2:
TABLE 2
(4) Results and analysis:
after the PCR is completed, the PCR product is electrophoretically detected, and the result is shown in FIG. 2. In FIG. 2, lane 1 is marker, lanes 2, 3 and 4 are electrophoresis results of amplification products corresponding to the modified primer pair, and lanes 5, 6 and 7 are electrophoresis results of amplification products corresponding to the conventional primer pair. As can be seen from FIG. 2, the electrophoresis bands of the amplification products corresponding to the improved primer pair are single, while the electrophoresis bands of the amplification products corresponding to the conventional primer pair are dispersed. This shows that many non-specific products, especially primer dimers, are produced during amplification with conventional primer pairs, and the improved primer pairs form stem-loop structures with themselves, reducing non-specific amplification, resulting in single amplified product and no primer dimers.
Example 2 multiplex PCR amplification of 14 high-risk HPV viral genes
A conventional PCR primer and an improved PCR primer are respectively adopted to amplify the L1 region fragment of 14 high-risk human papilloma virus genomes in a PCR reaction tube so as to verify the good amplification performance of the primer disclosed by the embodiment of the invention. The specific verification steps comprise:
(1) designing a primer:
a: the conventional primer pairs are shown in Table 3.
TABLE 3
B: the improved primer pair has underlined sequence for hybridizing with the template:
TABLE 4
(2) Preparing a PCR reaction solution:
PCR reaction systems (total system volume 25. mu.L) for the conventional primer pair and the improved primer pair were prepared, respectively, as shown in Table 5:
TABLE 5
(3) And (3) PCR amplification: vortex mixing, instantaneous centrifugation, and PCR reaction in a real-time fluorescent quantitative PCR instrument according to the reaction program set in the table 6:
TABLE 6
(4) Results and analysis:
after the PCR was completed, an amplification curve was obtained, and the amplification curve is shown in FIGS. 3 and 4. FIG. 3 is an amplification curve of the modified primer pair, and FIG. 4 is an amplification curve of the conventional primer pair.
As can be seen from FIGS. 3 and 4, the improved primer pairs have no non-specific amplification curves for pure water (no template sample) and negative sample (no HPV DNA sample), and the amplification curves for positive sample (HPV DNA-containing sample) are good, while the conventionally designed primer pairs have non-specific amplification curves for pure water (no template sample) and negative sample (no HPV DNA sample), indicating that primer dimers are present during amplification of the conventional primer pairs.
(5) And (3) increasing the PCR annealing temperature for amplification verification:
respectively preparing PCR reaction systems of a conventional primer pair and an improved primer pair according to the following table 5, performing vortex mixing, then performing instant centrifugation, and performing PCR reaction in a real-time fluorescent quantitative PCR instrument according to a reaction program set in the following table 7:
TABLE 7
As a result: after the annealing temperature was increased, the non-specific amplification curves of the conventional designed primer pair were still present in pure water (template-free sample) and negative sample (HPV-free DNA sample), and the changes were not significant, and the Ct values of the conventional primer pair and the improved primer pair are shown in Table 8 below.
TABLE 8
Example 3 multiplex PCR detection of tumor Gene methylation
The method of the present invention is used to design primer and amplify several methylated tumor gene segments in one PCR reaction tube to realize high sensitivity detection of tumor gene methylation. The method comprises the following specific steps:
(1) designing a primer:
underlined are sequences that hybridize to the template:
TABLE 9
(2) Preparing a PCR reaction solution:
a PCR reaction system (total system volume 25. mu.L) was prepared as shown in Table 10:
watch 10
(3) And (3) PCR amplification: vortex mixing, instantaneous centrifugation, and PCR reaction in a real-time fluorescent quantitative PCR instrument according to the reaction program set in the table 11:
TABLE 11
(4) Results and analysis:
after the PCR is finished, an amplification curve is obtained, and the amplification curve is shown in FIGS. 5 to 10.
As can be seen from FIGS. 5 to 10, the PCR reaction system showed a good amplification curve for the positive sample (tumor tissue sample) and no non-specific amplification curve for pure water (template-free sample) and the negative sample (healthy tissue sample). Sample Ct values are shown in Table 12 below.
