Shared sequence mediated enhanced single-tube nested multiplex PCR (Polymerase Chain Reaction) detection method and application

Abstract

The invention discloses a shared sequence mediated enhanced single-tube nested multiplex PCR (Polymerase Chain Reaction) detection method and application, the method comprises the following steps: carrying out a first round of target amplification by utilizing a plurality of pairs of specific primers (F1 / R1-Fn / Rn) of which 5'ends are modified with the same shared sequence to generate an intermediate product containing the shared sequence; a single enhanced primer FR which is completely consistent with a shared sequence is adopted for second-round amplification, so that the detection sensitivity is remarkably improved. According to the method, single-tube nested multiplex PCR is innovatively realized through a shared sequence, the whole process is closed, pollution is avoided, and 3-10 kinds of targets can be synchronously detected. The method can realize absolute quantification less than 10 copies / [mu] L in a digital PCR system, and is especially suitable for high-sensitivity synchronous detection of blood flow infection pathogens and drug-resistant genes. The kit has high throughput of multiple PCR and high sensitivity of nested PCR, and has important application value in the fields of infectious disease diagnosis and drug-resistant gene screening.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, specifically to a shared sequence-mediated enhanced single-tube nested multiplex PCR detection method, and its application in pathogen diagnosis, drug resistance gene screening, and digital PCR systems. Background Technology

[0002] In clinical diagnosis, epidemiological surveillance, and public health control, simultaneous detection of multiple pathogens or multiple targets has significant practical implications (see, for example, Cai Zhen et al., Multi-target Gene Parallel Detection Technology Provides a New Model for Personalized Tumor Treatment, 2013, Vol. 5, No. 6: 361-366). Many infectious diseases (such as respiratory and gastrointestinal infections) have similar clinical symptoms, but the pathogens are diverse (such as bacteria, viruses, and fungi). Relying solely on single-target detection may lead to missed or misdiagnosed cases, delaying treatment. Furthermore, some diseases (such as cancer and genetic diseases) involve multiple gene mutations or abnormal expression, and the detection of a single biomarker cannot comprehensively assess disease risk or prognosis.

[0003] Traditional detection methods (such as conventional PCR, immunoassay, or culture) typically detect only one or a few targets at a time, resulting in low throughput, long processing times, and large sample consumption, making it difficult to meet the clinical needs for efficient and accurate diagnosis. Multiplex PCR technology, due to its ability to amplify multiple targets simultaneously in a single reaction, significantly improves detection efficiency and reduces costs, becoming an important solution for multi-target detection (see, for example, Lai Junhua et al., Application of Multiplex Polymerase Chain Reaction in the Detection of Sepsis Pathogens, 2014, Vol. 24, No. 17: 4409-4411). However, existing multiplex PCR technologies still face problems such as primer interference, uneven amplification efficiency, and insufficient sensitivity, limiting their application in complex samples (see, for example, Gao Yang et al., Research Progress in Multiplex PCR Detection Technology for Pathogens, 2024, Vol. 35, No. 2, 243-247). Key technical problems existing in current multiplex PCR technology for detecting multiple target molecules include:

[0004] (1) The problem of inconsistent amplification efficiency of each target due to the difference in annealing temperature of different primer sets;

[0005] (2) Dimers are easily formed or non-specific amplification problems may result when multiple sets of high-concentration primers coexist;

[0006] (3) The uneven amplification efficiency caused by the difference in the initial concentration of different target templates leads to the problem that low-concentration targets are difficult to detect.

[0007] Therefore, developing a novel multiplex PCR detection method with high sensitivity and specificity is of great significance for improving the accuracy of disease diagnosis, achieving early screening, and precision medicine.

[0008] Chinese patent application CN116355997A discloses a multiplex PCR reaction system, but provides a method for detecting and distinguishing multiple different targets in a single-tube PCR reaction using ultra-multiplex PCR. This method can avoid false positive signals caused by primer dimers in the presence of multiple specific primers. The patent also provides primers and probes for the single-tube ultra-multiplex PCR, a reaction system, and a multiplex target detection kit containing the primers and probes and the reaction system. However, this method is cumbersome to operate and has poor detection accuracy, therefore requiring further improvement. Summary of the Invention

[0009] The technical problem to be solved by this invention is how to solve the problems of low efficiency, poor sensitivity and specificity of existing multiplex PCR technology when detecting multiple target molecules.

