WO2022141230A1 - 一种基于纳米金颗粒的多重核酸检测的方法 - Google Patents

一种基于纳米金颗粒的多重核酸检测的方法 Download PDF

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WO2022141230A1
WO2022141230A1 PCT/CN2020/141533 CN2020141533W WO2022141230A1 WO 2022141230 A1 WO2022141230 A1 WO 2022141230A1 CN 2020141533 W CN2020141533 W CN 2020141533W WO 2022141230 A1 WO2022141230 A1 WO 2022141230A1
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nucleic acid
primer
target nucleic
gold
nano
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French (fr)
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童贻刚
贺育敢
米志强
秦思
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北京化工大学
中国人民解放军军事科学院军事医学研究院
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/686—Polymerase chain reaction [PCR]

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  • the invention belongs to the field of biotechnology, and in particular relates to a method for multiple nucleic acid detection based on nano-gold particles; in particular, using nano-gold particles as a carrier to carry out bridge PCR amplification on the surface thereof, combined with test strips containing specific probes , multiplex nucleic acid detection by DNA molecular hybridization.
  • the multiplex nucleic acid detection technology itself has certain limitations: first, the real-time fluorescence quantitative PCR technology has limited ability to detect multiple targets, expensive instruments, and high cost of using gene chips; secondly, there are few choices of mature multiplex detection equipment, and practical applications greatly restricted. To this end, it is of great practical significance to develop a rapid, accurate, high-throughput, and widely applicable multiplex nucleic acid detection technology, especially in the screening of known pathogens in the early stage of outbreaks of infectious diseases with similar infection symptoms.
  • the purpose of the present invention is rapid, accurate and high-throughput multiplex detection of nucleic acids.
  • the present invention first protects the method of multiplex nucleic acid detection.
  • the method for multiple nucleic acid detection protected by the present invention is specifically method one, comprising the following steps:
  • Each kit includes upstream primers, downstream primers and probes;
  • the upstream primer sequentially includes linker1, 1 U and DNA fragment 1 from the 5' end to the 3' end;
  • the downstream primer sequentially includes linker2, 1 U and DNA fragment 2 from the 5' end to the 3' end;
  • DNA fragment 1 and DNA fragment 2 are identical or complementary to the two segments on the target nucleic acid, respectively;
  • the probe can be a single-stranded DNA molecule composed of 20-40 (such as 20-30, 30-40, 20, 30 or 40) nucleotides, and DNA fragment 1 and DNA fragment 2 are in the target nucleic acid.
  • the partial segment of the target sequence is identical or complementary;
  • the target sequences of DNA fragment 1 and DNA fragment 2 in each kit of reagents do not cross-react with probes in other kits of reagents;
  • the primer@nano-gold is where the upstream primer and the downstream primer are immobilized on the surface of the nano-gold particles in the nano-gold solution to form ;
  • step (3) using all primers prepared in step (3)@nano gold to perform bridge PCR amplification on the sample to be tested, and then digest with USER enzyme and denature to obtain a denatured product;
  • step (2) spotting all the probes prepared in step (2) on a nitrocellulose membrane respectively to form detection points separated from each other to obtain test strips;
  • step (6) Use the test strip obtained in step (5) to detect the denatured product obtained in step (4), and determine what kind of target nucleic acid the sample to be tested contains according to whether the detection point on the test strip shows red spots.
  • the steps of preparing the nano-gold solution can be as follows:
  • (1-1) Take an aqueous solution of HAuCl 4 and treat at 90-97°C (eg 90-95°C, 95-97°C, 90°C, 95°C or 97°C) for more than 3 minutes (eg 3min, 5min);
  • the concentration of the HAuCl 4 aqueous solution may be 0.005-0.015% (eg 0.005-0.010%, 0.010-0.015%, 0.005%, 0.010% or 0.015%) (m/v).
  • the concentration of the sodium citrate aqueous solution may be 0.5-1.5% (eg 0.5-1.0%, 1.0-1.5%, 0.5%, 1.0% or 1.5%).
  • the volume ratio of the HAuCl 4 aqueous solution and the sodium citrate aqueous solution can be 100 mL: (2-3) mL (such as 100 mL: 2 mL, 100 mL: 2.5 mL, 100 mL: 3 mL, 100 mL: (2-2.5 mL). ) mL, 100 mL: (2.5-3) mL).
  • preparing a complete set of reagents according to the nucleotide sequence of each target nucleic acid may be to prepare a complete set of reagents according to the nucleotide sequence of each target nucleic acid conserved region.
  • a kit of reagents is designed and synthesized based on the nucleotide sequence of each target nucleic acid conserved region.
  • each reagent kit consists of the upstream primer, the downstream primer and the probe.
  • any of the above-mentioned upstream primers sequentially includes linker1, 1 U and DNA fragment 1 from the 5' end to the 3' end.
  • any of the above-mentioned downstream primers include linker2, 1 U and DNA fragment 2 in sequence from the 5' end to the 3' end.
  • the DNA fragment 1 and the DNA fragment 2 are respectively identical or complementary to the two segments on the target nucleic acid.
  • the probe can be a single-stranded DNA molecule composed of 20-40 (eg, 20-30, 30-40, 20, 30 or 40) nucleotides, which is in the presence of DNA fragment 1 and DNA fragment 2. Partial segments of the target sequence of the nucleic acid of interest are identical or complementary.
  • the target sequences of DNA fragment 1 and DNA fragment 2 in each kit of reagents do not cross-react with probes in other kits.
  • the 5' ends of the upstream primer, the downstream primer and the probe can all be modified.
  • the 5' ends of the upstream primer and the downstream primer can be modified by thiol group, so that the primers can be stably fixed to the surface of the gold nanoparticles.
  • the 5' end of the probe can be modified by Biotin, so that the probe can be stably combined with the nitrocellulose membrane.
  • the linker1 or the linker2 can be a single-stranded DNA molecule composed of 10-20 (such as 10-15, 15-20, 10, 15 or 20) nucleotides for connecting sulfhydryl groups and PCR
  • the primers (DNA fragment 1 or DNA fragment 2) can better extend the primers on the surface of the gold nanoparticles to facilitate subsequent PCR amplification.
  • Linker1 or linker2 includes DNA fragment A and DNA fragment B in order from the 5' end to the 3' end.
  • DNA fragment A may consist of 8-10 (eg, 8, 9 or 10) Ts.
