WO2023098787A1 - 核酸组合产品、检测试剂盒和扩增靶核酸的方法 - Google Patents

核酸组合产品、检测试剂盒和扩增靶核酸的方法 Download PDF

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WO2023098787A1
WO2023098787A1 PCT/CN2022/135802 CN2022135802W WO2023098787A1 WO 2023098787 A1 WO2023098787 A1 WO 2023098787A1 CN 2022135802 W CN2022135802 W CN 2022135802W WO 2023098787 A1 WO2023098787 A1 WO 2023098787A1
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primer
nucleic acid
primer pair
whose sequence
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阳卫超
马淑燕
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广州滴纳生物科技有限公司
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Definitions

  • 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.
  • PCR Polymerase chain reaction
  • Denaturation-annealing-extension 1
  • Denaturation of template DNA After the template DNA is heated to about 93°C for a certain period of time, the double-stranded DNA of the template or the double-stranded DNA formed by PCR amplification is dissociated, Make it a single strand so that it can combine with the primer to prepare for the next round of reaction;
  • 2Annealing (annealing) of the template DNA and the primer After the template DNA is heated and denatured into a single strand, the temperature drops to about 55°C, and the primer and primer The complementary sequence of the template DNA single strand is paired and combined;
  • 3Primer extension The DNA template-primer conjugate is at 72°C under the action of DNA polymerase (such as Taq DNA polymerase), using dNTP
  • Multiplex PCR refers to a PCR reaction in which two or more pairs of primers are added to a tube reaction system to simultaneously amplify multiple nucleic acid fragments. It effectively solves the needs of multi-site and multi-gene targeted amplification.
  • Primer-dimer is a small molecular weight double-stranded DNA fragment formed by the complementary combination of a pair of primers or the 3' end part of the primer itself, which is extended from the 3' end under the action of Taq enzyme.
  • the generation of primer dimers will reduce or even eliminate the yield of the target product, and also affect the multiplex number of multiplex PCR. The more PCR multiples, the more severe the PCR primer-dimer. When the multiplex number of multiplex PCR is as high as hundreds or thousands, partial base complementation at the 3' end is theoretically inevitable.
  • primer-dimers such as adding dimethyl sulfoxide (DMSO) to the reaction system, using hot-start polymerase and increasing the annealing temperature, etc., but none of them has solved the complementary problem between primers at the root.
  • DMSO dimethyl sulfoxide
  • a detection kit and a method for amplifying target nucleic acid that are not prone to non-specific amplification are also provided.
  • a nucleic acid combination product comprising at least one set of primer pairs, the primer pair comprising an upstream primer and a downstream primer; both the upstream primer and the downstream primer are stem-loop structures comprising a loop portion and a stem portion, and during 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 hybridize with one of the strands of the template, and 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 hybridization, neither the nucleic acid fragment at the 5' end of the upstream primer nor the downstream primer hybridizes to the template.
  • Both the upstream primer and the downstream primer of the primer pair of the above nucleic acid combination product have a stem-loop structure under non-denaturing conditions, and when annealing, at least part of the loop part and at least part of the nucleic acid fragment at the 3' end of the primer that hybridizes with the template is complementary to the template The remaining part does not hybridize with the template, while other primers that do not hybridize with the template have their own nucleic acid fragments at the 5' end and the nucleic acid fragments at the 3' end complementary pairing combination to form a stem-loop structure again, so that it is not easy to form primer dimers. Not prone to non-specific amplification.
  • the upstream primer and/or the downstream primer further includes a modification part, the modification part is located between the nucleic acid fragment at the 5' end of the corresponding primer and the loop part or between the nucleic acid fragment at the 3' end of the corresponding primer and the Between the loops, during extension, the modification prevents the polymerase from moving forward.
  • the modified portion is selected from at least one of the intermediate arm and NTP.
  • the nucleic acid fragment at the 3' end of the upstream primer includes an upstream extension segment and an upstream specific segment, the upstream specific segment is located between the upstream loop and the upstream extension segment, and the upstream extension segment The segment can hybridize with one of the strands of the template, and the upstream specific segment cannot be complementary to the template;
  • the nucleic acid fragment at the 3' end of the downstream primer includes a downstream extension segment and a downstream specific segment, the downstream specific segment is located between the downstream loop and the downstream extension segment, and the downstream extension segment can be combined with The other strand of the template is hybridized, and the downstream specific segment cannot be combined with the template in complementary pairing.
  • the length of the upstream extension segment is 3nt to 20nt
  • the length of the upstream specific segment is 3nt to 50nt
  • the length of the downstream extension segment is 3nt to 20nt
  • the length of the downstream specific segment is It is 3nt ⁇ 50nt.
  • the upstream primer and/or the downstream primer further includes a tag segment, the tag segment is located between the nucleic acid segment at the 5' end of the corresponding primer and the loop or between the nucleic acid segment at the 3' end of the corresponding primer and the between rings.
  • the lengths of the loop portion of the upstream primer and the loop portion of the downstream primer are independently 10 nt to 100 nt.
  • the nucleic acid combination product further includes at least one fluorescent probe, 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 fluorescent group corresponding to the fluorescent group. the quenching group.
  • the nucleic acid combination product may further include at least one set of universal primer pairs, and the universal primer pairs are capable of complementary pairing and binding to the template.
  • the nucleic acid combination product includes at least two of the following primer pairs:
  • the nucleic acid combination product includes at least two of the following primer pairs:
  • the SEPT9 primer pair whose sequence is shown in SEQIDNO.62 and SEQIDNO.63, the VWC2 primer pair whose sequence is shown in SEQIDNO.64 and SEQIDNO.65, the ADD2 primer pair whose sequence is shown in SEQIDNO.66 and SEQIDNO.67, the sequence is shown in The INA primer pair shown in SEQIDNO.68 and SEQIDNO.69, the POU4F1 primer pair shown in SEQIDNO.70 and SEQIDNO.71, the HOXB4 primer pair shown in SEQIDNO.72 and SEQIDNO.73, and the sequence shown in SEQIDNO.
  • the GHSR primer pair shown in 87 the JAM3 primer pair shown in SEQIDNO.88 and SEQIDNO.89, the PRDM14 primer pair shown in SEQIDNO.90 and SEQIDNO.91, the sequence shown in SEQIDNO.
  • the UCHL1 primer pair shown, the IRF4 primer pair shown in SEQIDNO.94 and SEQIDNO.95, the ZNF583 primer pair shown in SEQIDNO.96 and SEQIDNO.97, the sequence shown in SEQIDNO.98 and SEQIDNO.99 A pair of FLT1 primers, a pair of BOLL primers whose sequences are shown in SEQ ID NO.100 and SEQ ID NO.101, and a pair of ACTB primers whose sequences are shown in SEQ ID NO.102 and SEQ ID NO.103.
  • the nucleic acid composition product also includes an internal reference probe whose sequence is shown in SEQ ID NO.104, a detection probe whose sequence is shown in SEQ ID NO.105, and a sequence such as SEQ ID NO.106 and SEQ ID NO.107.
  • a pair of universal primers is shown, the internal reference probe and the detection probe are connected with a fluorescent group, and the fluorescent group on the internal reference probe is different from the fluorescent group on the detection probe.
  • the nucleic acid combination product includes at least two of the following primer pairs:
  • the nucleic acid combination product further includes a pair of universal primers whose sequences are shown in SEQ ID NO.128 and SEQ ID NO.129.
  • a detection kit comprising the above-mentioned nucleic acid combination product.
  • a method of amplifying a target nucleic acid comprising the steps of:
  • Figure 1 is a schematic diagram of the working principle of a nucleic acid combination product in one embodiment
  • Fig. 2 is the electrophoresis result figure of embodiment 1;
  • Fig. 3 is the amplification curve of the improved primer pair in embodiment 2;
  • Fig. 4 is the amplification curve of conventional primer pair in embodiment 2;
  • Fig. 5 ⁇ Fig. 10 are the amplification curve result figure in embodiment 3;
  • FIG. 11 is a graph of electrophoresis results in Example 4.
  • the "positive strand” and “negative strand” in this article refer to the two nucleic acid single strands of the DNA template in the PCR amplification process, and the positive strand and the negative strand are complementary to each other, wherein: base sequence and DNA template
  • the nucleic acid single strand corresponding to the same mRNA (only the difference between T and U) is defined as the positive strand; the nucleic acid single strand whose sequence is complementary to the mRNA corresponding to the DNA template is defined as the negative strand.
  • upstream loop and downstream loop of the upstream primer in this paper have the same meaning, and both refer to the loop in the stem-loop structure of the upstream primer;
  • downstream loop has the same meaning as the "loop of the downstream primer”, both refers to the loop portion in the stem-loop structure of the downstream primer.
  • nucleic acid combination product includes at least one set of primer pairs, the primer pair includes an upstream primer and a downstream primer; both the upstream primer and the downstream primer are stem-loops that include a loop and a stem Structure, when annealing, at least part of the loop portion and at least part of the nucleic acid fragment at the 3’ end of the upstream primer hybridizes with one of the strands of the template (such as a negative strand), and at least a portion of the loop portion of the downstream primer and at least a portion of the nucleic acid fragment at the 3’ end are hybridized with The other strand of the template hybridizes (eg, the positive strand), and neither the nucleic acid fragment at the 5' end of the upstream primer nor the downstream primer hybridizes to the template.
  • the primer pair includes an upstream primer and a downstream primer
  • both the upstream primer and the downstream primer are stem-loops that include a loop and a stem Structure, when annealing, at least part of the loop portion and at least
  • the primer pair in the above-mentioned nucleic acid combination product is by designing the upstream primer and the downstream primer into a stem-loop structure, both the upstream primer and the downstream primer are: under non-denaturing (such as normal temperature) conditions, the nucleic acid fragment at the 5' end is aligned with the The nucleic acid fragment at the 3' end forms a stem through complementary pairing, and the nucleic acid fragment located between the nucleic acid fragment at the 5' end and the nucleic acid fragment at the 3' end forms a loop.
  • non-denaturing such as normal temperature
  • the nucleic acid fragment at the loop and the 3' end of the upstream primer is complementary to one strand of the template (hybridizes), but the nucleic acid fragment at the 5' end of the upstream primer does not hybridize with the template;
  • the nucleic acid fragment at the 'end hybridizes to the other template, but the nucleic acid fragment at the 5' end of the downstream primer does not hybridize to the template; at this time, the nucleic acid fragment at the 5' end of the other primer that is not combined with the template is complementary to the nucleic acid fragment at the 3' end
  • the 5' end and 3' end of the primer are closed, and it is not easy to form a primer dimer. It has been verified that the primers designed according to the above have good specificity and are not prone to non-specific amplification.