[0010] The present invention solves the above-mentioned technical problems through the following technical means:

[0011] This invention proposes a shared sequence-mediated enhanced single-tube nested multiplex PCR detection method, comprising the following steps:

[0012] S1. Target-specific amplification: Multiple pairs of upstream and downstream primers modified with shared sequences (F1, F2, ... Fn and R1, R2, ... Rn) are used to specifically recognize the target sequence and generate amplification products (P1, P2, ... Pn) containing the shared sequence through the first round of PCR reaction.

[0013] S2. Shared sequence-mediated enhanced amplification: Using a single shared sequence enhancement primer FR, and with the amplification products (P1, P2, ... Pn) from step S1 as templates, a second round of PCR reaction is used to generate amplification products (P1, P2, ... Pn) containing the shared sequence.

[0014] Steps S1 and S2 are carried out continuously in a closed single reaction tube without the need to open the cap or add samples a second time.

[0015] Preferably, steps S1 and S2 employ differentiated temperature programs:

[0016] The annealing / stretching temperature in step S1 is 52℃-62℃, preferably 55℃;

[0017] The annealing / stretching temperature for step S2 is 45℃-55℃, preferably 50℃.

[0018] Preferably, the number of cycles in steps S1 and S2 is controlled independently:

[0019] The number of iterations in step S1 is 5-20 times, preferably 10 times;

[0020] The number of iterations in step S2 is 20-40 times, preferably 30 times.

[0021] Preferably, the 5' ends of the upstream and downstream primers (F1, F2, ... Fn and R1, R2, ... Rn) all contain the same shared sequence M, and their general structural formula is:

[0022] MN(I)

[0023] Wherein, M is the shared sequence; N is the target-specific recognition sequence.

[0024] Preferably, the nucleotide sequence of the enhancing primer FR is completely identical to the shared sequence M.

[0025] Preferably, the primer concentration satisfies:

[0026] The final concentration of the upstream and downstream primers (F1, F2, ... Fn and R1, R2, ... Rn) is 0.1-0.5 μM, preferably 0.2 μM;

[0027] The final concentration of the enhancing primer FR is 0.2-1.0 μM, preferably 0.4 μM;

[0028] Furthermore, the final concentration of the enhancing primer FR is not less than 1.5 times the final concentration of any upstream or downstream primer.

[0029] Preferably, the target to be tested includes one or more of DNA, RNA, or their reverse transcriptase products.

[0030] This invention also proposes the application of the above method in the detection of bloodstream infections for non-diagnostic or therapeutic purposes, simultaneously detecting multiple targets.

[0031] Preferably, the target includes at least one of Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecalis.

[0032] This invention also proposes the application of the above method in screening for antimicrobial drug resistance genes, simultaneously detecting multiple targets.

[0033] Preferably, the target includes at least one of the mecA gene, vanA gene, and IMP gene.

[0034] This invention also proposes the application of the above method in a digital PCR system, comprising the following steps:

[0035] (1) Preparation of microdroplet reaction unit: The single-tube nested multiplex PCR reaction system is encapsulated in microdroplets on a biochip by using droplet-encapsulated oil to form a microdroplet single-molecule reaction system;

[0036] (2) Microdroplet reaction unit amplification reaction: Thermal cycling amplification reaction of microdroplet single molecule reaction system was carried out using a PCR amplification instrument;

[0037] (3) Fluorescence signal acquisition and quantitative analysis: The fluorescence signal in the microdroplets is detected by a digital PCR instrument or a biochip reader. The absolute concentration of multi-target molecules is calculated based on the Poisson distribution algorithm to achieve absolute quantification of molecules.

[0038] The beneficial effects of this invention are as follows:

[0039] 1. This invention utilizes multiple pairs of specific primers (F1 / R1 to Fn / Rn) with the same shared sequence modified at their 5' ends for the first round of target amplification, generating an intermediate product containing the shared sequence; a single enhancing primer FR, completely identical to the shared sequence, is used for the second round of amplification, significantly improving detection sensitivity. The provided enhancing primer plays a dominant role in the entire reaction process, and only this one primer participates in the second round of amplification, completely solving the problem of low amplification efficiency caused by competition among multiple sets of primers in traditional multiplex PCR systems, and solving the problem of difficulty in detecting low concentrations of targets.