  • DNA fragment B may consist of 6-10 (eg 6-8, 8-10, 6, 8 or 10) nucleotides, each nucleotide being A or T.
  • the linker1 and the linker2 may be the same or different.
  • any of the above-mentioned upstream primers are sequentially composed of linker1, 1 U and DNA fragment 1 from the 5' end to the 3' end.
  • any of the above-mentioned downstream primers are sequentially composed of linker2, 1 U and DNA fragment 2 from the 5' end to the 3' end.
  • step (3) the steps of preparing each primer@nano gold are as follows:
  • (3-1) Mix the nano-gold solution and the Tween80 solution, and let stand for more than 20min (such as 20min, 20min, 30min or 40min);
  • each primer @ nanogold prepared is also completely different.
  • the concentration of the Tween80 solution can be 8-12% (v/v) (such as 8-10% (v/v), 10-12% (v/v), 8% ( v/v), 10% (v/v) or 12% (v/v)).
  • the nano-gold solution can be nano-gold concentrate.
  • the nano-gold concentrate can be obtained by centrifuging any of the above-mentioned nano-gold solutions by 2-4 times (eg, 2 times, 3 times or 4 times).
  • the PBS buffer may be a pH 8.0, 1 mM PBS buffer.
  • the step of preparing each primer@nano-gold also includes step (3-3) washing with pH8.0, 1mM PBS buffer 3 times, then with nuclease-free water for 2 times, and finally dispersing in nuclease-free water. Water is the primer@nano gold.
  • step (4) all primers@gold nanoparticles are subjected to bridge PCR amplification, enzyme digestion and denaturation in the same reaction system.
  • the denaturation may be 95°C for 10 min.
  • the bridge PCR amplification system, the bridge PCR amplification procedure, the restriction enzyme digestion system and the restriction restriction restriction procedure are shown in the examples.
  • the steps of preparing the test strip can be as follows:
  • the incubation may be incubation at room temperature for 2-4h (eg, 2-3h, 3-4h or 3h).
  • the probe of each target nucleic acid is different, so several probe solutions are obtained.
  • drying can be performed at 35-39° C. for 1 hour.
  • the criterion for judging what kind of target nucleic acid the sample to be tested contains can be: if a certain detection point shows a red spot, then the sample to be tested contains the target nucleic acid corresponding to the probe spotted at the detection point, otherwise. The sample to be tested does not contain the target nucleic acid corresponding to the probe spotted at the detection point.
  • the invention also protects a method for multiple nucleic acid detection, which uses nano gold particles as a carrier, performs bridge PCR amplification on its surface, and performs multiple nucleic acid detection in combination with test strips containing probes.
  • primer A and primer B for bridge PCR amplification are fixed on the surface of each nano-gold particle; all or part of primer A and all or part of primer B are respectively the same as the two segments on the target nucleic acid or complementary;
  • the test strip contains several probes, and each probe is identical or complementary to the partial segment of the target sequence of the target nucleic acid with primer A and primer B;
  • the primers A and B immobilized on the surface of the gold nanoparticles are different, and the probes on the test strip are different, which can realize the simultaneous detection of multiple target nucleic acids.
  • the primer A can be any one of the above-mentioned upstream primers, and the said primer B can be any of the above-mentioned downstream primers.
  • the nucleic acid may be deoxyribonucleic acid.
  • the present invention also protects a multiplex nucleic acid detection kit, which can include component A and component B;
  • the component A can be at least one of the above-mentioned nano-gold solution and nano-gold particle;
  • the component B can be any one of the reagent kits described above.
  • the application of the multiplex nucleic acid detection kit can be rapid, accurate and high-throughput multiplex detection of nucleic acids.
  • test kits may further include at least one of a reagent for bridge PCR amplification, a USER enzyme and a nitrocellulose membrane.
  • a nanoparticle is an independent nanoreactor, and a pair of primers of a target is fixed on its surface to amplify a target; a plurality of nanoparticles constitute a plurality of independent nanoreactors, respectively in The primers of multiple targets are immobilized on their surfaces, which can realize the simultaneous amplification of multiple targets in one reaction system;
  • nano gold particles are used as the carrier for bridge PCR amplification , and PCR primers are fixed on the surface thereof.
  • a large amount of double-stranded DNA in the bridge state was amplified on the surface.
  • the double-stranded DNA becomes single-stranded DNA after denaturation, and the single-stranded DNA is hybridized with the specific oligonucleotide probe immobilized on the test strip or chip, and the detection is realized by the color development of the hybridization spot.
  • one nano-gold particle is equivalent to an independent nano-reactor, and a target nucleic acid is amplified on its surface; therefore, multiple nano-gold particles constitute multiple independent reactors, which can Simultaneous amplification of multiple target nucleic acids means simultaneous detection of multiple target nucleic acids.
  • the invention has important application value.
  • Figure 1 is a schematic diagram of simultaneous amplification of multiple targets in bridge PCR with gold nanoparticles as a carrier.
  • Figure 2 is a schematic diagram of simultaneous detection of multiple targets in bridge PCR based on gold nanoparticles.
  • FIG. 3 is the accuracy experiment of Example 2.
  • USER enzyme is a product of NEB Company, and the catalog number is M5505S. 10 ⁇ CutSmart Buffer is a component of USER enzyme.
  • Example 1 Establishment of a method for multiple nucleic acid detection based on gold nanoparticles
  • the inventors of the present invention established a method for multiple nucleic acid detection based on gold nanoparticles after extensive experiments. Specific steps are as follows:
  • a kit of reagents is designed and synthesized based on the nucleotide sequence of each target nucleic acid conserved region.
  • Kits consist of upstream primers, downstream primers and probes.
  • the upstream primer consists of linker1, 1 U and DNA fragment 1 in order from the 5' end to the 3' end.
  • the downstream primer consists of linker2, 1 U and DNA fragment 2 in order from the 5' end to the 3' end.
  • linker1 or linker2 is a single-stranded DNA molecule composed of 10-20 nucleotides, which is used to connect sulfhydryl groups and PCR primers (DNA fragment 1 or DNA fragment 2), so that the primers can be better stretched on the surface of gold nanoparticles to facilitate subsequent PCR Amplification.
  • Linker1 or linker2 includes DNA fragment A and DNA fragment B in order from the 5' end to the 3' end.
  • DNA fragment A is composed of 8-10 Ts.