  • the upstream primer has a stem-loop structure
  • the upstream primer includes an upstream stem and an upstream loop connected to the upstream stem, and the nucleic acid fragment at the 5' end of the upstream primer is complementary to the nucleic acid fragment at the 3' end of the upstream primer to form an upstream primer.
  • the stem, the upstream loop portion are located 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.
  • the downstream primer also has a stem-loop structure, and the downstream primer includes a downstream stem and a downstream loop connected to the downstream stem, and the 5' of the downstream primer
  • the nucleic acid fragment at the end and the nucleic acid fragment at the 3' end of the downstream primer are complementary paired to form a downstream stem, and the downstream loop is 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.
  • At least Part of the downstream loop and at least part of the nucleic acid fragment at the 3' end of the downstream primer hybridizes with the other strand of the template, and the nucleic acid fragment at the 5' end of the downstream primer cannot be complementary to this strand.
  • the nucleic acid fragment at the 5' end of the upstream primer is used for complementary pairing with the nucleic acid fragment at the 3' end of the upstream primer under non-denaturing conditions (such as normal temperature); the nucleic acid fragment at the 3' end of the upstream primer is used for annealing with the template One of the strands (such as the negative strand) hybridizes, thereby allowing the polymerase to use this strand as a template for extension.
  • the nucleic acid fragment at the 5' end of the downstream primer is used for complementary pairing with the nucleic acid fragment at the 3' end of the downstream primer under non-denaturing conditions (such as normal temperature); the nucleic acid fragment at the 3' end of the downstream primer is used for annealing with the other template
  • the strand eg, plus strand
  • the upstream primer binds to the negative strand of the template
  • the downstream primer binds to the positive strand of the template as an example for illustration.
  • the nucleic acid fragment at the 3' end of the upstream primer hybridizes to the negative strand of the template.
  • the nucleic acid fragment at the 3' end of the upstream primer includes an upstream extension segment and an upstream specific segment, the upstream specific segment is located between the upstream loop and the upstream extension segment, the upstream extension segment can hybridize with the negative strand in the template, and the upstream specific segment is connected to the negative strand in the template. Negative strands cannot bind in complementary pairs.
  • the complementary pairing combination of the 3' end of the upstream primer and the negative strand depends on the upstream extension segment, so that the polymerase can start the extension only after the upstream extension segment needs to be completely complementary to the negative strand. , thus improving the specificity of the upstream primer.
  • the length of the upstream extension segment is 3nt-20nt, and the length of the upstream specific segment is 3nt-50nt.
  • the length of the upstream extension segment is 6nt-15nt, and the length of the upstream specific segment is 15nt-35nt.
  • all nucleic acid fragments at the 3' end of the upstream primer hybridize to the negative strand.
  • the nucleic acid fragment at the 3' end of the downstream primer hybridizes to the positive strand of the template.
  • the nucleic acid fragment at the 3' end of the downstream primer includes a downstream extension segment and a downstream specific segment, the downstream specific segment is located between the downstream loop and the downstream extension segment, the downstream extension segment can hybridize with the positive strand, and the downstream specific segment and the positive strand cannot Complementary pair binding.
  • the length of the downstream extension segment is 3nt-20nt, and the length of the downstream specific segment is 3nt-50nt. Further, the length of the downstream extension segment is 6nt-15nt, and the length of the downstream specific segment is 15nt-35nt.
  • the nucleic acid fragment at the 3' end of the downstream primer upon annealing, fully hybridizes to the negative strand.
  • the upstream loop is used to combine with the negative strand of the template, which facilitates the binding of the nucleic acid fragment at the 3' end of the upstream primer to the negative strand. At least part of the upstream loop is capable of complementary pairing with the negative strand. In some embodiments, the length of the upstream loop is between 10 nt and 100 nt. Further, the length of the upstream ring portion is 18nt ⁇ 60nt. In one of the embodiments, part of the upstream loop is capable of complementary pairing with the negative strand. Specifically, in the upstream loop, the length of the nucleic acid fragment complementary to the negative strand is 10 nt to 50 nt.
  • the length of the nucleic acid fragment complementary to the negative strand is 15nt, 20nt, 30nt, 40nt or 45nt.
  • all of the upstream loops are capable of complementary pairing with the negative strand.
  • the downstream loop is used to combine with the positive strand of the template, which facilitates the binding of the nucleic acid fragment at the 3' end of the downstream primer to the positive strand.
  • At least part of the downstream loop is capable of complementary pairing with the negative strand.
  • the length of the downstream loop is between 10 nt and 100 nt.
  • the length of the downstream ring part is 18nt-60nt.
  • a portion of the downstream loop is capable of complementary pairing with the positive strand.
  • the length of the nucleic acid fragment complementary to the positive strand is 10 nt to 50 nt.
  • the length of the nucleic acid fragment complementary to the positive strand is 15nt, 20nt, 30nt, 40nt or 45nt.
  • all of the downstream loops are capable of complementary pairing with the plus strand.
  • the upstream primer and/or the downstream primer further includes a modification part, the modification part is located between the nucleic acid fragment at the 5' end of the corresponding primer and the loop part or between the nucleic acid fragment at the 3' end of the corresponding primer and the loop part, When , the modified portion can prevent the polymerase from moving forward.
  • the upstream primer further includes an upstream modification part, which is located between the nucleic acid fragment at the 5' end of the upstream primer and the upstream loop part; during extension, the upstream modification part can prevent the polymerase from moving forward.
  • DNA polymerase moves to the nucleic acid at the 5' end of the upstream primer on the template when a nucleic acid single strand containing the upstream primer is used as a template.
  • the fragment stops moving, and the extension is terminated, avoiding non-specific amplification by extending the complementary strand of the nucleic acid fragment at the 5' end of the upstream primer (that is, the 5' end of the negative strand at this time).
  • the polymerase herein refers to DNA polymerase; the forward direction of the polymerase is from the 5' end to the 3' end of the template.
  • the upstream modification part is selected from at least one of Spacer and NTP.
  • the intermediate arm is at least one of Spacer C3, Spacer C6, Spacer C9 and Spacer C18, wherein Spacer C3 refers to -CH 2 CH 2 CH 2 -, and Spacer C6 refers to 6 -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, Spacer C9 refers to the linker chain composed of 3 consecutive ethylene glycols (-CH 2 CH 2 O-), Spacer C18 refers to 6 consecutive ethylene glycols (-CH 2 The linker chain composed of CH 2 O-).
  • the specific composition of the upstream modification part is not limited to the above, and can also be other substances that can prevent the polymerase from continuing to move to the 5' end of the upstream primer.
  • the downstream primer further includes a downstream modification part, the downstream modification part is located between the nucleic acid fragment at the 5' end of the downstream primer and the downstream loop part, and during extension, the downstream modification part can prevent the polymerase from moving forward.
  • the downstream modification part By providing a downstream modification part between the nucleic acid fragment at the 5' end of the downstream primer and the downstream loop, when the nucleic acid single strand containing the downstream primer is used as a template, the DNA polymerase moves to the nucleic acid fragment at the 5' end of the downstream primer. The movement is stopped, the extension is terminated, and non-specific amplification occurs due to the extension of the complementary strand of the nucleic acid fragment at the 5' end of the synthetic downstream primer.
  • the downstream modification part is selected from at least one of Spacer and NTP.
  • the intermediate arm is at least one of Spacer C3, Spacer C6, Spacer C9 and Spacer C18, wherein Spacer C3 refers to -CH 2 CH 2 CH 2 -, and Spacer C6 refers to -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, Spacer C9 refers to the linker chain composed of 3 consecutive ethylene glycols (-CH 2 CH 2 O-), Spacer C18 refers to 6 consecutive ethylene glycols (-CH 2 CH 2 O-) linker chain.
  • the specific composition of the downstream modification part is not limited to the above, and can also be other substances that can prevent the polymerase from continuing to move to the 5' end of the downstream primer.
  • the upstream primer and/or the downstream primer further includes a tag segment, and the tag segment is located between the nucleic acid segment at the 5' end of the corresponding primer and the loop or between the nucleic acid segment at the 3' end of the corresponding primer and the loop.
  • the upstream primer further includes an upstream tag segment, which is located between the nucleic acid segment at the 5' end of the upstream primer and the upstream loop, and the upstream tag segment and the negative strand cannot be complementary paired and combined.
  • the upstream tag segment is used to introduce universal primer sequences and molecular tags into the newly synthesized chain, which facilitates subsequent construction of sequencing libraries.
  • the downstream primer further includes a downstream label segment, which is located between the nucleic acid fragment at the 5' end of the downstream primer and the downstream loop, and the downstream label segment cannot be complementary to the positive strand.
  • the downstream tag segment is used to introduce universal primer sequences and molecular tags into the newly synthesized chain, which facilitates subsequent construction of sequencing libraries.
  • the upstream primer has a fluorophore attached.
  • a fluorescent group is connected to the 5' end of the upstream primer, and a quenching group corresponding to the fluorescent group is connected to the 3' end of the upstream primer.
  • the degree of amplification can be characterized by the fluorescence intensity. It can be understood that the fluorescent group and the quencher group are not particularly limited.
  • the downstream primer has a fluorophore attached.
  • a fluorescent group is connected to the 5' end of the downstream primer, and a quencher group corresponding to the fluorescent group is connected to the 3' end of the downstream primer.
  • the degree of amplification can be characterized by the fluorescence intensity. It can be understood that the fluorescent group and the quencher group are not particularly limited.
  • At least one fluorescent probe is included in the nucleic acid combination.
  • a fluorescent group is connected to the 5' end of the fluorescent probe, and a quenching group corresponding to the fluorescent group is connected to the 3' end of the fluorescent probe.
  • the degree of amplification can be characterized by the fluorescence intensity by enzymatically cutting and degrading the probe to make it fluoresce during the amplification process. It can be understood that the fluorescent group and the quencher group are not particularly limited.
  • the nucleic acid combination product includes at least one set of universal primer pairs.
  • the universal primer pair can be combined with complementary templates for sequencing.