TABLE 12
Example 4 preparation of sequencing library for tumor Gene mutation detection Using multiplex PCR
The gene highly related to tumor individual administration is selected, the method is utilized to design a primer, and a plurality of gene segments are amplified in one PCR reaction tube, so that the tumor gene mutation can be detected with high sensitivity. The method comprises the following specific steps:
(1) designing a primer:
underlined are sequences that hybridize to the template:
watch 13
(2) Preparing a PCR reaction solution:
a PCR reaction system (total system volume 25. mu.L) was prepared as shown in Table 14:
TABLE 14
(3) And (3) PCR amplification: vortex mixing, instantaneous centrifugation, and PCR reaction in a PCR instrument according to the reaction program set in the table 15:
watch 15
(4) And (3) purification and detection:
the gel was recovered in a range of 200 to 300bp by electrophoresis using 2% agarose (120V) for 1 hour, and eluted with 40. mu.l of nuclease-free water using a gel recovery kit (Qiagen). Qubit (fluorescence quantification) quantification was performed and the library was diluted with nuclease-free water to 2 ng/. mu.l for use.
Detecting the size of the library insert by an Aglilent 2100 bioanalyzer, and detecting the concentration of the library by a Q-PCR (Q-polymerase chain reaction) instrument; the detection results are shown in fig. 11.
As can be seen from FIG. 11, the size of the library constructed in this example is about 250bp, the linker sequences at both ends are removed by 116bp, and the size of the insert is about 130bp, which meets the insert size required for double-end sequencing.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.
Sequence listing
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<210> 30
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cactgctctc cattgtgctg ggtgccagac accgcagtg 39
<210> 31
<211> 183
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tccttcctgg gcatggagtc ctgtggcatc cacgaaacta ccttcaactc catcatgaag 60
tgtgacgtgg acatccgcaa agacctgtac gccaacacag tgctgtctgg cggcaccacc 120
atgtaccctg gcattgccga caggatgcag aaggagatca ctgccctggc acccagcaca 180
atg 183
<210> 32
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
<210> 33
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
<210> 34
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ttgatactac acgcagtaca aat 23
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tattttgcac agttgaaaaa taaa 24
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tagataccac tcgcagtacc aat 23
<210> 37
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tacacaactg aaaaataaac tgca 24
<210> 38
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gtagatacca cacgtagtac caata 25
<210> 39
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tttgcataac tgaaatataa attgt 25
<210> 40
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ccactcgcag tactaatatg act 23
<210> 41
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
aaactgtaga tcatattctt caac 24
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tgatacaacc cgtagtacaa ata 23
<210> 43
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ctgtaaatca tattcttcac catg 24
<210> 44
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tacccgtagt accaacttta catt 24
<210> 45
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tgacagtaca cagttgaaat ataa 24
<210> 46
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
acactacccg cagtactaat tt 22
<210> 47
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gcacaactga aaaataaact gtaaa 25
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ttgatactac cagaagtaca aat 23
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
aaataaattg caattcatac tct 23
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cactcgtagc actaacatga c 21
<210> 51
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
cacaattgaa aaataaattg taaa 24
<210> 52
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gataccacca ggaatacaaa catg 24
<210> 53
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
ttacatagtt gaaacacaaa ttgt 24
<210> 54
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tactactaga agtactaaca tgac 24
<210> 55
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
tttgcataat tgaaaaacaa attg 24
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gataccactc gtagcactaa tatga 25
<210> 57
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tgcaaagctg aaaaacaaac tgta 24
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tactactcgc agcaccaatc t 21
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tttacacagt tgaaatataa ac 22
<210> 60
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
