A method, kit and use of emQPCR multi-target joint detection of trace DNA

Abstract

The present invention discloses a method, a kit and a use of emQPCR multi-target joint detection of trace DNA, the method comprising the following steps: 1) emulsion microdroplet nested multiple PCR pre-amplification, preparing a water phase and an oil phase in a certain ratio to generate an oil-in-water microdroplet system, and then performing 10-20 cycles of nested PCR pre-amplification on the prepared microdroplet system; after the microdroplet pre-amplification, the emulsion is broken to obtain a pre-amplification product of the water phase; 2) multiple nested real-time fluorescent PCR amplification detection, using a set of "nested" primer probes for each detection target, and realizing a highly specific single-tube multiple first-round nested pre-amplification and a second-round fluorescent PCR amplification multiple joint detection through this set of "nested" primer probes. The present invention provides a highly sensitive, effective and rapid molecular detection scheme for the detection of pathogens such as bloodstream infection and bone and joint infection.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to a method, a kit and a use of emQPCR multi-target joint detection of trace DNA, and more specifically, relates to a method, a kit and a use of high-sensitivity and high-specificity detection of multiple pathogen target nucleic acids in bloodstream infection samples. Background Art

[0002] At present, blood culture is the main method for identifying pathogenic microorganisms in sterile body fluid samples (such as whole blood, joint puncture fluid, cerebrospinal fluid, etc.). However, relevant data show that the positive rate of culture in patients with sepsis is low (about 1-2% in newborns and up to 20-30% in emergency departments). The low positive rate is mainly due to the widespread use of antibiotics and the low amount of pathogens in the blood of BSI patients. For patients with clinical manifestations of sepsis, the negative rate of blood culture results is high, and antibacterial treatment is mainly empirical treatment. Early detection and adequate treatment of pathogens within 6-12 hours are crucial for patients with bloodstream infections to achieve good treatment results. The development of molecular detection technology can further supplement the deficiencies of traditional culture methods. The development of rapid detection methods for bloodstream infections has been regarded as an important medical need to supplement traditional blood culture diagnosis.

[0003] At present, the molecular detection methods that can be widely used in clinical practice include real-time fluorescence PCR technology, capillary electrophoresis detection technology, isothermal amplification technology, etc., which have shortened the diagnosis time to a certain extent, but have low sensitivity and cannot quickly identify specimens with trace bacterial content in patient samples. In addition, the detection targets are limited and cannot meet the needs of rapid detection and early diagnosis of pathogens of clinical sterile body fluid infections.

[0004] The simultaneous detection of multiple gene targets is usually restricted by the following factors: first, the mutual interference between multiplex PCR primers and templates is serious, which limits the amplification efficiency of multiplex PCR; for multiplex PCR amplification of trace DNA, the template concentration is too low, the interaction between molecules is reduced, and the primer concentration is high and the number of molecules is excessive, which greatly increases the probability of the primers themselves forming dimers. The above reasons lead to the incorrect consumption of each component in the system, making it impossible to complete the amplification of the target product. Although a booster PCR method was developed in 1989, this method requires splitting an amplification cycle into steps, and the lid needs to be opened during the process. The operation is complicated, easy to cause contamination, and limited to single-site analysis.

[0005] In order to solve this problem, the present invention uses emulsion droplets combined with nested amplification technology. The small droplets formed by droplet PCR constitute an independent PCR reaction space, which effectively solves the problem of mutual interference between primers and primers, and between primers and templates in multiple PCR, especially when the template concentration is as low as dozens of copies. In addition, the combination of nested amplification and emulsion droplet technology achieves high-efficiency and high-specificity amplification of trace DNA through amplification with a relatively small number of cycles, because more amplification cycles will make it easier to produce non-specific amplification.