  • At least one of the upstream primer and the downstream primer is a stem-loop primer. Further, at least one of the upstream primer and the downstream primer is a molecular beacon primer.
  • the nucleic acid combination product includes at least two primer pairs, and different primer pairs are used to amplify different nucleic acid fragments.
  • the structures of each pair of primers are as described above, and the difference between each pair of primers lies in that different pairs of primers target different target regions.
  • different primer pairs are packaged as a mixture. In other embodiments, different primer pairs are packaged independently.
  • the above-mentioned nucleic acid combination product is a product for detecting HPV virus.
  • the nucleic acid combination product includes at least two groups of the following primer pairs: the HPV16 primer pair whose sequence is shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence such as SEQ ID NO.3 and SEQ ID NO.4.
  • the HPV18 primer pair shown the sequence such as the HPV31 primer pair shown in SEQ ID NO.5 and SEQ ID NO.6, the sequence such as the HPV33 primer pair shown in SEQ ID NO.7 and SEQ ID NO.8, the sequence such as SEQ ID The pair of HPV35 primers shown in NO.9 and SEQ ID NO.10, the pair of HPV39 primers shown in SEQ ID NO.11 and SEQ ID NO.12, the sequence shown in SEQ ID NO.13 and SEQ ID NO.14
  • the HPV45 primer pair shown the sequence such as the HPV51 primer pair shown in SEQ ID NO.15 and SEQ ID NO.16, the sequence such as the HPV52 primer pair shown in SEQ ID NO.17 and SEQ ID NO.18, the sequence such as SEQ ID The pair of HPV53 primers shown in NO.19 and SEQ ID NO.20, the pair of HPV56 primers shown in SEQ ID NO.21 and SEQ ID NO.22, the sequence shown in SEQ ID NO.23 and SEQ ID NO
  • the aforementioned nucleic acid combination product is a product for detecting tumor gene methylation.
  • the nucleic acid combination product includes at least two groups of the following primer pairs: a pair of SEPT9 primers with sequences as shown in SEQ ID NO.62 and SEQ ID NO.63, a sequence as shown in SEQ ID NO.64 and SEQ ID NO.65
  • the VWC2 primer pair shown the sequence such as the ADD2 primer pair shown in SEQ ID NO.66 and SEQ ID NO.67, the sequence such as the INA primer pair shown in SEQ ID NO.68 and SEQ ID NO.69, the sequence such as SEQ ID The POU4F1 primer pair shown in NO.70 and SEQ ID NO.71, the HOXB4 primer pair shown in SEQ ID NO.72 and SEQ ID NO.73, the sequence shown in SEQ ID NO.74 and SEQ ID NO.75 SHOX2 primer pair shown, sequence such as PTGER4 primer pair shown in SEQ ID NO.76 and SEQ
  • the nucleic acid composition product for detecting tumor gene methylation also includes an internal reference probe whose sequence is shown in SEQ ID NO.104, a detection probe whose sequence is shown in SEQ ID NO.105, and a sequence such as SEQ ID NO.105.
  • both the internal reference probe and the detection probe are connected with a fluorescent group, and the fluorescent group on the internal reference probe is different from the fluorescent group on the detection probe.
  • the aforementioned nucleic acid combination product is a product for detecting tumor gene mutations.
  • the nucleic acid combination product includes at least two groups of the following primer pairs: the EGFR18 primer pair whose sequence is shown in SEQ ID NO.108 and SEQ ID NO.109, the sequence such as SEQ ID NO.110 and SEQ ID NO.111.
  • the EGFR19 primer pair shown the sequence such as the EGFR20 primer pair shown in SEQ ID NO.112 and SEQ ID NO.113, the sequence such as the EGFR21 primer pair shown in SEQ ID NO.114 and SEQ ID NO.115, the sequence such as SEQ ID The KRAS2 primer pair shown in NO.116 and SEQ ID NO.117, the KRAS3 primer pair shown in SEQ ID NO.118 and SEQ ID NO.119, the sequence shown in SEQ ID NO.120 and SEQ ID NO.121
  • the KRAS4 primer pair shown the BRAF primer pair shown in the sequence such as SEQ ID NO.122 and SEQ ID NO.123, the PIK3CA primer pair shown in the sequence such as SEQ ID NO.124 and SEQ ID NO.125, and the sequence shown in SEQ ID NO.125
  • the nucleic acid combination product for detecting tumor gene mutations also includes a pair of universal primers whose sequences are shown in SEQ ID NO.128 and SEQ ID NO.129.
  • the primer pairs in the nucleic acid combination product are a set.
  • the primer pair is used to amplify a sequence on the human ACTB genome.
  • the primer pair of the nucleic acid combination product is as shown in SEQ ID NO.29 and SEQ ID NO.30.
  • an embodiment of the present application also provides a detection kit, including the nucleic acid combination product of any of the above embodiments.
  • the above detection kit also includes at least one of polymerase, buffer and universal primers for sequencing.
  • a polymerase is used to extend the 3' end of the primer during PCR.
  • Buffers provide a buffer system for the PCR reaction.
  • Universal primers are used to amplify products to prepare sequencing libraries. It can be understood that when the above-mentioned nucleic acid combination product contains a pair of universal primers for sequencing, the universal primers in the above-mentioned detection kit can be omitted.
  • the above-mentioned detection kit includes the above-mentioned nucleic acid combination product, and the primer pair of the above-mentioned nucleic acid combination product solves the problem that the primers are easily complementary and paired to form a dimer from the structure of the primers.
  • the primers Dimers and non-specific amplification are not easy to appear between them, and the non-specific amplification of multiplex PCR can be improved.
  • an embodiment of the present application also provides a method for amplifying a target nucleic acid, the method comprising the following steps: mixing the template, the nucleic acid combination product of any of the above examples, a polymerase, and a buffer to prepare a PCR reaction solution and performing a PCR reaction on the PCR reaction solution.
  • the method for amplifying target nucleic acid can be used as amplification of nucleic acid fragments before library preparation.
  • the method of amplifying target nucleic acid is used to construct a sequencing library.
  • the conditions of the PCR reaction are: pre-denaturation: 90°C-98°C for 1min-10min; denaturation: 90°C-98°C for 2s-60s; annealing: 48°C-68°C for 10s-120min; extension: 68°C °C ⁇ 75°C10s ⁇ 10min; number of cycles: 2 ⁇ 60.
  • the above method for amplifying target nucleic acid uses the above nucleic acid combination product, which has good specificity and is not prone to non-specific amplification.
  • Embodiment 1 Amplification of human ACTB gene
  • a segment of the human ACTB genome sequence (ie, target nucleic acid) was amplified using conventional PCR primer pairs and improved primer pairs, respectively, to verify the good amplification of the improved primer pairs.
  • the base sequence of the target nucleic acid is shown in SEQ ID NO.31.
  • the specific verification steps include:
  • lane 1 is the maker
  • lanes 2, 3 and 4 are the electrophoresis results of the amplification products corresponding to the improved primer pair
  • lanes 5, 6 and 7 are the electrophoresis results of the amplification products corresponding to the conventional primer pair. It can be seen from Figure 2 that the electrophoretic band of the amplification product corresponding to the improved primer pair is single, while the electrophoretic band of the amplification product corresponding to the conventional primer pair is diffuse.
  • Embodiment 2 Multiplex PCR amplification of 14 kinds of high-risk HPV virus genes
  • Hot start Taq enzyme (5U/ ⁇ L) 0.5 ⁇ L HPV16F (10 ⁇ mol) 0.35 ⁇ L dNTP (25mM) 0.5 ⁇ L HPV16R (10 ⁇ mol) 0.35 ⁇ L 10 ⁇ Buffer 2.5 ⁇ L HPV18F (10 ⁇ mol) 0.35 ⁇ L 10 ⁇ Evagreen 2.5 ⁇ L HPV18R (10 ⁇ mol) 0.35 ⁇ L HPV52F (10 ⁇ mol) 0.35 ⁇ L HPV31F (10 ⁇ mol) 0.35 ⁇ L HPV52R (10 ⁇ mol) 0.35 ⁇ L HPV31R (10 ⁇ mol) 0.35 ⁇ L HPV53F (10 ⁇ mol) 0.35 ⁇ L HPV33F (10 ⁇ mol) 0.35 ⁇ L HPV53R (10 ⁇ mol) 0.35 ⁇ L HPV33R (10 ⁇ mol) 0.35 ⁇ L HPV56F (10 ⁇ mol) 0.35 ⁇ L HPV
  • Fig. 3 is the amplification curve of the improved primer pair
  • Fig. 4 is the amplification curve of the conventional primer pair.
  • the improved primer pair has no non-specific amplification curve to pure water (no template sample) and negative sample (no HPV DNA sample), the positive sample (containing HPV DNA sample) amplification curve is good, and
  • the conventionally designed primer pair had non-specific amplification curves for pure water (no template sample) and negative sample (no HPV DNA sample), indicating that primer-dimers existed during the amplification of the conventional primer pair.
  • Select genes that are highly methylated in tumor samples use the method of the present invention to design primers, and amplify multiple methylated fragments of tumor genes in one PCR reaction tube, so as to achieve highly sensitive detection of tumor gene methylation.
  • Specific steps include:
  • the underlined part is the hybridization sequence with the template:
  • Hot start Taq enzyme (5U/ ⁇ L) 0.5 ⁇ L SDC2F (2 ⁇ mol) 0.1 ⁇ L dNTP (25mM) 0.5 ⁇ L SDC2R (2 ⁇ mol) 0.1 ⁇ L 10 ⁇ Buffer 2.5 ⁇ L PAX1F (2 ⁇ mol) 0.1 ⁇ L Methyl probe (10 ⁇ mol) 0.5 ⁇ L PAX1R (2 ⁇ mol) 0.1 ⁇ L Universal F (10 ⁇ mol) 1 ⁇ L SOX1F (2 ⁇ mol) 0.1 ⁇ L Universal R (10 ⁇ mol) 1 ⁇ L SOX1R (2 ⁇ mol) 0.1 ⁇ L Ultra-pure water 4.3 ⁇ L GHSRF (2 ⁇ mol) 0.1 ⁇ L sample DNA 10 ⁇ L GHSRR (2 ⁇ mol) 0.1 ⁇ L SEPT9F (2 ⁇ mol) 0.1 ⁇ L JAM3F (2 ⁇ mol) 0.1 ⁇ L SEPT9R (2 ⁇ mol) 0.1 ⁇ L JAM3R (2 ⁇ mol) 0.1 ⁇ L VWC2F (2 ⁇ mol) 0.1 ⁇ L PRDM14F (2 ⁇ mol) 0.1 ⁇ L VWC2F (2 ⁇
  • the amplification curve is obtained, and the amplification curve is shown in Fig. 5 to Fig. 10 .