[0040] 2. This invention innovatively achieves single-tube nested multiplex PCR through shared sequences, with a fully closed operation to avoid contamination, and can simultaneously detect 3-10 targets. This method can achieve absolute quantification of less than 10 copies / μL in a digital PCR system, and is particularly suitable for highly sensitive simultaneous detection of bloodstream pathogens (such as Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecalis) and drug resistance genes (mecA, vanA, IMP, etc.).

[0041] 3. This invention combines the high throughput of multiplex PCR with the high sensitivity of nested PCR, and has important application value in the fields of infectious disease diagnosis and drug resistance gene screening.

[0042] 4. The upstream and downstream primers provided by this invention use a low-concentration primer system in the first round of reaction, which can significantly reduce the problem of dimers within and between primers, thereby improving the specificity of multiplex PCR amplification.

[0043] 5. The technical solution provided by this invention can solve the risk of aerosol contamination caused by opening the nested PCR container twice, and at the same time, it eliminates the need for secondary sample addition, greatly improving the ease of operation.

[0044] 6. This invention provides an enhanced single-tube nested multiplex PCR detection method mediated by shared sequences. Through primer design modified with shared sequences and two-stage differential amplification, it achieves high sensitivity and high specificity multiplex target detection in a closed single tube and is compatible with digital PCR systems. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the reaction principle of the shared sequence-mediated enhanced single-tube nested multiplex PCR detection method provided in Embodiment 1 of the present invention;

[0046] Figure 2 To utilize the amplification curves for simultaneous detection of multiple pathogens provided in Example 1 of this invention;

[0047] Figure 3 To utilize the graph provided in Example 2 of this invention for screening genes for resistance to multiple antimicrobial drugs;

[0048] Figure 4 This is a diagram illustrating the use of Embodiment 3 of the present invention for achieving absolute quantification of multi-target copy numbers in a digital PCR system. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0050] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0051] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0052] The shared sequence-mediated enhanced single-tube nested multiplex PCR detection method provided by this invention is applicable to one or more of DNA, RNA, or their reverse transcription products, and also includes any artificially synthesized plasmid DNA molecules. The following examples use artificially synthesized plasmid DNA as the target to describe the method of this invention.

[0053] Example 1:

[0054] Simultaneous detection of three pathogens causing bloodstream infections:

[0055] This embodiment targets Staphylococcus aureus (SA), Enterococcus faecalis (EF), and Klebsiella pneumoniae (KP), and uses artificially synthesized pathogen 16S rRNA gene plasmids as sample templates, following the... Figure 1The technical principle illustrated demonstrates the construction of a single-tube nested multiplex PCR detection scheme. The prepared reaction system includes: 2× premix buffer, 3 pairs of target-specific upstream and downstream primers, 1 enhancement primer, 3 probes, 3 plasmid DNAs, and finally, water to a final volume of 20 μL. The primer and probe concentrations and their corresponding sequence information are as follows:

[0056] 0.4 μM enhancing primer: FR (SEQ ID NO:1),

[0057] 0.2μM upstream and downstream primers:

[0058] SA-F(SEQ ID NO:2), SA-R(SEQ ID NO:3),

[0059] EF-F (SEQ ID NO:4), EF-R (SEQ ID NO:5),

[0060] KP-F (SEQ ID NO:6), KP-R (SEQ ID NO:7);

[0061] 0.25 μM probes: SA-P (SEQ ID NO:8), EF-P (SEQ ID NO:9), KP-P (SEQ ID NO:10). The prepared reagent system was used for single-tube nested multiplex PCR in a quantitative PCR instrument. The reaction program was: 95℃ for 1 min; 10 cycles (95℃, 10 s; 55℃, 30 s); followed by 30 cycles (95℃, 10 s; 50℃, 30 s). Fluorescence signals were collected after each cycle, for a total of 40 measurements. The detection results are shown below. Figure 2 As shown, standard "S"-shaped amplification curves were observed for all three targets, indicating that the single-tube nested multiplex PCR detection method provided by this invention can simultaneously detect three bloodstream pathogens with high specificity and significantly improved detection efficiency.