[0006] Secondly, the current detection methods for PCR amplification products mainly include real-time fluorescence quantitative PCR, digital PCR, second-generation sequencing and capillary electrophoresis. The real-time fluorescence quantitative PCR (qPCR) technology of Taqman probe method is not sensitive enough for low starting sample detection. Although digital PCR technology has high detection sensitivity, the detection cost is high and the number of detection targets is limited. Although the second-generation sequencing has a large detection throughput, the cost of single sample detection is high and it is not suitable for universal clinical development. Therefore, a method is needed that can not only meet the requirements of high-sensitivity detection of body fluid samples with low pathogenic microorganism content and the needs of multi-target detection, but also can be suitable for widespread clinical development at low detection cost and instrument cost. Summary of the invention

[0007] In view of the problems of low sensitivity, poor specificity, and limited target detection in existing nucleic acid detection of sterile body fluid samples with low pathogen content, especially the requirement of both detection performance and timeliness for bloodstream infection samples, the present invention provides a method for multi-target joint detection of trace DNA by emQPCR (emulsion Quantitative real-time PCR, abbreviated as emQPCR), that is, the present invention combines emulsion droplet technology, nested PCR based on specially designed "nested" primer probe sets, and real-time fluorescence PCR technology to perform multi-target joint detection of trace pathogen nucleic acids. The characteristics of this method are as follows:

[0008] 1. Development of nested emulsion PCR pre-amplification technology for single tube ≥ 8-plex:

[0009] (1) In existing emulsion droplet amplification technologies such as digital PCR, single-tube amplification is generally 1 to 4-fold, and a cycle of no less than 40 cycles is required to achieve a low sensitivity. In the present invention, a specially designed "nested" primer set can achieve no less than 8-fold nested amplification in a single tube, that is, a single-tube ≥ 8-fold nested emulsion PCR low-cycle pre-amplification is achieved, and ultimately a sensitivity of the target bacteria as low as 10 CFU / mL is achieved.

[0010] (2) The realization of the above-mentioned single-tube ≥ 8-plex solution requires that the primer pairs designed for each target gene have high amplification efficiency and good specificity, there is no cross-reaction between primer pairs of different targets, and the first round of pre-amplification can still effectively obtain amplification products when the template concentration is very low (as low as 100 copies or even dozens of copies). The above technical difficulties were solved by combining emulsion droplet technology with a specially designed nested amplification method, strict primer and probe design and screening, and optimization of the optimal ratio of primer and probe group concentration system.

[0011] 2. Multiple nested fluorescent PCR multiple detection technology:

[0012] The effective products obtained from the first round of single-tube multiplex nested emulsion PCR pre-amplification were demulsified to obtain the aqueous phase, and then the nucleic acid recovered from the demulsification was used as a template for the second round of multiplex real-time fluorescence PCR amplification (abbreviated as "qPCR"). The nested multiplex micro-droplet pre-amplification combined with the fluorescent probe qPCR multiplex detection can improve the specificity and sensitivity of multi-target detection at the same time. The detection limit can reach 60 copies / mL or 10 CFU / mL, which is suitable for sterile body fluid samples with low pathogenic microorganism content, especially for multi-target joint detection of sterile body fluid samples such as whole blood, joint fluid, and cerebrospinal fluid.

[0013] The purpose of the present invention is to provide a method for emQPCR multi-target joint detection of trace DNA (the method can also be referred to as a method for realizing a single multiple pathogen target nucleic acid joint detection by emulsion droplet nested PCR pre-amplification combined with a real-time fluorescent PCR probe melting curve double discrimination method), and to provide a kit for joint detection and differentiation of pathogen multi-target nucleic acids comprising a specific primer probe set and its use.

[0014] According to a first aspect of the present invention, the present invention provides a method for emQPCR multi-target joint detection of trace DNA, wherein the method establishes an emulsion droplet independent amplification system to perform a first round of nested multiple emulsion PCR pre-amplification reaction; then the product of the first round of nested multiple emulsion PCR pre-amplification is demulsified to obtain an aqueous phase, and then the second round of multiple real-time fluorescence PCR amplification is directly performed using the first round of nested multiple emulsion PCR pre-amplification products of the demulsified aqueous phase as a template, and a highly sensitive multi-target fluorescence amplification analysis and detection of trace pathogen nucleic acids is performed.

[0015] Here, the "trace amount" refers to a very low concentration of the template, as low as 100 copies or even dozens of copies.

[0016] The method uses a specially designed "nested" primer probe set to perform the first round of single-tube 8-plex nested emulsion PCR pre-amplification.