  • the PCR reaction system has good amplification curves for positive samples (tumor tissue samples), and no non-specific amplification curves for pure water (no template samples) and negative samples (healthy tissue samples).
  • the sample Ct values are shown in Table 12 below.
  • Tumor sample 1 30.17 30.35 Tumor sample 1 30.14 30.31 Tumor sample 2 30.85 31.28 Tumor sample 2 30.77 31.22 Tumor sample 3 29.65 30.61 Tumor sample 3 29.58 30.54 Healthy sample 1 / 27.55 Healthy sample 1 / 27.62 Healthy sample 2 / 28.04 Healthy sample 2 / 28.12 pure water / / pure water / /
  • the underlined part is the hybridization sequence with the template:
  • Electrophoresis was performed using agarose with a concentration of 2%, 1 hour at 120 volts, the range of 200-300 bp was recovered by cutting the gel, recovered using a gel recovery kit (Qiagen), and eluted with 40 ⁇ l of nuclease-free water. Perform Qubit (fluorescence quantification instrument) quantification, and dilute the library to 2 ng/ ⁇ l with nuclease-free water for use.
  • Qubit fluorescence quantification instrument
  • the Aglilent 2100 bioanalyzer was used to detect the size of the library insert, and the Q-PCR instrument was used to detect the library concentration; the detection results are shown in Figure 11.
  • the size of the library constructed in this example is about 250 bp
  • the adapter sequence at both ends is removed 116 bp
  • the size of the insert is about 130 bp, which meets the requirement of paired-end sequencing.

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Abstract

本发明涉及一种核酸组合产品、检测试剂盒和扩增靶核酸的方法。该核酸组合产品包括至少一种引物对，该引物对包括上游引物和下游引物，上游引物呈茎环结构，在退火时，上游引物的环部和3'端的核酸片段与模板的负链杂交，上游引物的5'端的核酸片段不与负链杂交；下游引物也呈茎环结构，在退火时，下游引物的环部和3'端的核酸片段与模板的正链杂交，下游引物的5'端的核酸片段不与正链杂交。上述核酸组合产品的引物对可改善多重PCR的非特异性扩增。

Description

核酸组合产品、检测试剂盒和扩增靶核酸的方法
相关申请
本申请要求于2021年12月1日申请的，申请号为CN202111447510.X，名称为“核酸组合产品、检测试剂盒和扩增靶核酸的方法”的中国专利申请的优先权，在此将全文引入作为参考。
技术领域
本发明涉及分子生物技术领域，特别是涉及一种核酸组合产品、检测试剂盒和扩增靶核酸的方法。
背景技术
聚合酶链式反应(PCR)是一种用于放大扩增特定的DNA片段的分子生物学技术，是生物技术领域最有效的DNA序列获取方法。PCR由变性-退火-延伸三个基本反应步骤构成：①模板DNA的变性：模板DNA经加热至93℃左右一定时间后，使模板DNA双链或经PCR扩增形成的双链DNA解离，使之成为单链，以便它与引物结合，为下轮反应作准备；②模板DNA与引物的退火(复性)：模板DNA经加热变性成单链后，温度降至55℃左右，引物与模板DNA单链的互补序列配对结合；③引物的延伸：DNA模板-引物结合物在72℃、DNA聚合酶(如Taq DNA聚合酶)的作用下，以dNTP为反应原料，靶序列为模板，按碱基互补配对与半保留复制原理，合成一条新的与模板DNA链互补的半保留复制链，重复循环变性-退火-延伸三过程就可获得更多的“半保留复制链”，而且这种新链又可成为下次循环的模板。每完成一个循环需2～4分钟，2～3小时就能将待扩目的基因扩增放大几百万倍。随着技术的进步，PCR有了很多的更新和应用。
多重PCR是指在一管反应体系里加两对以上引物，同时扩增多个核酸片段的PCR反应。它有效的解决了多位点，多基因的靶向扩增需求。引物二聚体是一对引物或者引物自身的3’端部分碱基互补结合，在Taq酶的作用下从3’末端延伸所形成的小分子量的双链DNA片段。引物二聚体的产生，会使得目标产物产量降低甚至没有，同时也影响多重PCR的重数。当PCR重数越多，则PCR的引物二聚体越严重。当多重PCR重数高到几百上千时，3’端的部分碱基互补理论上常不可避免。
目前有很多解决引物二聚体的方法，例如在反应体系中加入二甲亚砜(DMSO)、采用热启动聚合酶和提高退火温度等，但都未从根源上解决引物间互补问题，多重PCR的非特异性扩增仍比较明显。
发明内容
基于此，有必要提供一种可改善多重PCR的非特异性扩增的核酸组合产品。
此外，还提供了一种不容易出现非特异性扩增的检测试剂盒和种扩增靶核酸的方法。
一种核酸组合产品，包括至少一组引物对，所述引物对包括上游引物和下游引物；所述上游引物和所述下游引物均为包括环部和茎部的茎环结构，在退火时，所述上游引物的至少部分环部和至少部分3’端的核酸片段与模板的其中一条链杂交，所述下游引物的至少部分环部和至少部分3’端的核酸片段与所述模板的另一条链杂交，所述上游引物和所述下游引物的5’端的核酸片段均不与所述模板杂交。
上述核酸组合产品的引物对的上游引物和下游引物在非变性条件下均呈茎环结构，在退火时，与模板杂交的引物的至少部分环部和至少部分3’端的核酸片段与模板互补配对结合，其余部分不与模板杂交，同时其他不与模板杂交的引物则自身的5’端的核酸片段与3’端的核酸片段互补配对结合而再次形成茎环结构，从而不容易形成引物二聚体，不容易出现非特异性扩增。
在其中一个实施例中，所述上游引物和/或所述下游引物还包括修饰部，所述修饰部位于相应引物的5’端的核酸片段与环部之间或相应引物的3’端的核酸片段与环部之间，在延伸时，所述修饰部能阻止聚合酶继续向前移动。
在其中一个实施例中，所述修饰部选自间臂及NTP中的至少一种。
在其中一个实施例中，所述上游引物的3’端的核酸片段包括上游延伸段和上游特异段，所述上游特异段位于所述上游环部和所述上游延伸段之间，所述上游延伸段能与所述模板的其中一条链杂交，所述上游特异段与所述模板不能互补配对结合；
及/或，所述下游引物的3’端的核酸片段包括下游延伸段和下游特异段，所述下游特异段位于所述下游环部和所述下游延伸段之间，所述下游延伸段能与所述模板的另一条链杂交，所述下游特异段与所述模板不能互补配对结合。
在其中一个实施例中，所述上游延伸段的长度为3nt～20nt，所述上游特异段的长度为3nt～50nt；所述下游延伸段的长度为3nt～20nt，所述下游特异段的长度为3nt～50nt。
在其中一个实施例中，所述上游引物和/或所述下游引物还包括标签段，所述标签段位于相应引物的5’端的核酸片段与环部之间或相应引物的3’端的核酸片段与环部之间。
在其中一个实施例中，所述上游引物的环部和所述下游引物的环部的长度分别独立地为10nt～100nt。
在其中一个实施例中，所述核酸组合产品中还包括至少一条荧光探针，所述荧光探针的5’端连接有荧光基团，荧光探针的3’端连接有与荧光基团对应的淬灭基团。
在其中一个实施例中，所述核酸组合产品中还可包括至少一组通用引物对，所述通用引物对能与所述模板互补配对结合。
在其中一个实施例中，所述核酸组合产品包括以下引物对中的至少两组：
序列如SEQIDNO.1及SEQIDNO.2所示的HPV16引物对、序列如SEQIDNO.3及SEQIDNO.4所示的HPV18引物对、序列如SEQIDNO.5及SEQIDNO.6所示的HPV31引物对、序列如SEQIDNO.7及SEQIDNO.8所示的HPV33引物对、序列如SEQIDNO.9及SEQIDNO.10所示的HPV35引物对、序列如SEQIDNO.11及SEQIDNO.12所示的HPV39引物对、序列如SEQIDNO.13及SEQIDNO.14所示的HPV45引物对、序列如SEQIDNO.15及SEQIDNO.16所示的HPV51引物对、序列如SEQIDNO.17及SEQIDNO.18所示的HPV52引物对、序列如SEQIDNO.19及SEQIDNO.20所示的HPV53引物对、序列如SEQIDNO.21及SEQIDNO.22所示的HPV56引物对、序列如SEQIDNO.23及SEQIDNO.24所示的HPV58引物对、序列如SEQIDNO.25及SEQIDNO.26所示的HPV59引物对和序列如SEQIDNO.27及SEQIDNO.28所示的HPV66引物对。
在其中一个实施例中，所述核酸组合产品包括以下引物对中的至少两组：