<210> 61
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
tttacaaagt tgaaacacaa actg 24
<210> 62
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gaaaaaacca ctgcagtact cgcacggtgt tttcggtggg tgggagttgc acagacgcag 60
tctcactgag cacggttttt c 81
<210> 63
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
cgttttatgc tacgcaccag tagagcaaat cccgacctat aaactcaaca caaaaacg 58
<210> 64
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
gcgaatacca ctgcagtact cgcaccgtta gtttttatgt tggtttcgca cagacgcagt 60
<210> 65
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cggttgatgc tacgcaccag tagagccccg acaaccgaaa tatctaacac acaaccg 57
<210> 66
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
gaattcacca ctgcagtact cgcaccggta gggttgtgag ggatcacaga cgcagtctca 60
ctgagcaccg ggaattc 77
<210> 67
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
cgtcgcatgc tacgcaccag tagagctccc ccaacaatac aacgaacaca gcgacg 56
<210> 68
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
gaaaacacca ctgcagtact cgcacgaggg gatagggtta agggcacaga cgcagtctca 60
ctgagcacgt gtggttttc 79
<210> 69
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
cggaaaatgc tacgcaccag tagagccatc tacatcgaaa tcgccacaca tttccg 56
<210> 70
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
gcgctcacca ctgcagtact cgcactagac gtacggggtc gtcgcacaga cgcagtctca 60
ctgagcacag gagagcgc 78
<210> 71
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
cgagttatgc tacgcaccag tagagccgtc ccgaaaaact ctcgacacaa actcg 55
<210> 72
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
gaataaacca ctgcagtact cgcacattta tagagcgatt atttacacag acgcagtctc 60
actgagcacg cgattattc 79
<210> 73
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cgtgtaatgc tacgcaccag tagagcatcc cgacaaaccg cgtaacacat acacg 55
<210> 74
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gaacgcacca ctgcagtact cgcaccgtgc ggcggcgatg gaacacagac gcagtctcac 60
tgagcactta cggcgttc 78
<210> 75
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
cgggagatgc tacgcaccag tagagctgtt ggagagcggg tcgacacact cccg 54
<210> 76
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gcgtacacca ctgcagtact cgcaccgttg ggcgtattta agttcacaga cgcagtctca 60
<210> 77
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
cgatttatgc tacgcaccag tagagcctat ccgcgaaccg caacaacaca aaatcg 56
<210> 78
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gaatttggag ggagggtcgg gtatcggtag ggttgtgagg gatcacagac gcagtctcac 60
tgagcactta taaattc 77
<210> 79
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
cggtaaatgc tacgcaccag tagagctatc cgtcccaacc caaacacaca ttaccg 56
<210> 80
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
gaacgaacca ctgcagtact cgcactatat cggagattcg ttgggcacag acgcagtctc 60
actgagcact gcgttcgttc 80
<210> 81
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
cggtttatgc tacgcaccag tagagctcaa aaacctctac gactacacaa aaccg 55
<210> 82
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gccctaacca ctgcagtact cgcacggata gaaggagggg gtacacagac gcagtctcac 60
tgagcacgag ttttagggc 79
<210> 83
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
cgggaaatgc tacgcaccag tagagcacgc gcgaattaac aactcaacac attcccg 57
<210> 84
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gcgaaaacca ctgcagtact cgcacattcg ttttgaggtt tcgtcacaga cgcagtctca 60
ctgagcacta tgtggtttcg c 81
<210> 85
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
cgtgggatgc tacgcaccag tagagcaata taacgcacgc ttttctaaca cacccacg 58
<210> 86
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gactctacca ctgcagtact cgcacgtcgg tagtatgtgg aacgcacaga cgcagtctca 60
ctgagcacag cgaagagtc 79
<210> 87
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
cgattcatgc tacgcaccag tagagcaact acaacaactc gtcgcacaca gaatcg 56
<210> 88
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gaatacacca ctgcagtact cgcacaaaga gaatttatgt gtcgcacaga cgcagtctca 60
ctgagcacta tcgttgtatt c 81
<210> 89
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
cggtcgatgc tacgcaccag tagagctacg acccgcccca attacacaca cgaccg 56
<210> 90
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
gctacgacca ctgcagtact cgcactcgag ttcgagtggt ttatcgcaca gacgcagtct 60
cactgagcac ggtttcgtag c 81
<210> 91
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cgcggtatgc tacgcaccag tagagccacc acctccaaaa acgacacaac cgcg 54
<210> 92
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gaccgaacca ctgcagtact cgcactgaga ttgtaaggtt tgcacagacg cagtctcact 60
gagcacgggg ttcggtc 77
<210> 93
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
cgtacgatgc tacgcaccag tagagcgcga ccaaataaat acgaaacaca cgtacg 56
<210> 94
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