  • DNA fragment B is composed of 6-10 nucleotides, each nucleotide is A or T.
  • linker1 and linker2 can be the same or different.
  • DNA Fragment 1 and DNA Fragment 2 are identical or complementary to the two segments on the nucleic acid of interest, respectively.
  • the probe is a single-stranded DNA molecule consisting of 20-40 nucleotides, which is identical to or complementary to the partial segment of the target sequence of the target nucleic acid with DNA fragment 1 and DNA fragment 2.
  • the 5' ends of both upstream primers and downstream primers are modified, such as S-S-C6 modification (thiol modification), so that the primers are robustly immobilized on the surface of gold nanoparticles.
  • the 5' end of the probe is modified, such as Biotin modification (biotin modification), so that the probe can be stably bound to the nitrocellulose membrane.
  • the target sequences of DNA fragment 1 and DNA fragment 2 in each kit of reagents do not cross-react with probes in other kits.
  • the preparation method of primer@gold nanoparticles is as follows:
  • step (1) After completing step (1), add 100 ⁇ L of the upstream primer aqueous solution with a concentration of 10 ⁇ M, 100 ⁇ L of the downstream primer aqueous solution with a concentration of 10 ⁇ M and 500 ⁇ L of pH8.0, 1mM PBS buffer into the EP tube, and vortex to mix. , 50 °C for 4h.
  • step (3) After completing step (2), first wash three times with 1mL pH8.0, 1mM PBS buffer, then wash twice with 500 ⁇ L nuclease-free water, and finally disperse in 200 ⁇ L nuclease-free water, which is the primer@ Nano gold.
  • the upstream and downstream primers are different for each nucleic acid of interest. Therefore, the prepared primers@nano-gold are also completely different.
  • the reaction system is 50 ⁇ L, including the solution to be tested, several primers@gold nanoparticles, 5 ⁇ L 10 ⁇ EasyTaq Buffer, 4 ⁇ L 2.5mM dNTPs, 1 ⁇ L DNA Polymerase, 1 ⁇ L of 2% BSA solution, and nuclease-free water.
  • Reaction program pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; final extension at 72 °C for 5 min.
  • the bridge PCR amplification product obtained in step 4 was taken, digested with USER enzyme, and then denatured at 95°C for 10 min to obtain the denatured product by enzyme digestion.
  • the digestion system was 30 ⁇ L, including 24 ⁇ L bridge PCR amplification product, 3 ⁇ L USER enzyme and 3 ⁇ L 10 ⁇ CutSmart Buffer.
  • Enzyme digestion procedure incubate at 37°C for 20min.
  • the probe of each target nucleic acid is different, so several probe solutions are obtained
  • the solution to be tested contains the target nucleic acid corresponding to the probe, otherwise the solution to be tested does not contain the target nucleic acid corresponding to the probe ;
  • the nucleic acid is deoxyribonucleic acid (eg, genomic DNA, cDNA)).
  • FIG. 1 A schematic diagram of the simultaneous amplification of multiple targets in bridge PCR using gold nanoparticles as a carrier is shown in Figure 1.
  • thermostable nuclease the GeneID of thermostable nuclease is: 3919380
  • the complete set of reagent A consists of primers NFU, Primer NR and probe NP are composed.
  • a complete set of reagent B was designed and synthesized.
  • the complete set of reagent B consists of It consists of primer KFU, primer KR and probe KP.
  • primer NFU The nucleotide sequences of primer NFU, primer NR, probe NP, primer KFU, primer KR and probe KP are shown in Table 1.
  • Biotin means biotin modification
  • -S-S-C6 means sulfhydryl modification
  • step (1) After completing step (1), add 100 ⁇ L primer NFU aqueous solution with a concentration of 10 ⁇ M, 100 ⁇ L primer NR aqueous solution with a concentration of 10 ⁇ M and 500 ⁇ L pH8.0, 1mM PBS buffer into the EP tube, vortex and mix well , 50 °C for 4h.
  • step (3) After completing step (2), first wash 3 times with 1mL pH8.0, 1mM PBS buffer, then wash 2 times with 500 ⁇ L nuclease-free water, and finally disperse in 200 ⁇ L nuclease-free water, which is NFUR@ Au.
  • the primer NFU was replaced with the primer KFU, and the primer NR was replaced with the primer KR, and the other steps remained unchanged to obtain KFUR@Au.
  • the reaction system is 50 ⁇ L, including 3 ⁇ L test solution, 15 ⁇ L NFUR@Au, 15 ⁇ L KFUR@Au, 5 ⁇ L 10 ⁇ EasyTaq Buffer, 4 ⁇ L 2.5mM dNTPs, 1 ⁇ L DNA Polymerase, 1 ⁇ L of 2% BSA solution, and 6 ⁇ L of nuclease-free water.
  • Reaction program pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; final extension at 72 °C for 5 min.
  • the solution to be tested consists of 1.5 ⁇ L of the genomic DNA solution of Staphylococcus aureus (containing 0.5 ng of the genomic DNA of Staphylococcus aureus) and 1.5 ⁇ L of the genomic DNA solution of Klebsiella pneumoniae (containing 0.5 ng of the genomic DNA of Klebsiella pneumoniae). )composition.
  • the bridge PCR amplification product obtained in step 4 was taken, digested with USER enzyme, and then denatured at 95°C for 10 min to obtain the denatured product by enzyme digestion.
  • the digestion system was 30 ⁇ L, including 24 ⁇ L bridge PCR amplification product, 3 ⁇ L USER enzyme and 3 ⁇ L 10 ⁇ CutSmart Buffer.
  • Enzyme digestion procedure incubate at 37°C for 20min.
  • probe NP aqueous solution with a concentration of 100 ⁇ M and 15.4 ⁇ L streptavidin aqueous solution with a concentration of 75.76 ⁇ M, and incubate at room temperature for 3 hours; then add 64.6 ⁇ L pH8.0, 1mM PBS buffer, mix again Homogenize to obtain a probe NP solution.
  • probe KP aqueous solution with a concentration of 100 ⁇ M and 15.4 ⁇ L streptavidin aqueous solution with a concentration of 75.76 ⁇ M, and incubate at room temperature for 3 hours; then add 64.6 ⁇ L pH8.0, 1mM PBS buffer, mix again Homogenize to obtain probe KP solution.