序列如SEQIDNO.62及SEQIDNO.63所示的SEPT9引物对、序列如SEQIDNO.64及SEQIDNO.65所示的VWC2引物对、序列如SEQIDNO.66及SEQIDNO.67所示的ADD2引物对、序列如SEQIDNO.68及SEQIDNO.69所示的INA引物对、序列如SEQIDNO.70及SEQIDNO.71所示的POU4F1引物对、序列如SEQIDNO.72及SEQIDNO.73所示的HOXB4引物对、序列如SEQIDNO.74及SEQIDNO.75所示的SHOX2引物对、序列如SEQIDNO.76及SEQIDNO.77所示的PTGER4引物对、序列如SEQIDNO.78及SEQIDNO.79所示的THBD引物对、序列如SEQIDNO.80及SEQIDNO.81所示的SDC2引物对、序列如SEQIDNO.82及SEQIDNO.83所示的PAX1引物对、序列如SEQIDNO.84及SEQIDNO.85所示的SOX1引物对、序列如SEQIDNO.86及SEQIDNO.87所示的GHSR引物对、序列如SEQIDNO.88及SEQIDNO.89所示的JAM3引物对、序列如SEQIDNO.90及SEQIDNO.91所示的PRDM14引物对、序列如SEQIDNO.92及SEQIDNO.93所示的UCHL1引物对、序列如SEQIDNO.94及SEQIDNO.95所示的IRF4引物对、序列如SEQIDNO.96及SEQIDNO.97所示的ZNF583引物对、序列如SEQIDNO.98及SEQIDNO.99所示的FLT1引物对、序列如SEQIDNO.100及SEQIDNO.101所示的BOLL引物对、和序列如SEQIDNO.102及SEQIDNO.103所示的ACTB引物对。
在其中一个实施例中，所述核酸组合物产品还包括序列如SEQIDNO.104所示的内参探针、序列如SEQIDNO.105所示的检测探针、和序列如SEQIDNO.106及SEQIDNO.107所示的通用引物对，所述内参探针和所述检测探针上均连接有荧光基团，所述内参探针上的荧光基团与所述检测探针上的荧光基团不同。
在其中一个实施例中，所述核酸组合产品包括以下引物对中的至少两组：
序列如SEQIDNO.108及SEQIDNO.109所示的EGFR18引物对、序列如SEQIDNO.110及SEQIDNO.111所示的EGFR19引物对、序列如SEQIDNO.112及SEQIDNO.113所示的EGFR20引物对、序列如SEQIDNO.114及SEQIDNO.115所示的EGFR21引物对、序列如SEQIDNO.116及SEQIDNO.117所示的KRAS2引物对、序列如SEQIDNO.118及SEQIDNO.119所示的KRAS3引物对、序列如SEQIDNO.120及SEQIDNO.121所示的KRAS4引物对、序列如SEQIDNO.122及SEQIDNO.123所示的BRAF引物对、序列如SEQIDNO.124及SEQIDNO.125所示的PIK3CA引物对、和序列如SEQIDNO.126及SEQIDNO.127所示的PIK3CA引物对。
在其中一个实施例中，所述核酸组合产品中还包括序列如SEQIDNO.128及SEQIDNO.129所示的通用引物对。
一种检测试剂盒，包括上述的核酸组合产品。
一种扩增靶核酸的方法，所述方法包括以下步骤：
将模板、上述的核酸组合产品、聚合酶和缓冲剂混合，制成PCR反应液；及将所述PCR反应液进行PCR 反应。
附图说明
图1为一实施方式的核酸组合产品的工作原理示意图；
图2为实施例1的电泳结果图；
图3为实施例2中改进后的引物对的扩增曲线；
图4为实施例2中常规引物对的扩增曲线；
图5～图10为实施例3中的扩增曲线结果图；
图11为实施例4中的电泳结果图。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使本发明公开内容更加透彻全面。术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。需要说明的是，本文中的“正链”和“负链”是指PCR扩增过程中DNA模板的两条核酸单链，正链与负链互补配对，其中：将碱基序列与DNA模板所对应的mRNA一致(只是T和U的区别)的核酸单链定为正链；将序列与DNA模板所对应的mRNA互补的核酸单链定为负链。本文中的“上游环部”与“上游引物的环部”含义相同，均是指上游引物的茎环结构中的环部；“下游环部”与“下游引物的环部”含义相同，均是指下游引物的茎环结构中的环部。
本申请一实施方式提供了一种的核酸组合产品，该核酸组合产品包括至少一组引物对，引物对包括上游引物和下游引物；上游引物和下游引物均为包括环部和茎部的茎环结构，在退火时，上游引物的至少部分环部和至少部分3’端的核酸片段与模板的其中一条链杂交(例如负链)，下游引物的至少部分环部和至少部分3’端的核酸片段与模板的另一条链杂交(例如正链)，上游引物和下游引物的5’端的核酸片段均不与模板杂交。
请参阅图1，上述核酸组合产品中的引物对通过将上游引物和下游引物设计成茎环结构，上游引物和下游引物均是：在非变性(例如常温)条件下，5’端的核酸片段与3’端的核酸片段互补配对结合而形成茎部，位于5’端的核酸片段与3’端的核酸片段之间的核酸片段形成环部。在退火时，上游引物的环部和3’端的核酸片段与模板的其中一条链互补配对结合(杂交)，但上游引物的5’端的核酸片段不与模板杂交；同时下游引物的环部和3’端的核酸片段与模板的另一条杂交，但下游引物的5’端的核酸片段与模板不杂交；此时，没有与模板结合的其他引物则自身的5’端的核酸片段与3’端的核酸片段互补配对结合而再次形成茎环结构的引物，引物的5’端与3’端封闭，不容易形成引物二聚体。经验证，按照上述设计的引物的特异性好，不容易出现非特异性扩增。
具体地，上游引物呈茎环结构，上游引物包括上游茎部和与上游茎部连接的上游环部，上游引物的5’端的核酸片段与上游引物的3’端的核酸片段互补配对结合而形成上游茎部，上游环部位于上游引物的5’端的核酸片段与上游引物的3’端的核酸片段之间，在退火时，至少部分上游环部和至少部分上游引物的3’端的核酸片段与模板的其中一条链杂交，上游引物的5’端的核酸片段与该链不能互补配对；下游引物也呈茎环结构，下游引物包括下游茎部和与下游茎部连接的下游环部，下游引物的5’端的核酸片段与下游引物的3’端的核酸片段互补配对结合而形成下游茎部，下游环部位于下游引物的5’端的核酸片段与下游引物的3’端的核酸片段之间，在退火时，至少部分下游环部和至少部分下游引物的3’端的核酸片段与模板的另一条链杂交，下游引物的5’端的核酸片段与该链不能互补配对。
具体地，上游引物的5’端的核酸片段用于在非变性条件(例如常温)下与上游引物的3’端的核酸片段互补配对结合；上游引物的3’端的核酸片段用于在退火时与模板的其中一条链(例如负链)杂交，进而使得聚合酶能以该链为模板进行延伸。下游引物的5’端的核酸片段用于在非变性条件(例如常温)下与下游引物的3’端的核酸片段互补配对结合；下游引物的3’端的核酸片段用于在退火时与模板的另一条链(例如正链)杂交，进而使得聚合酶能以该链为模板进行延伸。下文以上游引物结合模板的负链，下游引物结合模板的正链为例进行说明。
在一些实施例中，在退火时，部分上游引物的3’端的核酸片段与模板的负链杂交。具体地，上游引物的 3’端的核酸片段包括上游延伸段和上游特异段，上游特异段位于上游环部和上游延伸段之间，上游延伸段能与模板中的负链杂交，上游特异段与负链不能互补配对结合。通过设置不能与负链杂交的上游特异段而使得上游引物的3’端与负链的互补配对结合依赖于上游延伸段，从而使得上游延伸段需要与负链完全互补配对后聚合酶才能启动延伸，因而提高了上游引物的特异性。可选地，上游延伸段的长度为3nt～20nt，上游特异段的长度为3nt～50nt。进一步地，上游延伸段的长度为6nt～15nt，上游特异段的长度为15nt～35nt。在另一些实施例中，在退火时，上游引物的3’端的核酸片段全部与负链杂交。
在一些实施例中，在退火时，部分下游引物的3’端的核酸片段与模板的正链杂交。具体地，下游引物的3’端的核酸片段包括下游延伸段和下游特异段，下游特异段位于下游环部和下游延伸段之间，下游延伸段能与正链杂交，下游特异段与正链不能互补配对结合。通过设置不能与正链杂交的下游特异段而使得下游引物的3’端与正链的互补配对结合依赖于下游延伸段，从而使得下游延伸段需要与正链完全互补配对后聚合酶才能启动延伸，因而提高了下游引物的特异性。可选地，下游延伸段的长度为3nt～20nt，下游特异段的长度为3nt～50nt。进一步地，下游延伸段的长度为6nt～15nt，下游特异段的长度为15nt～35nt。在另一些实施例中，在退火时，下游引物的3’端的核酸片段完全与负链杂交。
具体地，上游环部用于与模板的负链结合，利于上游引物的3’端的核酸片段与负链的结合。至少部分上游环部能与负链互补配对。在一些实施例中，上游环部的长度为10nt～100nt。进一步地，上游环部的长度为18nt～60nt。在其中一个实施例中，部分上游环部能与负链互补配对。具体地，在上游环部中，与负链互补配对的核酸片段的长度为10nt～50nt。可选地，在上游环部中，与负链互补配对的核酸片段的长度为15nt、20nt、30nt、40nt或45nt。在另一个实施例中，上游环部全部能与负链互补配对。
具体地，下游环部用于与模板的正链结合，利于下游引物的3’端的核酸片段与正链的结合。至少部分下游环部能与负链互补配对。在一些实施例中，下游环部的长度为10nt～100nt。进一步地，下游环部的长度为18nt～60nt。在其中一个实施例中，部分下游环部能与正链互补配对。具体地，在下游环部中，与正链互补配对的核酸片段的长度为10nt～50nt。可选地，在下游环部中，与正链互补配对的核酸片段的长度为15nt、20nt、30nt、40nt或45nt。在另一个实施例中，下游环部全部能与正链互补配对。
在一些实施例中，上游引物和/或下游引物还包括修饰部，修饰部位于相应引物的5’端的核酸片段与环部之间或相应引物的3’端的核酸片段与环部之间，在延伸时，所述修饰部能阻止聚合酶继续向前移动。
在其中一个实施例中，上游引物还包括上游修饰部，上游修饰部位于上游引物的5’端的核酸片段与上游环部之间；在延伸时，上游修饰部能阻止聚合酶继续向前移动。通过在上游引物的5’端的核酸片段与上游环部之间设置上游修饰部，使得在以含有上游引物的核酸单链作为模板时，DNA聚合酶移动到模板上的上游引物的5’端的核酸片段处就停止移动，延伸终止，避免延伸合成上游引物的5’端(也即是此时的负链的5’端)的核酸片段的互补链而出现非特异性扩增。需要说明的是，本文中的聚合酶是指DNA聚合酶；聚合酶的前进方向是从模板的5’端向3’端。