gaatctacca ctgcagtact cgcacttgat tggttttttg aggtcacaga cgcagtctca 60
ctgagcacag gcgagattc 79
<210> 95
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cggtttatgc tacgcaccag tagagcgctt cgaaaactat caacacaaaa ccg 53
<210> 96
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
gcgctcacca ctgcagtact cgcactagcg atggatttat agttcacaga cgcagtctca 60
ctgagcaccg ttggagcgc 79
<210> 97
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
cgcgttatgc tacgcaccag tagagcaaac taccaaaccg aaatacacac aaacgcg 57
<210> 98
<211> 82
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
gaccgcacca ctgcagtact cgcacaggaa atgatttggg cgggtgcaca gacgcagtct 60
cactgagcac attaatgcgg tc 82
<210> 99
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
cgttagatgc tacgcaccag tagagcacac taaacctaaa acgcctacac actaacg 57
<210> 100
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
gaatccacca ctgcagtact cgcacagagg ttgtgttatt gttggacaca gacgcagtct 60
cactgagcac ggtcgggatt c 81
<210> 101
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
cgattgatgc tacgcaccag tagagcactt aatcaaaacc taccatacac acaatcg 57
<210> 102
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
gaatccacca ctgcagtact cgcactcgta ataggtaaag atgagttagt ctactatgga 60
att 63
<210> 103
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
tttgaagatg ctacgcacca gtagagccaa actataacct ctacaaacac acttcaaa 58
<210> 104
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
aggttgtttg ttttagttta cggtataagg 30
<210> 105
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
cacagacgca gtctcactga gcac 24
<210> 106
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
accactgcag tactcgcac 19
<210> 107
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
<210> 108
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
tggttgtctt tccctacacg acgctcttcc gatctgtgga gcctcttaca cccagtggag 60
nnnnnnnncc caacca 76
<210> 109
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
gtgtatgtga ctggagttca gacgtgtgct cttccgatct cccagcccag aggcctgtgc 60
nnnnnnnnct tatacac 77
<210> 110
<211> 71
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
cgggaatctt tccctacacg acgctcttcc gatctctctg gatcccagaa ggtgannnnn 60
nnnaattccc g 71
<210> 111
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
ctttgcgtga ctggagttca gacgtgtgct cttccgatct ggtgggcctg aggttcagag 60
nnnnnnnnac agcaaag 77
<210> 112
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
gagaggtctt tccctacacg acgctcttcc gatctctcct tctggccacc atgcgaagnn 60
nnnnnncgtg cctctc 76
<210> 113
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
cctggagtga ctggagttca gacgtgtgct cttccgatct agccaatatt gtctttgtgt 60
nnnnnnnnta gtccagg 77
<210> 114
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
tgctgctctt tccctacacg acgctcttcc gatctggcag ccaggaacgt actggnnnnn 60
nnnaccgcag ca 72
<210> 115
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gccagcgtga ctggagttca gacgtgtgct cttccgatct tagtgggaag gcagcctggt 60
nnnnnnnnaa tgctggc 77
<210> 116
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
ccacaatctt tccctacacg acgctcttcc gatctttata aggcctgctg aaaatgnnnn 60
nnnnaacttg tgg 73
<210> 117
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gtggacgtga ctggagttca gacgtgtgct cttccgatct caagatttac ctctattgtt 60
nnnnnnnntc gtccac 76
<210> 118
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
tctccatctt tccctacacg acgctcttcc gatctccttc tcaggattcc tacaggaann 60
nnnnnnttga tggaga 76
<210> 119
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
gaccaggtga ctggagttca gacgtgtgct cttccgatct agaaagccct ccccagtcnn 60
nnnnnngtac tggtc 75
<210> 120
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
actaggtctt tccctacacg acgctcttcc gatctgagtt aaggactctg aagatnnnnn 60
nnnggtccta gt 72
<210> 121
<211> 81
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gtaacagtga ctggagttca gacgtgtgct cttccgatct tatgattttg cagaaaacag 60
atcnnnnnnn ntcagtgtta c 81
<210> 122
<211> 74
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
ggtctttctt tccctacacg acgctcttcc gatctttact tactacacct cagatannnn 60
nnnncatgaa gacc 74
<210> 123
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
gtctgggtga ctggagttca gacgtgtgct cttccgatct cctcaattct taccatccan 60
nnnnnnnatc cagac 75
<210> 124
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ctcttgtctt tccctacacg acgctcttcc gatctacatt cgaaagaccc tagccttann 60
nnnnnngagc aagag 75
<210> 125
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
atggatgtga ctggagttca gacgtgtgct cttccgatct tgcatgctgt ttaattgtgt 60
gnnnnnnnnc caatccat 78
<210> 126
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gttacttctt tccctacacg acgctcttcc gatctaagaa agctatataa gatattattn 60
nnnnnnncag agtaac 76
<210> 127