[0017] The method specifically comprises the following steps:

[0018] Step 1: The first round of single-tube nested multiplex emulsion PCR pre-amplification based on the emulsion droplet reaction system

[0019] The water phase and the oil phase are mixed to prepare a sufficient amount of water-in-oil emulsion droplets (not less than 40,000), and then the prepared emulsion droplets are collected into eight PCR tubes with sealed rubber caps, and then a 10-20 cycle first round of single-tube nested multiple emulsion PCR pre-amplification is performed;

[0020] The aqueous phase contains nucleic acid templates extracted and purified from pathogenic microorganisms of the sample to be tested, specially designed "nested" primer pairs for nested multiple pre-amplification reactions of detection targets, and multiple PCR reaction solution Mix (including multiple reaction buffers, multiple hot-start Taq DNA polymerases, etc.).

[0021] Among them, the pathogens (pathogenic microorganisms) include Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

[0022] The volume ratio of the water phase to the oil phase is 1:5 to 1:10;

[0023] As a preferred embodiment of the present invention, the final concentration of the forward and reverse outer primers in the aqueous phase is 0.1-0.9 μM, and the fragment size of the amplified product of the forward and reverse primer pairs is 150-450 bp; the first round of nested multiplex PCR pre-amplification program is 95° C. for 10-100 s; 94° C. for 5-30 s, 52-62° C. for 10-60 s, and 10-20 cycles.

[0024] Preferably, the final concentration of forward and reverse primers in the aqueous phase is 0.15-0.75 μM, and the fragment size of the amplified product of the forward and reverse primer pairs is 190-410 bp; the first round of nested multiple emulsion PCR pre-amplification program is 95°C 30s; 94°C 10s, 53°C 30s, 15 cycles.

[0025] Step 2: Demulsification to separate the water phase and the oil phase

[0026] In the step 1), after the first round of single-tube nested multiple emulsion PCR pre-amplification, the product is demulsified to separate the water phase and the oil phase, thereby obtaining the first round of nested multiple emulsion PCR pre-amplification products in the water phase.

[0027] The dosage of the microdroplet demulsifier after amplification is at least 1 to 3 times the reaction volume before microdroplet amplification to ensure that the oil phase and the water phase are completely separated after demulsification to achieve a stable recovery rate.

[0028] Step 3: Second round of multiplex real-time PCR multiplex detection

[0029] Using the first round pre-amplification product of the aqueous nested multiplex emulsion PCR obtained in step 2) as a template, add 8 specific modified primer pairs and probe sets of the detection target and internal standard gene, and perform the second round of multiplex real-time fluorescence PCR amplification (double discrimination of probe melting curve) to perform multi-target joint detection of trace pathogen nucleic acids.

[0030] The test results are directly judged according to the discrimination principle of the corresponding target. The primer set of the first round of single-tube nested multiple emulsion PCR pre-amplification and the modified primer probe set of the second round of multiple real-time fluorescence PCR multiple detection are used to perform a two-step nested multiple PCR reaction. That is, the primer set used in the first round of single-tube multiple amplification is the outer primer for target-specific gene amplification, and the modified primer probe set of the second round of multiple real-time fluorescence PCR multiple detection is the inner primer.

[0031] Preferably, the upstream or downstream end of the inner primer can be shared with the upstream or downstream end of the outer primer, but it is required that no additional cross or non-specific amplification can be generated, so as to further improve the amplification efficiency and system stability. The advantage of this design is that the second round of amplifications each binds to its first stage primer with the same kinetics, and the complexity of the first round of single-tube multiplex PCR reaction can be minimized.

[0032] As a preferred embodiment of the present invention, the specific amplification primer probe set for the detection target is "nested" or designed inside the specific product of the first round of nested multiplex PCR reaction. Preferably, after multiple rounds of screening, it is optimal to share only one upstream or downstream primer among the target-specific outer primer and inner primer pairs, thereby improving the sensitivity and specificity of the reaction.

[0033] As a preferred embodiment of the present invention, the final concentrations of the specific modified inner primers and modified probe sets of the eight detection target genes involved are 0.05-0.5 μM and 0.05-0.3 μM, respectively. The second round of multiplex real-time fluorescence PCR amplification program is 37°C 2-5 min, 95°C 1-5 min, 95°C 10-30 s, 63-55°C 30-90 s, 30-45 cycles, and fluorescence collection at 60°C→90°C.