  • the solution to be tested contains the genomic DNA of Staphylococcus aureus, otherwise the solution to be tested does not contain the genomic DNA of Staphylococcus aureus ;
  • the solution to be tested contains the genomic DNA of Klebsiella pneumoniae, otherwise the solution to be tested does not contain Klebsiella pneumoniae. genomic DNA;
  • test results are shown in Figure 3.
  • the results showed that the solution to be tested contained the genomic DNA of Staphylococcus aureus and the genomic DNA of Klebsiella pneumoniae. Exactly as expected.
  • Example 1 It can be seen that the method established in Example 1 can simultaneously detect multiple nucleic acids with high accuracy.
  • the invention establishes a multiple nucleic acid detection method based on nano gold particles.
  • one nano gold particle is equivalent to an independent nano reactor, and a target nucleic acid is amplified on its surface;
  • the gold nanoparticles constitute multiple independent reactors, which can simultaneously amplify multiple target nucleic acids, that is, to realize the simultaneous detection of multiple target nucleic acids.

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Abstract

本发明公开了一种基于纳米金颗粒的多重核酸检测的方法。该方法是以纳米金颗粒为载体，在其表面进行桥式PCR扩增，结合含有探针的试纸条进行多重核酸检测；每个纳米金颗粒表面固定有进行桥式PCR扩增的引物；由于纳米金颗粒表面固定的引物不同，试纸条上的探针不同，可以实现多个目的核酸同时检测。本发明的桥式PCR反应体系中，一个纳米金颗粒相当于一个独立的纳米反应器，在其表面扩增一种目的核酸；因此，多个纳米金颗粒便构成多个独立的反应器，可以同时扩增多种目的核酸，即实现多个目的核酸的同时检测。

Description

一种基于纳米金颗粒的多重核酸检测的方法 技术领域
本发明属于生物技术领域，具体涉及一种基于纳米金颗粒的多重核酸检测的方法；特别涉及以纳米金颗粒为载体，在其表面进行桥式PCR扩增，结合含有特异探针的试纸条，通过DNA分子杂交进行多重核酸检测。
背景技术
一直以来，新发、突发传染病频频发生，尤其是诸如近年SARS-CoV及SARS-CoV-2的突然爆发，对人类的生活、生产以及发展带来了巨大影响。而引起人类感染的病原体种类繁多，不同的病原体可引起相同或相似的临床症状，此时对这些疾病的诊断就变得复杂。临床治疗中，临床症状也很少特异地由单一病原体引起，若感染所致疾病得不到快速诊断，对于后续的治疗有严重的影响。为此，发展多种病原体同时检测的策略，以及病原体的快速、准确识别在疾病管理中尤为重要。同时，生物恐怖因子检测、遗传疾病中易感基因的筛查、SNP检测等对现有的生物检测技术提出了更高的要求。多重核酸检测技术的快速、准确、高通量的特点，使病原体的检测与鉴定时间大大缩短，在1-2小时即可完成；而当面对多个靶点或大量样品时，多重核酸检测技术的应用所带来的时效性更是使其优势尽显。
但是，多重核酸检测技术本身存在一定的自身局限：首先，实时荧光定量PCR技术检测多重靶点能力有限、仪器昂贵，基因芯片的使用成本高等；其次，成熟的多重检测设备选择很少，实际应用受到很大制约。为此，开发快速、准确、高通量、应用性广的多重核酸检测技术，尤其是在应对有相似感染症状的传染病爆发早期的已知病原体筛查中，具有重要的实际意义。
发明公开
本发明的目的是快速、准确、高通量的多重检测核酸。
本发明首先保护多重核酸检测的方法。
本发明所保护的多重核酸检测的方法，具体可为方法一，包括如下步骤：
(1)制备纳米金溶液；
(2)根据每个目的核酸的核苷酸序列制备成套试剂；
每个成套试剂包括上游引物、下游引物和探针；
上游引物从5’末端到3’末端依次包括由linker1、1个U和DNA片段1；
下游引物从5’末端到3’末端依次包括由linker2、1个U和DNA片段2；
DNA片段1和DNA片段2分别与目的核酸上的两个区段相同或互补；
探针可为20-40个(如20-30个、30-40个、20个、30个或40个)核苷酸组成的单链DNA分子，与DNA片段1和DNA片段2在目的核酸的靶序列的部分区段相同或互补；
各个成套试剂之间没有交叉反应；
每个成套试剂中DNA片段1和DNA片段2在目的核酸的靶序列与其它成套试剂中的探针没有交叉反应；
(3)完成步骤(1)和(2)后，每个目的核酸制备一个引物@纳米金；引物@纳米金为所述上游引物和所述下游引物固定至纳米金溶液中纳米金颗粒表面形成；
(4)采用步骤(3)制备的所有引物@纳米金对待测样本进行桥式PCR扩增，之后用USER酶酶切，变性，得到酶切变性产物；
(5)将步骤(2)制备的所有探针分别点样于硝酸纤维素膜，形成相互分离的检测点，得到试纸条；