[0062] Example 2:

[0063] Simultaneous screening of resistance genes for three antimicrobial drugs:

[0064] This embodiment targets the mecA, vanA, and IMP genes. First, a 20 μL single-tube nested multiplex PCR reagent system was prepared. This system included: 2× premix, 3 pairs of target-specific upstream and downstream primers, 1 enhancement primer, 3 probes, 3 plasmid DNAs, and finally, water was added to a final volume of 20 μL. The primer and probe concentrations and their corresponding sequence information are as follows:

[0065] 0.4 μM enhancing primer: FR (SEQ ID NO:1),

[0066] 0.2μM upstream and downstream primers:

[0067] mecA-F (SEQ ID NO:11), mecA-R (SEQ ID NO:12),

[0068] vanA-F (SEQ ID NO:13), vanA-R (SEQ ID NO:14),

[0069] IMP-F (SEQ ID NO:15), IMP-R (SEQ ID NO:16);

[0070] 0.25μM probes: mecA-P (SEQ ID NO:17), vanA-P (SEQ ID NO:18), IMP-P (SEQ ID NO:19).

[0071] The prepared reagent system was subjected to single-tube nested multiplex PCR in a quantitative PCR instrument. The reaction program was as follows: heating at 95℃ for 1 min; 10 cycles (95℃, 10 s; 55℃, 30 s); followed by 30 cycles (95℃, 10 s; 50℃, 30 s). Fluorescence signals were collected once after each cycle, for a total of 40 times. The detection results are shown below. Figure 3 As shown, the experimental groups containing target plasmid DNA all exhibited standard "S"-shaped amplification curves, while the negative control groups (NTC-1, NTC-2, NTC-3) without target plasmid DNA did not show any amplification curves. This indicates that the single-tube nested multiplex PCR detection method provided by this invention can simultaneously screen for three antimicrobial drug resistance genes (mecA, vanA, and IMP), demonstrating high specificity and significantly improving detection efficiency.

[0072] Example 3:

[0073] Adapting to digital PCR systems for absolute copy number quantification of multiple targets:

[0074] This embodiment uses Staphylococcus aureus, Enterococcus faecalis, and Klebsiella pneumoniae as detection targets. A 20 μL single-tube nested multiplex PCR reagent system was prepared according to the method described in Example 1. Then, on a biochip, the reaction system was uniformly divided into more than 20,000 microdroplets using a water-in-oil technique, with each microdroplet constituting an independent PCR reaction unit. The biochip was then thermally cycled on a PCR amplification instrument to achieve single-molecule-level target gene amplification. Finally, the fluorescence signal of the amplified microdroplets was acquired using a biochip reader, and the detection results are as follows: Figure 4As shown, the microdroplet fluorescence signal clearly distinguishes positive signals (bright spots) from negative background (dark spots) in the detection systems for the three pathogens, indicating that this scheme successfully achieves simultaneous and specific detection of SA, KP, and EF. The absolute copy numbers of the three pathogens calculated based on the Poisson distribution algorithm are: Staphylococcus aureus 4 copies / μL, Enterococcus faecalis 5 copies / μL, and Klebsiella pneumoniae 8 copies / μL, verifying that the described single-tube nested multiplex PCR method can be adapted to digital PCR systems to achieve absolute quantitative detection of multiple targets, accurately detecting even low-concentration targets, significantly improving detection sensitivity.