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
gagtcagtga ctggagttca gacgtgtgct cttccgatct gaaacagaga atctccattt 60
tagnnnnnnn nctgtgactc 80
<210> 128
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctc 49
<210> 129
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtg 46
Claims (14)
1. A nucleic acid combination comprising at least one primer pair comprising an upstream primer and a downstream primer; the upstream primer and the downstream primer are stem-loop structures comprising a loop part and a stem part, when annealing, at least part of the loop part and at least part of the 3 'end of the upstream primer is hybridized with one strand of a template, at least part of the loop part and at least part of the 3' end of the downstream primer is hybridized with the other strand of the template, the 5 'end of the upstream primer and the 5' end of the downstream primer are not hybridized with the template, the upstream primer and/or the downstream primer further comprise a modified part, the modified part is positioned between the 5 'end of the corresponding primer and the loop part or between the 3' end of the corresponding primer and the loop part, and the modified part can prevent polymerase from moving forwards continuously when extending;
the nucleic acid segment at the 3' end of the upstream primer comprises an upstream extension segment and an upstream specific segment, the upstream specific segment is positioned between the upstream ring part and the upstream extension segment, the upstream extension segment can be hybridized with one strand of the template, and the upstream specific segment cannot be bound with the template in a complementary pairing way;
the nucleic acid segment at the 3' end of the downstream primer comprises a downstream extension segment and a downstream specific segment, the downstream specific segment is positioned between the downstream loop part and the downstream extension segment, the downstream extension segment can be hybridized with the other strand of the template, and the downstream specific segment cannot be bound with the template in a complementary pairing mode.
2. The nucleic acid combination according to claim 1, wherein the modified portion is at least one selected from the group consisting of a spacer and NTP.
3. The nucleic acid combination product of claim 1, wherein the upstream extension is 3nt to 20nt long, and the upstream specific segment is 3nt to 50nt long; the length of the downstream extension section is 3 nt-20 nt, and the length of the downstream specific section is 3 nt-50 nt.
4. The nucleic acid combination product of claim 1, wherein the forward primer and/or the reverse primer further comprises a tag segment between the nucleic acid fragment at the 5 'end of the respective primer and the loop portion or between the nucleic acid fragment at the 3' end of the respective primer and the loop portion.
5. The nucleic acid composition as claimed in any one of claims 1 to 4, wherein the loop portion of the forward primer and the loop portion of the reverse primer are each independently 10nt to 100nt in length.
6. The nucleic acid composition of claim 5, further comprising at least one fluorescent probe, wherein the fluorescent probe has a fluorophore attached to its 5 'end and a quencher corresponding to the fluorophore attached to its 3' end.
7. A nucleic acid combination according to claim 5, further comprising at least one set of universal primer pairs which bind to complementary pairs of said templates.
8. A nucleic acid combination according to claim 5, wherein the nucleic acid combination comprises at least two of the following primer pairs:
HPV16 primer pairs with sequences shown as SEQ ID No.1 and SEQ ID No.2, HPV18 primer pairs with sequences shown as SEQ ID No.3 and SEQ ID No.4, HPV31 primer pairs with sequences shown as SEQ ID No.5 and SEQ ID No.6, HPV33 primer pairs with sequences shown as SEQ ID No.7 and SEQ ID No.8, HPV35 primer pairs with sequences shown as SEQ ID No.9 and SEQ ID No.10, HPV39 primer pairs with sequences shown as SEQ ID No.11 and SEQ ID No.12, HPV45 primer pairs with sequences shown as SEQ ID No.13 and SEQ ID No.14, HPV51 primer pairs with sequences shown as SEQ ID No.15 and SEQ ID No.16, HPV52 primer pairs with sequences shown as SEQ ID No.17 and SEQ ID No.18, HPV53 primer pairs with sequences shown as SEQ ID No.19 and SEQ ID No.20, and HPV 5824 primer pairs with sequences shown as SEQ ID No.21 and SEQ ID No. 56, and SEQ ID No.24, An HPV59 primer pair with sequences shown as SEQ ID No.25 and SEQ ID No.26 and an HPV66 primer pair with sequences shown as SEQ ID No.27 and SEQ ID No. 28.