(6)用步骤(5)得到的试纸条检测步骤(4)得到的酶切变性产物，根据试纸条上的检测点是否显出的红色斑点，判断待测样本含有何种目的核酸。
所述步骤(1)中，制备纳米金溶液的步骤依次可如下：
(1-1)取HAuCl 4水溶液，90-97℃(如90-95℃、95-97℃、90℃、95℃或97℃)处理3min以上(如3min、5min)；
(1-2)加入柠檬酸钠水溶液，混匀，90-97℃(如90-95℃、95-97℃、90℃、95℃或97℃)处理30min以上(如30min、40min)；自然冷却，得到纳米金溶液。
所述步骤(1-1)中，HAuCl 4水溶液浓度可为0.005-0.015％(如0.005-0.010％、0.010-0.015％、0.005％、0.010％或0.015％)(m/v)。
所述步骤(1-2)中，柠檬酸钠水溶液的浓度可为0.5-1.5％(如0.5-1.0％、1.0-1.5％、0.5％、1.0％或1.5％)。
所述步骤(1)中，HAuCl 4水溶液和柠檬酸钠水溶液的体积比可为100mL：(2-3)mL(如100mL：2mL、100mL：2.5mL、100mL：3mL、100mL：(2-2.5)mL、100mL：(2.5-3)mL)。
所述步骤(2)中，根据每个目的核酸的核苷酸序列制备成套试剂可为根据每个目的核酸保守区的核苷酸序列制备成套试剂。根据每个目的核酸保守区的核苷酸序列设计并合成一个成套试剂。
所述步骤(2)中，每个成套试剂由所述上游引物、所述下游引物和所述探针组成。
上述任一所述上游引物从5’末端到3’末端依次包括linker1、1个U和DNA片段1。
上述任一所述下游引物从5’末端到3’末端依次包括linker2、1个U和DNA片段2。
所述DNA片段1和所述DNA片段2分别与目的核酸上的两个区段相同或互补。
所述探针可为20-40个(如20-30个、30-40个、20个、30个或40个)核苷酸组成的单链DNA分子，与DNA片段1和DNA片段2在目的核酸的靶序列的部分区段相同或互补。
各个成套试剂之间没有交叉反应；
每个成套试剂中DNA片段1和DNA片段2在目的核酸的靶序列与其它成套试剂中的探针没有交叉反应。
所述上游引物、所述下游引物和所述探针的5’末端均可进行修饰。
所述上游引物和所述下游引物的5’末端进行修饰可为巯基修饰，以使引物稳健固定至纳米金颗粒表面。
所述探针的5’末端进行修饰可为Biotin修饰，以使探针和硝酸纤维素膜稳定结合。
所述linker1或所述linker2可为10-20个(如10-15个、15-20个、10个、15个或20个)核苷酸组成的单链DNA分子，用于连接巯基和PCR引物(DNA片段1或DNA片段2)，使引物更好的在纳米金颗粒表面伸展以利于后续PCR 扩增。linker1或linker2从5’末端到3’末端依次包括DNA片段甲和DNA片段乙。DNA片段甲可由8-10个(如8个、9个或10个)T组成。DNA片段乙可由6-10个(如6-8个、8-10个、6个、8个或10个)核苷酸组成，每个核苷酸为A或T。
所述linker1和所述linker2可以相同，也可以不同。
上述任一所述上游引物从5’末端到3’末端依次由linker1、1个U和DNA片段1组成。
上述任一所述下游引物从5’末端到3’末端依次由linker2、1个U和DNA片段2组成。
所述步骤(3)中，制备每个引物@纳米金的步骤依次如下：
(3-1)将纳米金溶液和Tween80溶液混匀，静置20min以上(如20min、20min、30min或40min)；
(3-2)加入所述上游引物、所述下游引物和PBS缓冲液，混匀，40-60℃(如40-50℃、50-60℃、40℃、50℃或60℃)保温3-5h(如3-4h、4-5h、3h、4h或5h)；
每个目的核酸的上游引物和下游引物不同，制备的每个引物@纳米金也完全不同。
所述步骤(3-1)中，Tween80溶液的浓度可为8-12％(v/v)(如8-10％(v/v)、10-12％(v/v)、8％(v/v)、10％(v/v)或12％(v/v))。所述纳米金溶液可为纳米金浓缩液。所述纳米金浓缩液可为将上述任一所述纳米金溶液离心浓缩2-4倍(如2倍、3倍或4倍)获得。
所述步骤(3-2)中，PBS缓冲液可为pH8.0、1mM的PBS缓冲液。
所述制备每个引物@纳米金的步骤还包括步骤(3-3)先用pH8.0、1mM的PBS缓冲液洗涤3次，再用无核酸酶水洗涤2次，最后分散于无核酸酶水，即为引物@纳米金。
每个目的核酸制备一个引物@纳米金。
所述步骤(4)中，所有引物@纳米金在同一个反应体系中进行桥式PCR扩增、酶切和变性。所述变性可为95℃变性10min。
桥式PCR扩增体系、桥式PCR扩增程序、酶切体系和酶切程序见实施例。
所述步骤(5)中，制备试纸条的步骤可依次如下：
(5-1)分别将各个探针和链霉亲和素溶液混匀，孵育，得到探针溶液；
(5-2)分别将各个探针溶液和链霉亲和素溶液混匀，均匀点样于硝酸纤维素膜，干燥，得到同时检测多个目的核酸的试纸条。
所述步骤(5-1)中，孵育可为室温孵育2-4h(如2-3h、3-4h或3h)。
所述步骤(5-1)中，每个目的核酸的探针不同，因此获得了若干个探针溶液。
所述步骤(5-2)中，干燥可为35-39℃干燥1h。
所述步骤(6)中，判断待测样本含有何种目的核酸的标准可为：如果某检测点显出红色斑点，则待测样本中含有该检测点点样的探针对应的目的核酸，否则待测样本中不含有该检测点点样的探针对应的目的核酸。
本发明还保护一种多重核酸检测的方法，是以纳米金颗粒为载体，在其表面进行桥式PCR扩增，结合含有探针的试纸条进行多重核酸检测。
上述方法中，每个纳米金颗粒表面固定有进行桥式PCR扩增的引物A和引物B；引物A的全部或部分和引物B的全部或部分分别与目的核酸上的两个区段相同或互补；
试纸条上含有若干探针，每个探针与引物A和引物B在目的核酸的靶序列的部分区段相同或互补；
纳米金颗粒表面固定的引物A和引物B不同，试纸条上的探针不同，可以实现多个目的核酸同时检测。
上述方法中，所述引物A可为上述任一所述上游引物，所述引物B可为上述任一所述下游引物。
上述任一所述的方法中，所述核酸可为脱氧核糖核酸。
本发明还保护一种多重核酸检测试剂盒，可包括组分A和组分B；
所述组分A可为上述任一所述纳米金溶液和纳米金颗粒中的至少一种；