可选地，上游修饰部选自间臂(Spacer)及NTP中的至少一种。在其中一个实施例中，间臂为Spacer C3、Spacer C6、Spacer C9和Spacer C18中的至少一种，其中Spacer C3是指-CH 2CH 2CH 2-，Spacer C6是指6个-CH 2CH 2CH 2CH 2CH 2CH 2-，Spacer C9是指3个连续乙二醇(-CH 2CH 2O-)组成的linker链，Spacer C18是指6个连续乙二醇(-CH 2CH 2O-)组成的linker链。可以理解的是，上游修饰部的具体组成不限于上述，还可以是其他能够阻止聚合酶继续向上游引物的5’端移动的其他物质。
在其中一个实施例中，下游引物还包括下游修饰部，下游修饰部位于下游引物的5’端的核酸片段与下游环部之间，在延伸时，下游修饰部能阻止聚合酶继续向前移动。通过在下游引物的5’端的核酸片段与下游环部之间设置下游修饰部，使得在以含有下游引物的核酸单链作为模板时，DNA聚合酶移动到下游引物的5’端的核酸片段处就停止移动，延伸终止，避免延伸合成下游引物的5’端的核酸片段的互补链而出现非特异性扩增。
可选地，下游修饰部选自间臂(Spacer)及NTP中的至少一种。在其中一个实施例中，间臂为Spacer C3、Spacer C6、Spacer C9和Spacer C18中的至少一种，其中Spacer C3是指-CH 2CH 2CH 2-，Spacer C6是指-CH 2CH 2CH 2CH 2CH 2CH 2-，Spacer C9是指3个连续乙二醇(-CH 2CH 2O-)组成的linker链，Spacer C18是指6个连续乙二醇(-CH 2CH 2O-)组成的linker链。可以理解的是，下游修饰部的具体组成不限于上述，还可以是其他能够阻止聚合酶继续向下游引物的5’端移动的其他物质。
在一些实施例中，上游引物和/或下游引物还包括标签段，标签段位于相应引物的5’端的核酸片段与环部 之间或相应引物的3’端的核酸片段与环部之间。
在其中一个实施例中，上游引物还包括上游标签段，上游标签段位于上游引物的5’端的核酸片段与上游环部之间，上游标签段与负链不能互补配对结合。上游标签段用于向新合成的链中引入通用引物序列和分子标签等，便于后续构建测序文库。
在其中一个实施例中，下游引物还包括下游标签段，下游标签段位于下游引物的5’端的核酸片段与下游环部之间，下游标签段与正链不能互补配对结合。下游标签段用于向新合成的链中引入通用引物序列和分子标签等，便于后续构建测序文库。
在一些实施例中，上游引物连接有荧光基团。在一个可选地具体示例中，上游引物的5’端连接有荧光基团，上游引物的3’端连接有与荧光基团对应的淬灭基团。通过在上游引物上连接荧光基团，则通过荧光强度可以表征扩增程度。可以理解的是，荧光基团和淬灭基团没有特别限制。
在一些实施例中，下游引物连接有荧光基团。在一个可选地具体示例中，下游引物的5’端连接有荧光基团，下游引物的3’端连接有与荧光基团对应的淬灭基团。通过在下游引物上连接荧光基团，则通过荧光强度可以表征扩增程度。可以理解的是，荧光基团和淬灭基团没有特别限制。
在一些实施例中，核酸组合产品中包括至少一条荧光探针。在一个可选地具体示例中，荧光探针的5’端连接有荧光基团，荧光探针的3’端连接有与荧光基团对应的淬灭基团。通过在扩增过程中酶切降解探针使其发出荧光，则通过荧光强度可以表征扩增程度。可以理解的是，荧光基团和淬灭基团没有特别限制。
在其中一个实施例中，核酸组合产品中包括至少一组通用引物对。通用引物对能与模板互补配对结合，用于测序。
在其中一个实施例中，上游引物和下游引物中的至少一个为茎环形型引物。进一步地，上游引物和下游引物中的至少一个为分子信标型引物。
在一些实施例中，核酸组合产品中包括至少两组引物对，不同的引物对用于扩增不同的核酸片段。具体地，各引物对的结构均如上文描述，各引物对的差别在于，不同引物对针对的靶区域不同。在一些实施例中，不同引物对以混合物的形式包装。在另一些实施例中，不同引物对独立包装。
在其中一个实施例中，上述核酸组合产品为用于检测HPV病毒的产品。具体地，核酸组合产品包括以下引物对中的至少两组：序列如SEQ ID NO.1及SEQ ID NO.2所示的HPV16引物对、序列如SEQ ID NO.3及SEQ ID NO.4所示的HPV18引物对、序列如SEQ ID NO.5及SEQ ID NO.6所示的HPV31引物对、序列如SEQ ID NO.7及SEQ ID NO.8所示的HPV33引物对、序列如SEQ ID NO.9及SEQ ID NO.10所示的HPV35引物对、序列如SEQ ID NO.11及SEQ ID NO.12所示的HPV39引物对、序列如SEQ ID NO.13及SEQ ID NO.14所示的HPV45引物对、序列如SEQ ID NO.15及SEQ ID NO.16所示的HPV51引物对、序列如SEQ ID NO.17及SEQ ID NO.18所示的HPV52引物对、序列如SEQ ID NO.19及SEQ ID NO.20所示的HPV53引物对、序列如SEQ ID NO.21及SEQ ID NO.22所示的HPV56引物对、序列如SEQ ID NO.23及SEQ ID NO.24所示的HPV58引物对、序列如SEQ ID NO.25及SEQ ID NO.26所示的HPV59引物对和序列如SEQ ID NO.27及SEQ ID NO.28所示的HPV66引物对。
在其中一个实施例中，上述核酸组合产品为用于检测肿瘤基因甲基化的产品。具体地，核酸组合产品包括以下引物对中的至少两组：序列如SEQ ID NO.62及SEQ ID NO.63所示的SEPT9引物对、序列如SEQ ID NO.64及SEQ ID NO.65所示的VWC2引物对、序列如SEQ ID NO.66及SEQ ID NO.67所示的ADD2引物对、序列如SEQ ID NO.68及SEQ ID NO.69所示的INA引物对、序列如SEQ ID NO.70及SEQ ID NO.71所示的POU4F1引物对、序列如SEQ ID NO.72及SEQ ID NO.73所示的HOXB4引物对、序列如SEQ ID NO.74及SEQ ID NO.75所示的SHOX2引物对、序列如SEQ ID NO.76及SEQ ID NO.77所示的PTGER4引物对、序列如SEQ ID NO.78及SEQ ID NO.79所示的THBD引物对、序列如SEQ ID NO.80及SEQ ID NO.81所示的SDC2引物对、序列如SEQ ID NO.82及SEQ ID NO.83所示的PAX1引物对、序列如SEQ ID NO.84及SEQ ID NO.85所示的SOX1引物对、序列如SEQ ID NO.86及SEQ ID NO.87所示的GHSR引物对、序列如SEQ ID NO.88及SEQ ID NO.89所示的JAM3引物对、序列如SEQ ID NO.90及SEQ ID NO.91所示的PRDM14引物对、序列如SEQ ID NO.92及SEQ ID NO.93所示的UCHL1引物对、序列如SEQ ID NO.94及SEQ ID NO.95所示的IRF4引物对、序列如SEQ ID NO.96及SEQ ID NO.97所示的ZNF583引物对、序列如SEQ ID NO.98及SEQ ID NO.99所示的FLT1引物对、序列如SEQ ID NO.100及SEQ ID NO.101所示的BOLL引物对、和序列如SEQ ID NO.102及SEQ ID NO.103所示的ACTB引物对。
进一步地，用于检测肿瘤基因甲基化的核酸组合物产品还包括序列如SEQ ID NO.104所示的内参探针、序列如SEQ ID NO.105所示的检测探针、和序列如SEQ ID NO.106及SEQ ID NO.107所示的通用引物对。其中，内参探针和检测探针上均连接有荧光基团，内参探针上的荧光基团与及检测探针上的荧光基团不同。
在其中一个实施例中，上述核酸组合产品为用于检测肿瘤基因突变的产品。具体地，核酸组合产品包括以下引物对中的至少两组：序列如SEQ ID NO.108及SEQ ID NO.109所示的EGFR18引物对、序列如SEQ ID NO.110及SEQ ID NO.111所示的EGFR19引物对、序列如SEQ ID NO.112及SEQ ID NO.113所示的EGFR20引物对、序列如SEQ ID NO.114及SEQ ID NO.115所示的EGFR21引物对、序列如SEQ ID NO.116及SEQ ID NO.117所示的KRAS2引物对、序列如SEQ ID NO.118及SEQ ID NO.119所示的KRAS3引物对、序列如SEQ ID NO.120及SEQ ID NO.121所示的KRAS4引物对、序列如SEQ ID NO.122及SEQ ID NO.123所示的BRAF引物对、序列如SEQ ID NO.124及SEQ ID NO.125所示的PIK3CA引物对、和序列如SEQ ID NO.126及SEQ ID NO.127所示的PIK3CA引物对。
进一步地，用于检测肿瘤基因突变的核酸组合产品还包括序列如SEQ ID NO.128及SEQ ID NO.129所示的通用引物对。
在一些实施例中，核酸组合产品中的引物对为一组。在其中一个实施例中，引物对用于扩增人ACTB基因组上的一段序列。在一个具体的示例中，核酸组合产品的引物对如SEQ ID NO.29及SEQ ID NO.30所示。
此外，本申请一实施方式还提供了一种检测试剂盒，包括上述任一实施例的核酸组合产品。
可选地，上述检测试剂盒还包括聚合酶、缓冲剂和用于测序的通用引物中的至少一种。
具体地，聚合酶用于在PCR时在引物的3’端进行延伸。缓冲剂为PCR反应提供缓冲体系。通用引物用于扩增产物制备测序文库。可以理解的是，在上述核酸组合产品中含有用于测序的通用引物对时，上述检测试剂盒中的通用引物可以省略。
上述检测试剂盒包括上述核酸组合产品，上述核酸组合产品的引物对从引物的结构上解决了引物间容易互补配对而形成二聚体的问题，在上述核酸组合产品用于多重PCR检测时，引物间不容易出现二聚体，不容易出现非特异性扩增，可以改善多重PCR的非特异性扩增。
此外，本申请一实施方式还提供了一种扩增靶核酸的方法，该方法包括以下步骤：将模板、上述任一实施例的核酸组合产品、聚合酶和缓冲剂混合，制成PCR反应液；及将PCR反应液进行PCR反应。
具体地，该扩增靶核酸的方法可以作为制备文库前的核酸片段的扩增。在其中一个实施例中，扩增靶核酸的方法用于构建测序文库。
在其中一个实施例中，PCR反应的条件为：预变性：90℃～98℃1min～10min；变性：90℃～98℃2s～60s；退火：48℃～68℃10s～120min；延伸：68℃～75℃10s～10min；循环数：2～60。
上述扩增靶核酸的方法采用了上述核酸组合产品，特异性好，不容易出现非特异性扩增。