9. A nucleic acid combination according to claim 5, wherein the nucleic acid combination comprises at least two of the following primer pairs:
SEPT9 primer pair with sequences shown as SEQ ID No.62 and SEQ ID No.63, VWC2 primer pair with sequences shown as SEQ ID No.64 and SEQ ID No.65, ADD2 primer pair with sequences shown as SEQ ID No.66 and SEQ ID No.67, INA primer pair with sequences shown as SEQ ID No.68 and SEQ ID No.69, POU4F1 primer pair with sequences shown as SEQ ID No.70 and SEQ ID No.71, HOXB4 primer pair with sequences shown as SEQ ID No.72 and SEQ ID No.73, SHOX2 primer pair with sequences shown as SEQ ID No.74 and SEQ ID No.75, PTGER4 primer pair with sequences shown as SEQ ID No.76 and SEQ ID No.77, THBD primer pair with sequences shown as SEQ ID No.78 and SEQ ID No.79, THPT 2 primer pair with sequences shown as SEQ ID No.80 and SEQ ID No.81, SOX 3582 and SOX 3584 primer pair with sequences shown as SEQ ID No.85 and SEQ ID No.83, GHSR primer pair with sequences shown as SEQ ID No.86 and SEQ ID No.87, JAM3 primer pair with sequences shown as SEQ ID No.88 and SEQ ID No.89, PRDM14 primer pair with sequences shown as SEQ ID No.90 and SEQ ID No.91, UCHL1 primer pair with sequences shown as SEQ ID No.92 and SEQ ID No.93, IRF4 primer pair with sequences shown as SEQ ID No.94 and SEQ ID No.95, ZNF583 primer pair with sequences shown as SEQ ID No.96 and SEQ ID No.97, FLT1 primer pair with sequences shown as SEQ ID No.98 and SEQ ID No.99, BOLL primer pair with sequences shown as SEQ ID No.100 and SEQ ID No.101, and ACTB primer pair with sequences shown as SEQ ID No.102 and SEQ ID No. 103.
10. The nucleic acid composition product of claim 9, further comprising an internal reference probe having a sequence shown in SEQ ID No.104, a detection probe having a sequence shown in SEQ ID No.105, and a universal primer pair having sequences shown in SEQ ID nos. 106 and 107, wherein both the internal reference probe and the detection probe are connected to a fluorophore, and the fluorophore on the internal reference probe is different from the fluorophore on the detection probe.
11. A nucleic acid combination according to claim 10, wherein the nucleic acid combination comprises at least two of the following primer pairs:
an EGFR18 primer pair with sequences shown as SEQ ID No.108 and SEQ ID No.109, an EGFR19 primer pair with sequences shown as SEQ ID No.110 and SEQ ID No.111, an EGFR20 primer pair with sequences shown as SEQ ID No.112 and SEQ ID No.113, an EGFR21 primer pair with sequences shown as SEQ ID No.114 and SEQ ID No.115, a KRAS2 primer pair with sequences shown as SEQ ID No.116 and SEQ ID No.117, a KRAS3 primer pair with sequences shown as SEQ ID No.118 and SEQ ID No.119, a KRAS4 primer pair with sequences shown as SEQ ID No.120 and SEQ ID No.121, a BRAF primer pair with sequences shown as SEQ ID No.122 and SEQ ID No.123, a PIK3CA primer pair with sequences shown as SEQ ID No.124 and SEQ ID No.125, and a PIK3CA primer pair with sequences shown as SEQ ID No.126 and SEQ ID No. 127.
12. The nucleic acid composition product of claim 11, further comprising a pair of universal primers having the sequences shown in SEQ ID nos. 128 and 129.
13. A test kit comprising the nucleic acid composition of any one of claims 1 to 12.
14. A method for amplifying a target nucleic acid, which method does not aim at diagnosis and treatment of a disease, comprising the steps of:
mixing a template, the nucleic acid composition product of any one of claims 1 to 12, a polymerase and a buffer to prepare a PCR reaction solution; and carrying out PCR reaction on the PCR reaction solution.