所述组分B可为上述任一所述成套试剂。
所述多重核酸检测试剂盒的用途可为快速、准确、高通量的多重检测核酸。
上述任一所述试剂盒还可包括进行桥式PCR扩增的试剂、USER酶和硝 酸纤维素膜中的至少一种。
本发明取得的有益效果如下：
(1)建立了一种以纳米颗粒为载体的桥式PCR扩增体系，扩增产物以弯曲的双链DNA形态存在并固定于纳米颗粒表面；
(2)在反应体系中，一个纳米颗粒是一个独立的纳米反应器，在其表面固定一个靶标的一对引物，扩增一个靶标；多个纳米颗粒构成多个独立的纳米反应器，分别在它们的表面固定多个靶标的引物，便可实现在一个反应体系中对多个靶标同时扩增；
(3)将上述的多个靶标扩增产物酶切变性后和固定在试纸条或芯片上的不同位置的寡核苷酸探针杂交，根据杂交斑点是否显色即可实现多靶标同时检测。
本发明以纳米金颗粒作为桥式PCR扩增的载体，在其表面固定PCR引物，在一定的缓冲溶液中，在dNTPs、Mg 2+、DNA聚合酶、核酸模板的参与下，在纳米金颗粒表面扩增出大量的呈桥式状态的双链DNA。双链DNA变性后变为单链DNA，单链DNA与固定在试纸条或芯片上的特异性寡核苷酸探针杂交，通过杂交斑点显色而实现检测。本发明的桥式PCR反应体系中，一个纳米金颗粒相当于一个独立的纳米反应器，在其表面扩增一种目的核酸；因此，多个纳米金颗粒便构成多个独立的反应器，可以同时扩增多种目的核酸，即实现多个目的核酸的同时检测。本发明具有重要的应用价值。
附图说明
图1为以纳米金为载体的桥式PCR多靶标同时扩增示意图。
图2为基于纳米金颗粒的桥式PCR多靶标同时检测示意图。
图3为实施例2的准确性实验。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中 所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
USER酶为NEB公司的产品，产品目录号为M5505S。10×CutSmart Buffer为USER酶中的组件。
实施例1、基于纳米金颗粒的多重核酸检测的方法的建立
本发明的发明人经过大量实验，建立了基于纳米金颗粒的多重核酸检测的方法。具体步骤如下：
1、纳米金溶液的制备
取规格为250mL的三口烧瓶，加入100mL 0.01％(m/v)HAuCl 4水溶液，水浴加热至95℃，保温5min(目的为平衡HAuCl 4水溶液温度至95℃)；之后快速加入2.5mL 1％(m/v)柠檬酸钠水溶液，在快速搅拌下于95℃保温40min，室温冷却，得到纳米金溶液。
2、成套试剂的制备
根据每个目的核酸保守区的核苷酸序列设计并合成一个成套试剂。
成套试剂由上游引物、下游引物和探针组成。
上游引物从5’末端到3’末端依次包括由linker1、1个U和DNA片段1。
下游引物从5’末端到3’末端依次包括由linker2、1个U和DNA片段2。
linker1或linker2为10-20个核苷酸组成的单链DNA分子，用于连接巯基和PCR引物(DNA片段1或DNA片段2)，使引物更好的在纳米金颗粒表面伸展以利于后续PCR扩增。linker1或linker2从5’末端到3’末端依次包括DNA片段甲和DNA片段乙。DNA片段甲为8-10个T组成。DNA片段乙为6-10个核苷酸组成，每个核苷酸为A或T。
linker1和linker2可以相同，也可以不同。
DNA片段1和DNA片段2分别与目的核酸上的两个区段相同或互补。
探针为20-40个核苷酸组成的单链DNA分子，与DNA片段1和DNA片段2在目的核酸的靶序列的部分区段相同或互补。
上游引物和下游引物的5’末端均进行修饰，如S-S-C6修饰(巯基修饰)，以使引物稳健固定至纳米金颗粒表面。
探针的5’末端均进行修饰，如Biotin修饰(生物素修饰)，以使探 针和硝酸纤维素膜稳定结合。
各个成套试剂之间没有交叉反应。
每个成套试剂中DNA片段1和DNA片段2在目的核酸的靶序列与其它成套试剂中的探针没有交叉反应。
3、引物@纳米金的制备
引物@纳米金的制备方法如下：
(1)取规格为2.0mL的EP管，加入500μL纳米金浓缩液(步骤1得到的纳米金溶液离心浓缩3倍获得)和500μL 10％(v/v)Tween80水溶液，涡旋混匀后室温放置30min。
(2)完成步骤(1)后，向所述EP管中加入100μL浓度为10μM的上游引物水溶液、100μL浓度为10μM的下游引物水溶液和500μL pH8.0、1mM的PBS缓冲液，涡旋混匀，50℃保温4h。
(3)完成步骤(2)后，先用1mL pH8.0、1mM的PBS缓冲液洗涤3次，再用500μL无核酸酶水洗涤2次，最后分散于200μL无核酸酶水，即为引物@纳米金。
每个目的核酸制备一个引物@纳米金。
每个目的核酸的上游引物和下游引物不同。因此，制备的引物@纳米金也完全不同。
4、桥式PCR扩增
采用Transgen的
Figure PCTCN2020141533-appb-000001
DNA Polymerase进行PCR扩增，得到桥式PCR扩增产物。10×EasyTaq Buffer为
Figure PCTCN2020141533-appb-000002
DNA Polymerase中的组件。
反应体系为50μL，包括待测溶液、若干引物@纳米金、5μL 10×EasyTaq Buffer、4μL 2.5mM dNTPs、1μL 
Figure PCTCN2020141533-appb-000003
DNA Polymerase、1μL 2％BSA溶液和无核酸酶水。
反应程序：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸1min，35个循环；72℃终延伸5min。
5、桥式PCR扩增产物酶切变性
取步骤4获得的桥式PCR扩增产物，用USER酶酶切，然后95℃变性10min，获得酶切变性产物。
酶切体系为30μL，包括24μL桥式PCR扩增产物、3μL USER酶和3μ L10×CutSmart Buffer。
酶切程序：37℃孵育20min。
6、同时检测多个目的核酸的硝酸纤维素膜试纸条的制备
(1)将20μL浓度为100μM的探针水溶液和15.4μL浓度为75.76μM的链霉亲和素水溶液混匀，室温孵育3h；之后加入64.6μL pH8.0、1mM的PBS缓冲液，再次混匀，得到探针溶液。