具体实施例
以下结合具体实施例进行详细说明。以下实施例如未特殊说明，则不包括除不可避免的杂质外的其他组分。实施例中采用试剂和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，按照常规条件，例如文献、书本中所述的条件或者生产厂家推荐的方法实现。在以下实施例中，引物中的“Spacer”均为Spacer C9。
实施例1人ACTB基因的扩增
分别采用常规PCR引物对与改进后的引物对扩增人ACTB基因组一段序列(即靶核酸)，以验证改进后的引物对的良好扩增性。靶核酸的碱基序列如SEQ ID NO.31所示。具体验证步骤包括：
(1)引物设计：
A：常规引物对：F：5’-TCCTTCCTGGGCATGGAG-3’(SEQ ID NO.32)；R：
5’-CATTGTGCTGGGTGCCAG-3’(SEQ ID NO.33)；
B：改进后的引物对，有下划线部分为与模板杂交的序列：F：
5’-ACAGGACACA TCCTTCCTGGGCATGGAGAACCA TCCTGT-3’(SEQ ID NO.29)；R：
5’-CACTGCTCTC CATTGTGCTGGGTGCCAGACACC GCAGTG-3’(SEQ ID NO.30)。
(2)PCR反应液配制：
按照表1进行分别配制常规引物对和改进引物对的PCR反应体系(体系总体积为25μL)：
表1
组分 体积 组分 体积
热启动Taq酶 0.5μL dNTP(25mM) 0.5μL
Primer F(10μmol) 1μL 10×Buffer 2.5μL
Primer R(10μmol) 1μL 处理后的DNA样品 10μL
超纯水 9.5μL    
(3)PCR扩增：涡旋混匀后瞬时离心，在PCR仪中按表2设定反应程序进行PCR反应：
表2
Figure PCTCN2022135802-appb-000001
(4)结果及分析：
PCR结束后，将PCR产物进行电泳检测，结果如图2所示。在图2中，泳道1为maker，泳道2、3和4为改进后的引物对对应的扩增产物的电泳结果，泳道5、6和7为常规引物对对应的扩增产物的电泳结果。由图2可知，改进后的引物对对应的扩增产物的电泳条带单一，而常规引物对对应的扩增产物的电泳条带呈弥散状。这说明采用常规引物对扩增时会产生很多非特异性产物，特别是引物二聚体，而改进后的引物对利用自身形成茎环结构，降低了非特异性扩增，所扩增的产物较为单一，无引物二聚体。
实施例2多重PCR扩增14种高危型HPV病毒基因
分别采用常规PCR引物与改进后的PCR引物，在一个PCR反应管中扩增14种高危型人乳头瘤病毒基因组的L1区片段，以验证本发明实施例引物的良好扩增性。具体验证步骤包括：
(1)引物设计：
A：常规引物对如表3所示。
表3
Figure PCTCN2022135802-appb-000002
B：改进后的引物对，有下划线部分为与模板杂交序列：
表4
Figure PCTCN2022135802-appb-000003
(2)PCR反应液配制：
按照表5进行分别配制常规引物对和改进引物对的PCR反应体系(体系总体积为25μL)：
表5
组分 体积 组分 体积
热启动Taq酶(5U/μL) 0.5μL HPV16F(10μmol) 0.35μL
dNTP(25mM) 0.5μL HPV16R(10μmol) 0.35μL
10×Buffer 2.5μL HPV18F(10μmol) 0.35μL
10×Evagreen 2.5μL HPV18R(10μmol) 0.35μL
HPV52F(10μmol) 0.35μL HPV31F(10μmol) 0.35μL
HPV52R(10μmol) 0.35μL HPV31R(10μmol) 0.35μL
HPV53F(10μmol) 0.35μL HPV33F(10μmol) 0.35μL
HPV53R(10μmol) 0.35μL HPV33R(10μmol) 0.35μL
HPV56F(10μmol) 0.35μL HPV|35F(10μmol) 0.35μL
HPV56R(10μmol) 0.35μL HPV35R(10μmol) 0.35μL
HPV58F(10μmol) 0.35μL HPV39F(10μmol) 0.35μL
HPV58R(10μmol) 0.35μL HPV39R(10μmol) 0.35μL
HPV59F(10μmol) 0.35μL HPV45F(10μmol) 0.35μL
HPV59R(10μmol) 0.35μL HPV45R(10μmol) 0.35μL
HPV66F(10μmol) 0.35μL HPV51F(10μmol) 0.35μL
HPV66R(10μmol) 0.35μL HPV51R(10μmol) 0.35μL
HPV DNA样品 9.2μL    
(3)PCR扩增：涡旋混匀后瞬时离心，在实时荧光定量PCR仪中按表6设定反应程序进行PCR反应：
表6
Figure PCTCN2022135802-appb-000004
(4)结果及分析：
PCR结束后，获得扩增曲线，扩增曲线如图3和图4所示。图3为改进后的引物对的扩增曲线，图4为常规引物对的扩增曲线。
由图3和图4可知，改进后的引物对对纯水(无模板样本)和阴性样本(无HPV DNA样本)无非特异扩增曲线，阳性样本(含HPV DNA样本)扩增曲线良好，而常规设计引物对对纯水(无模板样本)和阴性样本(无HPVDNA样本)有非特异扩增曲线，说明常规引物对扩增时有引物二聚体存在。
(5)提高PCR退火温度进行扩增验证：
按照表5分别配制常规引物对和改进引物对的PCR反应体系，涡旋混匀后瞬时离心，在实时荧光定量PCR仪中按表7设定反应程序进行PCR反应：
表7
Figure PCTCN2022135802-appb-000005
结果：提高退火温度后，常规设计引物对对纯水(无模板样本)和阴性样本(无HPV DNA样本)仍有非特异扩增曲线，改变不明显，常规引物对和改进后的引物对的Ct值如下表8所示。
表8
Figure PCTCN2022135802-appb-000006
实施例3肿瘤基因甲基化多重PCR检测
选择在肿瘤样本中呈高度甲基化的基因，利用本发明方法设计引物，在一个PCR反应管中扩增多个肿瘤基因甲基化片段，以实现高灵敏度检测肿瘤基因甲基化。具体步骤包括：
(1)引物设计：
有下划线部分为与模板杂交序列：
表9
Figure PCTCN2022135802-appb-000007
Figure PCTCN2022135802-appb-000008
(2)PCR反应液配制：
按照表10配制PCR反应体系(体系总体积25μL)：
表10
组分 体积 组分 体积
热启动Taq酶(5U/μL) 0.5μL SDC2F(2μmol) 0.1μL
dNTP(25mM) 0.5μL SDC2R(2μmol) 0.1μL
10×Buffer 2.5μL PAX1F(2μmol) 0.1μL
甲基探针(10μmol) 0.5μL PAX1R(2μmol) 0.1μL
通用F(10μmol) 1μL SOX1F(2μmol) 0.1μL
通用R(10μmol) 1μL SOX1R(2μmol) 0.1μL
超纯水 4.3μL GHSRF(2μmol) 0.1μL
样本DNA 10μL GHSRR(2μmol) 0.1μL
SEPT9F(2μmol) 0.1μL JAM3F(2μmol) 0.1μL
SEPT9R(2μmol) 0.1μL JAM3R(2μmol) 0.1μL
VWC2F(2μmol) 0.1μL PRDM14F(2μmol) 0.1μL
VWC2R(2μmol) 0.1μL PRDM14R(2μmol) 0.1μL
ADD2F(2μmol) 0.1μL UCHL1F(2μmol) 0.1μL
ADD2R(2μmol) 0.1μL UCHL1R(2μmol) 0.1μL
INAF(2μmol) 0.1μL IRF4F(2μmol) 0.1μL
INAR(2μmol) 0.1μL IRF4R(2μmol) 0.1μL
POU4F1F(2μmol) 0.1μL ZNF583F(2μmol) 0.1μL
POU4F1R(2μmol) 0.1μL ZNF583R(2μmol) 0.1μL
HOXB4F(2μmol) 0.1μL FLT1F(2μmol) 0.1μL
HOXB4R(2μmol) 0.1μL FLT1R(2μmol) 0.1μL
SHOX2F(2μmol) 0.1μL BOLLF(2μmol) 0.1μL
SHOX2R(2μmol) 0.1μL BOLLR(2μmol) 0.1μL
PTGER4F(2μmol) 0.1μL ACTBF(2μmol) 0.1μL
PTGER4R(2μmol) 0.1μL ACTBR(2μmol) 0.1μL
THBDF(2μmol) 0.1μL ACTB-P(10μmol) 0.5μL
THBDR(2μmol) 0.1μL    
(3)PCR扩增：涡旋混匀后瞬时离心，在实时荧光定量PCR仪中按表11设定反应程序进行PCR反应：
表11
Figure PCTCN2022135802-appb-000009
(4)结果及分析：
PCR结束后，获得扩增曲线，扩增曲线如图5～图10所示。
由图5～图10可知，PCR反应体系对阳性样本(肿瘤组织样本)扩增曲线良好，对纯水(无模板样本)和阴性样本(健康组织样本)无非特异扩增曲线。样本Ct值如下表12所示。
表12
样本类型 FAM荧光通道的Ct值 HEX荧光通道的Ct值
肿瘤样本1 30.17 30.35
肿瘤样本1 30.14 30.31
肿瘤样本2 30.85 31.28
肿瘤样本2 30.77 31.22
肿瘤样本3 29.65 30.61
肿瘤样本3 29.58 30.54
健康样本1 / 27.55
健康样本1 / 27.62
健康样本2 / 28.04
健康样本2 / 28.12
纯水 / /
纯水 / /
实施例4利用多重PCR制备肿瘤基因突变检测的测序文库
选择与肿瘤个体化用药高度相关的基因，利用本发明方法设计引物，在一个PCR反应管中扩增多个基因片段，以实现高灵敏度检测肿瘤基因突变。具体步骤包括：
(1)引物设计：
有下划线部分为与模板杂交序列：
表13
Figure PCTCN2022135802-appb-000010
(2)PCR反应液配制：
按照表14进行配制PCR反应体系(体系总体积25μL)：
表14
组分 体积 组分 体积
2×Phusion PCR Master Mix 12.5μL KR3F(2μmol) 0.1μL