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| CN202111447510.XA CN113862339B (en) | 2021-12-01 | 2021-12-01 | Nucleic acid combination, detection kit and method for amplifying target nucleic acid |
| PCT/CN2022/135802 WO2023098787A1 (en) | 2021-12-01 | 2022-12-01 | Nucleic acid combination product, detection kit and method for amplifying target nucleic acid |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002027988A (en) * | 2000-07-14 | 2002-01-29 | Unitech Kk | Method for designing primer for expressing gene |
| CN1639352A (en) * | 2002-01-31 | 2005-07-13 | 犹他州大学 | Amplifying repetitive nucleic acid sequences |
| CN100999763A (en) * | 2006-01-12 | 2007-07-18 | 张曼 | Process of detecting cell splitting periodic protein gene 6 in tumor using modified primer |
| CN108699595A (en) * | 2016-02-25 | 2018-10-23 | 豪夫迈·罗氏有限公司 | The elimination that primer-primer interacts during primer extend |
| CN112899350A (en) * | 2018-05-14 | 2021-06-04 | 北京艾克伦医疗科技有限公司 | Nucleic acid detection method and kit |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277607B1 (en) * | 1999-05-24 | 2001-08-21 | Sanjay Tyagi | High specificity primers, amplification methods and kits |
| EP1924704B1 (en) * | 2005-08-02 | 2011-05-25 | Rubicon Genomics, Inc. | Compositions and methods for processing and amplification of dna, including using multiple enzymes in a single reaction |
| US20090023190A1 (en) * | 2007-06-20 | 2009-01-22 | Kai Qin Lao | Sequence amplification with loopable primers |
| CA3152038A1 (en) * | 2019-08-20 | 2021-02-25 | Fluent Biosciences Inc. | Hairpin primer design for sequential pcr production of targeted sequencing libraries |
| CN111534569A (en) * | 2020-05-29 | 2020-08-14 | 安徽安龙基因科技有限公司 | Oligonucleotide primer, kit and application |
| CN112980928B (en) * | 2021-04-02 | 2023-07-18 | 川北医学院附属医院 | Stem-loop primer-assisted isothermal nucleic acid amplification method |
| CN113862339B (en) * | 2021-12-01 | 2022-03-22 | 广州滴纳生物科技有限公司 | Nucleic acid combination, detection kit and method for amplifying target nucleic acid |
-
2021
- 2021-12-01 CN CN202111447510.XA patent/CN113862339B/en active Active
-
2022
- 2022-12-01 WO PCT/CN2022/135802 patent/WO2023098787A1/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002027988A (en) * | 2000-07-14 | 2002-01-29 | Unitech Kk | Method for designing primer for expressing gene |
| CN1639352A (en) * | 2002-01-31 | 2005-07-13 | 犹他州大学 | Amplifying repetitive nucleic acid sequences |
| CN100999763A (en) * | 2006-01-12 | 2007-07-18 | 张曼 | Process of detecting cell splitting periodic protein gene 6 in tumor using modified primer |
| CN108699595A (en) * | 2016-02-25 | 2018-10-23 | 豪夫迈·罗氏有限公司 | The elimination that primer-primer interacts during primer extend |
| CN112899350A (en) * | 2018-05-14 | 2021-06-04 | 北京艾克伦医疗科技有限公司 | Nucleic acid detection method and kit |
| CN112941155A (en) * | 2018-05-14 | 2021-06-11 | 北京艾克伦医疗科技有限公司 | DNA primer pair with stem-loop structure and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| "A new self-passivating template with the phosphorothioate strategy to effectively improve the detection limit and applicability of exponential amplification reaction";Jikai Mao等;《Anal. Methods》;20210628;第13卷;第3947-3953页 * |
| "OmegaPrimer";1234mb摩羯;《百度百科》;20210623;第1页 * |
| "二代测序平台进行核酸检测的新技术研究";陈科;《中国学位论文全文数据库》;20190118;第64-65页 * |
| 陈科."二代测序平台进行核酸检测的新技术研究".《中国学位论文全文数据库》.2019,第64-65页. * |
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