每个目的核酸的探针不同，因此获得了若干个探针溶液
(2)分别取各个探针溶液和浓度为75.76μM的链霉亲和素水溶液(作为阴性对照)各1μL，均匀涂在硝酸纤维素膜试纸条(宽为1cm)上，形成相互分离的检测点(各检测点相隔1cm以上)，37℃干燥1h，得到同时检测多个目的核酸的硝酸纤维素膜试纸条。
7、检测
取规格为1.5mL的EP管，加入20μL步骤5获得的酶切变性产物，之后垂直放入步骤6制备的同时检测多个目的核酸的硝酸纤维素膜试纸条使试纸条的下端浸没于酶切变性产物中；反应10分钟。
根据膜上显出的红色斑点情况进行如下判断：
(1)如果试纸条上在涂某探针溶液的斑点处显出红色斑点，则待测溶液中含有该探针对应的目的核酸，否则待测溶液中不含有该探针对应的目的核酸；
(2)如果试纸条上的阴性对照显出红色斑点，则为无效实验结果，需重新测定。
上述方法中，核酸为脱氧核糖核酸((如基因组DNA、cDNA))。
以纳米金颗粒为载体的桥式PCR多靶标同时扩增示意图见图1。
基于纳米金颗粒的桥式PCR多靶标同时检测示意图见图2。
实施例2、实施例1建立的方法的准确性实验
1、纳米金溶液的制备
取规格为250mL的三口烧瓶，加入100mL 0.01％(m/v)HAuCl 4水溶液，水浴加热至95℃，保温5min(目的为平衡HAuCl 4水溶液温度至95℃)；之后快速加入2.5mL 1％(m/v)柠檬酸钠水溶液，在快速搅拌下于95℃保温40min，室温冷却，得到纳米金溶液。
2、成套试剂的制备
根据金黄色葡萄球菌高度保守的nuc基因(即耐热核酸酶的编码基因，耐热核酸酶的GeneID为：3919380)的核苷酸序列，设计并合成成套试剂甲，成套试剂甲由引物NFU、引物NR和探针NP组成。
根据肺炎克雷伯菌高度保守的khe基因(即特异性毒力蛋白的编码基因，特异性毒力蛋白的GeneID为：11849371)的核苷酸序列，设计并合成成套试剂乙，成套试剂乙由引物KFU、引物KR和探针KP组成。
引物NFU、引物NR、探针NP、引物KFU、引物KR和探针KP的核苷酸序列见表1。
表1
名称 序列(5’→3’)
引物NFU 5’-S-S-C6-TTTTTTTTTTATATAUAAGCGATTGATGGTGATACGGT
引物NR 5’-S-S-C6-TTTTTTTTTTCGTAAATGCACTTGCTTCAGGA
探针NP 5’-Biotin-TGTACAAAGGTCAACCAATGACATTCAGAC
引物KFU 5’-S-S-C6-TTTTTTTTTTATATAUCACCTCTTATCCACACGCGG
引物KR 5’-S-S-C6-TTTTTTTTTTCACACTTCCGGATAGCCCTC
探针KP 5’-Biotin-AGAGCGATGAGGAAGAGTTCATCTACGTGCTG
注：Biotin表示生物素修饰，-S-S-C6表示巯基修饰。
3、引物@纳米金(具体为NFUR@Au和KFUR@Au)的制备
(1)取规格为2.0mL的EP管，加入500μL纳米金浓缩液(步骤1得到的纳米金溶液离心浓缩3倍获得)和500μL 10％(v/v)Tween80水溶液，涡旋混匀后室温放置30min。
(2)完成步骤(1)后，向所述EP管中加入100μL浓度为10μM的引物NFU水溶液、100μL浓度为10μM的引物NR水溶液和500μL pH8.0、1mM的PBS缓冲液，涡旋混匀，50℃保温4h。
(3)完成步骤(2)后，先用1mL pH8.0、1mM的PBS缓冲液洗涤3次，再用500μL无核酸酶水洗涤2次，最后分散于200μL无核酸酶水，即为NFUR@Au。
按照上述步骤，将引物NFU替换为引物KFU，引物NR替换为引物KR，其它步骤均不变，得到KFUR@Au。
4、桥式PCR扩增
采用Transgen的
Figure PCTCN2020141533-appb-000004
DNA Polymerase进行PCR扩增，得到桥式PCR扩增产物。10×EasyTaq Buffer为
Figure PCTCN2020141533-appb-000005
DNA Polymerase中的组件。
反应体系为50μL，包括3μL待测溶液、15μL NFUR@Au、15μL KFUR@Au、5μL 10×EasyTaq Buffer、4μL 2.5mM dNTPs、1μL 
Figure PCTCN2020141533-appb-000006
DNA Polymerase、1μL 2％BSA溶液和6μL无核酸酶水。
反应程序：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸1min，35个循环；72℃终延伸5min。
待测溶液由1.5μL金黄色葡萄球菌的基因组DNA溶液(含0.5ng金黄色葡萄球菌的基因组DNA)和1.5μL肺炎克雷伯菌的基因组DNA溶液(含0.5ng肺炎克雷伯菌的基因组DNA)组成。
5、桥式PCR扩增产物酶切变性
取步骤4获得的桥式PCR扩增产物，用USER酶酶切，然后95℃变性10min，获得酶切变性产物。
酶切体系为30μL，包括24μL桥式PCR扩增产物、3μL USER酶和3μL10×CutSmart Buffer。
酶切程序：37℃孵育20min。
6、同时检测金黄色葡萄球菌和肺炎克雷伯菌的硝酸纤维素膜试纸条的制备
(1)将20μL浓度为100μM的探针NP水溶液和15.4μL浓度为75.76μM的链霉亲和素水溶液混匀，室温孵育3h；之后加入64.6μL pH8.0、1mM的PBS缓冲液，再次混匀，得到探针NP溶液。
(2)将20μL浓度为100μM的探针KP水溶液和15.4μL浓度为75.76μM的链霉亲和素水溶液混匀，室温孵育3h；之后加入64.6μL pH8.0、1mM的PBS缓冲液，再次混匀，得到探针KP溶液。
(3)分别取探针NP溶液、探针KP溶液和浓度为75.76μM的链霉亲和素水溶液(作为阴性对照)各1μL，均匀涂在硝酸纤维素膜试纸条(宽为1cm)上，形成相互分离的检测点(各检测点相隔1cm以上)，37℃干燥1h，得到同时检测金黄色葡萄球菌和肺炎克雷伯菌的硝酸纤维素膜试纸条。
7、检测
取规格为1.5mL的EP管，加入20μL步骤5获得的酶切变性产物，之后垂直放入步骤6制备的同时检测金黄色葡萄球菌和肺炎克雷伯菌的硝酸纤维素膜试纸条，使试纸条的下端浸没于酶切变性产物中；反应10分钟。