[0075] SEQ ID NO:1

[0076] gcactgcgag gtgc

[0077] SEQ ID NO:2

[0078] gcactgcgag gtgcgataaa tattttgaacc gcatggttc

[0079] SEQ ID NO:3

[0080] gcactgcgag gtgcgccgtt accttaccaa ctagc

[0081] SEQ ID NO:4

[0082] gcactgcgag gtgccacttg gaaacaggtg ctaatacc

[0083] SEQ ID NO:5

[0084] gcactgcgag gtgctggcct tggtgagccg t

[0085] SEQ ID NO:6

[0086] gcactgcgag gtgccaagcc tgatgcagcc atg

[0087] SEQ ID NO:7

[0088] gcactgcgag gtgcctgctg gcacggagtt agc

[0089] SEQ ID NO:8

[0090] acggtcttgc tgtcact

[0091] SEQ ID NO:9

[0092] cgctttcggg tgtcg

[0093] SEQ ID NO:10

[0094] aggaaggcga tgaggt

[0095] SEQ ID NO:11

[0096] tccttgtttc atttgagtct ctgc

[0097] SEQ ID NO:12

[0098] gcaacaagtc gtaaataaaa caca

[0099] SEQ ID NO:13

[0100] gtatcccttt tgtaggctgc ga

[0101] SEQ ID NO:14

[0102] ataacccaaa aggcggggagt ag

[0103] SEQ ID NO:15

[0104] tagtcacttg gtttgtggag cg

[0105] SEQ ID NO:16

[0106] tgctgtcgct atgaaaatga ga

[0107] SEQ ID NO:17

[0108] atttgccaat taagtttgc

[0109] SEQ ID NO:18

[0110] ttcaaagctc agcaatt

[0111] SEQ ID NO:19

[0112] tggctataaa ataaaaggc

[0113] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A shared sequence-mediated enhanced single-tube nested multiplex PCR detection method, characterized in that, Includes the following steps: S1. Target-specific amplification: Multiple pairs of upstream and downstream primers modified with shared sequences (F1, F2, ... Fn and R1, R2, ... Rn) are used to specifically recognize the target sequence and generate amplification products (P1, P2, ... Pn) containing the shared sequence through the first round of PCR reaction. S2. Shared sequence-mediated enhanced amplification: Using a single shared sequence enhancement primer FR, and with the amplification products (P1, P2, ... Pn) from step S1 as templates, a second round of PCR reaction is used to generate amplification products (P1, P2, ... Pn) containing the shared sequence. Steps S1 and S2 are performed continuously within a closed, single reaction tube.

2. The method according to claim 1, characterized in that, Steps S1 and S2 employ different temperature programs: The annealing / stretching temperature in step S1 is 52℃-62℃; The annealing / extending temperature for step S2 is 45℃-55℃.

3. The method according to claim 1, characterized in that, The number of cycles in steps S1 and S2 is controlled independently: The number of iterations in step S1 is 5-20. The number of iterations in step S2 is 20-40.

4. The method according to claim 1, characterized in that, The 5' ends of the upstream and downstream primers (F1, F2, ... Fn and R1, R2, ... Rn) all contain the same shared sequence M, and their general structural formula is as follows: MN(I) Wherein, M is the shared sequence; N is the target-specific recognition sequence.

5. The method according to claim 1, characterized in that, The nucleotide sequence of the enhancing primer FR is completely identical to the shared sequence M.

6. The method according to claim 1, characterized in that, Primer concentrations satisfy: The final concentration of the upstream and downstream primers (F1, F2, ... Fn and R1, R2, ... Rn) is 0.1-0.5 μM; The final concentration of the enhancing primer FR is 0.2-1.0 μM; Furthermore, the final concentration of the enhancing primer FR is not less than 1.5 times the final concentration of any upstream or downstream primer.

7. The method according to claim 1, characterized in that, The target to be tested includes one or more of DNA, RNA, or their reverse transcription products.

8. The application of the method according to any one of claims 1-7 in the detection of bloodstream infections for non-diagnostic or therapeutic purposes, characterized in that, Targets for simultaneous detection include: At least one of Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecalis.

9. The application of the method according to any one of claims 1-7 in screening for antimicrobial drug resistance genes, characterized in that, Targets for simultaneous detection include: At least one of the mecA gene, vanA gene, and IMP gene.

10. The application of the method according to any one of claims 1-7 in a digital PCR system, characterized in that, Includes the following steps: (1) Preparation of microdroplet reaction unit: The single-tube nested multiplex PCR reaction system is encapsulated in microdroplets on a biochip by using droplet-encapsulated oil to form a microdroplet single-molecule reaction system; (2) Microdroplet reaction unit amplification reaction: Thermal cycling amplification reaction of microdroplet single molecule reaction system was carried out using a PCR amplification instrument; (3) Fluorescence signal acquisition and quantitative analysis: The fluorescence signal in the microdroplets is detected by a digital PCR instrument or a biochip reader. The absolute concentration of multi-target molecules is calculated based on the Poisson distribution algorithm to achieve absolute quantification of molecules.

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