ER18F(2μmol) 0.1μL KR3R(2μmol) 0.1μL
ER18R(2μmol) 0.1μL KR4F(2μmol) 0.1μL
ER19F(2μmol) 0.1μL KR4R(2μmol) 0.1μL
ER19R(2μmol) 0.1μL BRF(2μmol) 0.1μ1L
ER20F(2μmol) 0.1μL BRR(2μmol) 0.1μL
ER20R(2μmol) 0.1μL PK1F(2μmol) 0.1μL
ER21F(2μmol) 0.1μL PK1R(2μmol) 0.1μL
ER21R(2μmol) 0.1μL PK2F(2μmol) 0.1μL
KR2F(2μmol) 0.1μL PK2R(2μmol) 0.1μL
KR2R(2μmol) 0.1μL P5(10μmol) 1μL
样本DNA 8.5μL P7(10μmol) 1μL
(3)PCR扩增：涡旋混匀后瞬时离心，在PCR仪中按表15设定反应程序进行PCR反应：
表15
Figure PCTCN2022135802-appb-000011
(4)纯化和检测：
使用浓度为2％的琼脂糖进行电泳，120伏电泳1个小时，切胶回收200～300bp范围，使用凝胶回收试剂盒(Qiagen)进行回收，使用40μl无核酸酶水进行洗脱。进行Qubit(荧光定量仪)定量，将文库用无核酸酶水稀释到2ng/μl备用。
用Aglilent 2100生物分析仪对文库插入片段大小进行检测，用Q-PCR仪对文库浓度进行检测；检测结果如图11所示。
从图11可以看出，本实施例所构建的文库的大小在250bp左右，去掉两端的接头序列116bp，插入片段的大小在130bp左右，符合双端测序要求的插入片段大小。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，便于具体和详细地理解本发明的技术方案，但并不能因此而理解为对发明专利保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。应当理解的是，在本领域技术人员在本发明提供的技术方案的基础上，通过合乎逻辑的分析、推理或有限的试验得到的技术方案，均在本发明所附权利要求的保护范围内。因此，本发明专利的保护范围应以所附权利要求的内容为准，说明书及附图可以用于解释权利要求的内容。

Claims (16)

  1. 一种核酸组合产品，其特征在于，包括至少一组引物对，所述引物对包括上游引物和下游引物；所述上游引物和所述下游引物均为包括环部和茎部的茎环结构，在退火时，所述上游引物的至少部分环部和至少部分3’端的核酸片段与模板的其中一条链杂交，所述下游引物的至少部分环部和至少部分3’端的核酸片段与所述模板的另一条链杂交，所述上游引物和所述下游引物的5’端的核酸片段均不与所述模板杂交。
  2. 根据权利要求1所述的核酸组合产品，其特征在于，所述上游引物和/或所述下游引物还包括修饰部，所述修饰部位于相应引物的5’端的核酸片段与环部之间或相应引物的3’端的核酸片段与环部之间，在延伸时，所述修饰部能阻止聚合酶继续向前移动。
  3. 根据权利要求1或2所述的核酸组合产品，其特征在于，所述修饰部选自间臂及NTP中的至少一种。
  4. 根据权利要求1～3任一项所述的核酸组合产品，其特征在于，所述上游引物的3’端的核酸片段包括上游延伸段和上游特异段，所述上游特异段位于所述上游环部和所述上游延伸段之间，所述上游延伸段能与所述模板的其中一条链杂交，所述上游特异段与所述模板不能互补配对结合；
    及/或，所述下游引物的3’端的核酸片段包括下游延伸段和下游特异段，所述下游特异段位于所述下游环部和所述下游延伸段之间，所述下游延伸段能与所述模板的另一条链杂交，所述下游特异段与所述模板不能互补配对结合。
  5. 根据权利要求4所述的核酸组合产品，其特征在于，所述上游延伸段的长度为3nt～20nt，所述上游特异段的长度为3nt～50nt；所述下游延伸段的长度为3nt～20nt，所述下游特异段的长度为3nt～50nt。
  6. 根据权利要求1～4任一项所述的核酸组合产品，其特征在于，所述上游引物和/或所述下游引物还包括标签段，所述标签段位于相应引物的5’端的核酸片段与环部之间或相应引物的3’端的核酸片段与环部之间。
  7. 根据权利要求1～4或6任一项所述的核酸组合产品，其特征在于，所述上游引物的环部和所述下游引物的环部的长度分别独立地为10nt～100nt。
  8. 根据权利要求1～7任一项所述的核酸组合产品，其特征在于，所述核酸组合产品中还包括至少一条荧光探针，所述荧光探针的5’端连接有荧光基团，荧光探针的3’端连接有与荧光基团对应的淬灭基团。
  9. 根据权利要求1～8任一项所述的核酸组合产品，其特征在于，所述核酸组合产品中还包括至少一组通用引物对，所述通用引物对能与所述模板互补配对结合。
  10. 根据权利要求1～9任一项所述的核酸组合产品，其特征在于，所述核酸组合产品包括以下引物对中的至少两组：
    序列如SEQ ID NO.1及SEQ ID NO.2所示的HPV16引物对、序列如SEQ ID NO.3及SEQ ID NO.4所示的HPV18引物对、序列如SEQ ID NO.5及SEQ ID NO.6所示的HPV31引物对、序列如SEQ ID NO.7及SEQ ID NO.8所示的HPV33引物对、序列如SEQ ID NO.9及SEQ ID NO.10所示的HPV35引物对、序列如SEQ ID NO.11及SEQ ID NO.12所示的HPV39引物对、序列如SEQ ID NO.13及SEQ ID NO.14所示的HPV45引物对、序列如SEQ ID NO.15及SEQ ID NO.16所示的HPV51引物对、序列如SEQ ID NO.17及SEQ ID NO.18所示的HPV52引物对、序列如SEQ ID NO.19及SEQ ID NO.20所示的HPV53引物对、序列如SEQ ID NO.21及SEQ ID NO.22所示的HPV56引物对、序列如SEQ ID NO.23及SEQ ID NO.24所示的HPV58引物对、序列如SEQ ID NO.25及SEQ ID NO.26所示的HPV59引物对和序列如SEQ ID NO.27及SEQ ID NO.28所示的HPV66引物对。
  11. 根据权利要求1～10任一项所述的核酸组合产品，其特征在于，所述核酸组合产品包括以下引物对中的至少两组：
    序列如SEQ ID NO.62及SEQ ID NO.63所示的SEPT9引物对、序列如SEQ ID NO.64及SEQ ID NO.65所示的VWC2引物对、序列如SEQ ID NO.66及SEQ ID NO.67所示的ADD2引物对、序列如SEQ ID NO.68及SEQ ID NO.69所示的INA引物对、序列如SEQ ID NO.70及SEQ ID NO.71所示的POU4F1引物对、序列如SEQ ID NO.72及SEQ ID NO.73所示的HOXB4引物对、序列如SEQ ID NO.74及SEQ ID NO.75所示的SHOX2引物对、序列如SEQ ID NO.76及SEQ ID NO.77所示的PTGER4引物对、序列如SEQ ID NO.78及SEQ ID NO.79所示的THBD引物对、序列如SEQ ID NO.80及SEQ ID NO.81所示的SDC2引物对、序列如SEQ ID NO.82及SEQ ID NO.83所示的PAX1引物对、序列如SEQ ID NO.84及SEQ ID NO.85所示的SOX1引物对、序列如SEQ ID NO.86及SEQ ID NO.87所示的GHSR引物对、序列如SEQ ID NO.88 及SEQ ID NO.89所示的JAM3引物对、序列如SEQ ID NO.90及SEQ ID NO.91所示的PRDM14引物对、序列如SEQ ID NO.92及SEQ ID NO.93所示的UCHL1引物对、序列如SEQ ID NO.94及SEQ ID NO.95所示的IRF4引物对、序列如SEQ ID NO.96及SEQ ID NO.97所示的ZNF583引物对、序列如SEQ ID NO.98及SEQ ID NO.99所示的FLT1引物对、序列如SEQ ID NO.100及SEQ ID NO.101所示的BOLL引物对、和序列如SEQ ID NO.102及SEQ ID NO.103所示的ACTB引物对。
  12. 根据权利要求1～11任一项所述的核酸组合产品，其特征在于，所述核酸组合产品还包括序列如SEQ ID NO.104所示的内参探针、序列如SEQ ID NO.105所示的检测探针、和序列如SEQ ID NO.106及SEQ ID NO.107所示的通用引物对，所述内参探针和所述检测探针上均连接有荧光基团，所述内参探针上的荧光基团与所述检测探针上的荧光基团不同。
  13. 根据权利要求1～12任一项所述的核酸组合产品，其特征在于，所述核酸组合产品包括以下引物对中的至少两组：
    序列如SEQ ID NO.108及SEQ ID NO.109所示的EGFR18引物对、序列如SEQ ID NO.110及SEQ ID NO.111所示的EGFR19引物对、序列如SEQ ID NO.112及SEQ ID NO.113所示的EGFR20引物对、序列如SEQ ID NO.114及SEQ ID NO.115所示的EGFR21引物对、序列如SEQ ID NO.116及SEQ ID NO.117所示的KRAS2引物对、序列如SEQ ID NO.118及SEQ ID NO.119所示的KRAS3引物对、序列如SEQ ID NO.120及SEQ ID NO.121所示的KRAS4引物对、序列如SEQ ID NO.122及SEQ ID NO.123所示的BRAF引物对、序列如SEQ ID NO.124及SEQ ID NO.125所示的PIK3CA引物对、和序列如SEQ ID NO.126及SEQ ID NO.127所示的PIK3CA引物对。
  14. 根据权利要求1～13任一项所述的核酸组合产品，其特征在于，所述核酸组合产品中还包括序列如SEQ ID NO.128及SEQ ID NO.129所示的通用引物对。
  15. 一种检测试剂盒，其特征在于，包括权利要求1～14任一项所述的核酸组合产品。
  16. 一种扩增靶核酸的方法，其特征在于，所述方法包括以下步骤：
    将模板、权利要求1～14任一项所述的核酸组合产品、聚合酶和缓冲剂混合，制成PCR反应液；及将所述PCR反应液进行PCR反应。
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