根据膜上显出的红色斑点情况进行如下判断：
(1)如果试纸条上在涂探针NP溶液的斑点处显出红色斑点，则待测溶液中含有金黄色葡萄球菌的基因组DNA，否则待测溶液中不含有金黄色葡萄球菌的基因组DNA；
(2)如果试纸条上在涂探针KP溶液的斑点处显出红色斑点，则待测溶液中含有肺炎克雷伯菌的基因组DNA，否则待测溶液中不含有肺炎克雷伯菌的基因组DNA；
(3)如果试纸条上的阴性对照显出红色斑点，则为无效实验结果，需重新测定。
检测结果见图3。结果表明，待测溶液中含有金黄色葡萄球菌的基因组DNA和肺炎克雷伯菌的基因组DNA。与预期完全一致。
由此可见，实施例1建立的方法可以同时检测多重核酸，且具有较高的准确性。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明基于纳米金颗粒建立了多重核酸检测方法，该方法的桥式PCR反应体系中，一个纳米金颗粒相当于一个独立的纳米反应器，在其表面扩增一种目的核酸；因此，多个纳米金颗粒便构成多个独立的反应器，可以同时扩增多种目的核酸，即实现多个目的核酸的同时检测。

Claims (15)

  1. 一种多重核酸检测的方法，包括如下步骤：
    (1)制备纳米金溶液；
    (2)根据每个目的核酸的核苷酸序列制备成套试剂；
    每个成套试剂包括上游引物、下游引物和探针；
    上游引物从5’末端到3’末端依次包括linker1、1个U和DNA片段1；
    下游引物从5’末端到3’末端依次包括linker2、1个U和DNA片段2；
    DNA片段1和DNA片段2分别与目的核酸上的两个区段相同或互补；
    探针为20-40个核苷酸组成的单链DNA分子，与DNA片段1和DNA片段2在目的核酸的靶序列的部分区段相同或互补；
    各个成套试剂之间没有交叉反应；
    每个成套试剂中DNA片段1和DNA片段2在目的核酸的靶序列与其它成套试剂中的探针没有交叉反应；
    (3)完成步骤(1)和(2)后，每个目的核酸制备一个引物@纳米金；引物@纳米金为所述上游引物和所述下游引物固定至纳米金溶液中纳米金颗粒表面形成；
    (4)采用步骤(3)制备的所有引物@纳米金对待测样本进行桥式PCR扩增，之后用USER酶酶切，变性，得到酶切变性产物；
    (5)将步骤(2)制备的所有探针分别点样于硝酸纤维素膜，形成相互分离的检测点，得到试纸条；
    (6)用步骤(5)得到的试纸条检测步骤(4)得到的酶切变性产物，根据试纸条上的检测点是否显出的红色斑点，判断待测样本含有何种目的核酸。
  2. 如权利要求1所述的方法，其特征在于：
    所述步骤(1)中，制备纳米金溶液的步骤依次如下：
    (1-1)取HAuCl 4水溶液，90-97℃处理3min以上；
    (1-2)加入柠檬酸钠水溶液，混匀，90-97℃处理30min以上；自然冷却，得到纳米金溶液。
  3. 如权利要求2所述的方法，其特征在于：
    HAuCl 4水溶液的浓度为0.005-0.015％；
    柠檬酸钠水溶液的浓度为0.5-1.5％；
    HAuCl 4水溶液和柠檬酸钠水溶液的比例为100mL：2-3mL。
  4. 如权利要求1所述的方法，其特征在于：所述步骤(2)中，根据每个目的核酸的核苷酸序列制备成套试剂为根据每个目的核酸保守区的核苷酸序列制备成套试剂。
  5. 如权利要求1所述的方法，其特征在于：所述步骤(2)中，上游引物、下游引物和探针的5’末端均进行修饰。
  6. 如权利要求5所述的方法，其特征在于：
    上游引物和下游引物的5’末端进行修饰为巯基修饰；
    探针的5’末端进行修饰为Biotin修饰。
  7. 如权利要求1所述的方法，其特征在于：所述步骤(2)中，linker1或linker2为10-20个核苷酸组成的单链DNA分子；linker1或linker2从5’末端到3’末端依次包括DNA片段甲和DNA片段乙；DNA片段甲由8-10个T组成；DNA片段乙由6-10个核苷酸组成，每个核苷酸为A或T。
  8. 如权利要求7所述的方法，其特征在于：linker1和linker2可以相同，也可以不同。
  9. 如权利要求1所述的方法，其特征在于：所述步骤(3)中，制备每个引物@纳米金的步骤依次如下：
    (3-1)将纳米金溶液和Tween80溶液混匀，静置20min以上；
    (3-2)加入上游引物、下游引物和PBS缓冲液，混匀，40-60℃保温3-5h；
    每个目的核酸的上游引物和下游引物不同，制备的每个引物@纳米金也完全不同。
  10. 如权利要求1所述的方法，其特征在于：所述步骤(5)中，制备试纸条的步骤依次如下：
    (5-1)分别将各个探针和链霉亲和素溶液混匀，孵育，得到探针溶液；
    (5-2)分别将各个探针溶液和链霉亲和素溶液混匀，均匀点样于硝酸纤维素膜，干燥，得到同时检测多个目的核酸的试纸条。
  11. 如权利要求1所述的方法，其特征在于：所述步骤(6)中，判断 待测样本含有何种目的核酸的标准为：如果某检测点显出红色斑点，则待测样本中含有该检测点涂布的探针对应的目的核酸，否则待测样本中不含有该检测点涂布的探针对应的目的核酸。
  12. 如权利要求1所述的方法，其特征在于：所述核酸为脱氧核糖核酸。
  13. 一种多重核酸检测的方法，是以纳米金颗粒为载体，在其表面进行桥式PCR扩增，结合含有探针的试纸条进行多重核酸检测。
  14. 如权利要求13所述的方法，其特征在于：
    每个纳米金颗粒表面固定有进行桥式PCR扩增的引物A和引物B；引物A的全部或部分和引物B的全部或部分分别与目的核酸上的两个区段相同或互补；
    试纸条上含有若干探针，每个探针与引物A和引物B在目的核酸的靶序列的部分区段相同或互补；
    纳米金颗粒表面固定的引物A和引物B不同，试纸条上的探针不同，可以实现多个目的核酸同时检测。
  15. 一种多重核酸检测试剂盒，包括组分A和组分B；
    所述组分A为权利要求1-3中任一所述纳米金溶液和纳米金颗粒中的至少一种；
    所述组分B为权利要求1、4、5、6、7和8中任一所述成套试剂。
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