[0034] Preferably, the final concentrations of the specific modified inner primers and modified probe sets of the eight detection target genes involved in the present invention are 0.15-0.3 μM and 0.06-0.15 μM, respectively. The second round of multiplex real-time fluorescence PCR amplification program is 37°C for 2 min, 95°C for 1 min; 95°C for 15 s, 63-55°C for 30 s, 10 cycles of TD amplification; 95°C for 15 s, 55°C for 30 s, 30 cycles; 68°C→90°C to collect fluorescence.

[0035] In the present invention, a primer probe set is required for detecting the target, and the primer probe set is a nested primer probe set; the sequence of the nested primer probe set includes:

[0036] An outer primer pair, an inner primer pair and a probe for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 1 to 5;

[0037] An outer primer pair, an inner primer and a probe for detecting Staphylococcus aureus as shown in SEQ ID NOs: 6 to 9;

[0038] An outer primer pair, an inner primer and a probe for detecting Escherichia coli as shown in SEQ ID NOs: 10 to 13;

[0039] An outer primer pair, an inner primer and a probe for detecting Enterococcus faecium as shown in SEQ ID NOs: 14 to 17;

[0040] An outer primer pair, an inner primer pair and a probe for detecting Enterococcus faecalis as shown in SEQ ID NOs: 18 to 22;

[0041] An outer primer pair, an inner primer pair and a probe for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 23 to 27;

[0042] An outer primer pair, an inner primer pair and a probe for detecting Pseudomonas aeruginosa as shown in SEQ ID NOs: 28 to 32;

[0043] The outer primer pair, inner primer pair and probe for detecting Stenotrophomonas maltophilia are shown in SEQ ID NOs: 33-36.

[0044] It should be noted that the second round of multiplex real-time fluorescent PCR amplification can be achieved by the following two methods:

[0045] Method 1: In the second round of amplification, 1 to 4 targets (including internal reference genes) are amplified in two reaction tubes respectively, and the test results are judged according to the discrimination principle of the targets contained in each reaction tube;

[0046] Method 2: The second round amplification method provides multiple reaction wells / tubes (each reaction well has a primer probe set for amplifying a locus of a different sequence from a plurality of target nucleic acid sequences), provides a portion of the sample into each of the multiple reaction wells, and simultaneously subjects the multiple reaction wells to several amplification cycles, detects whether amplification occurs in each of the multiple reaction wells in the second round of reaction, and interprets the result by identifying the corresponding targets present in the sample by identifying the corresponding wells in which amplification occurs.

[0047] It should be noted that the Mix of the first round of single-tube nested multiple emulsion PCR pre-amplification reaction is different from the Mix of the second round of multiple real-time fluorescence PCR multiple detection reaction. The Mix of the first round of single-tube nested multiple emulsion PCR pre-amplification reaction should not only meet the specificity and efficiency of PCR amplification, but also ensure the stable generation of the emulsion micro-droplet system and the stability of subsequent micro-droplet PCR amplification.

[0048] The present invention also provides a primer-probe composition for the joint detection of multiple targets of nucleic acid of bloodstream infection pathogens, comprising one or more probes and one or more primer sets, wherein the primer-probe set for each target detection is a nested primer-probe set; and the joint detection of multiple targets of nucleic acid of bloodstream infection pathogens is performed.

[0049] Specifically, the primer-probe combination (a specially designed nested primer-probe combination) comprises:

[0050] SEQ ID NOs: 1 to 5 are the first round pre-amplification outer primer pair, the second round multiplex fluorescent PCR amplification inner primer pair and the probe set for detecting Klebsiella pneumoniae;

[0051] 5'-GGACATCATCCGTTCCAATACC-3' (SEQ ID NO:1)

[0052] 5'-TTCGCTTCCACGACTTCG-3' (SEQ ID NO:2)

[0053] 5'-CCGTTCCAATACCTTTTGTGG-3' (SEQ ID NO:3)

[0054] 5'-GCGACAGTAAAGGCAGAAT-3' (SEQ ID NO:4)

[0055] 5'-CCGGTCATTGGTGGTCACTCC-3' (SEQ ID NO:5)

[0056] SEQ ID NOs: 6 to 9 are the first round pre-amplification outer primer pair, the second round multiplex fluorescent PCR amplification inner primer pair and the probe set for detecting Staphylococcus aureus;

[0057] 5'-TGTTTCGAAAGGGCAATACGC-3' (SEQ ID NO:6)

[0058] 5'-ATGCACTTGCTTCAGGACCAT-3' (SEQ ID NO:7)

[0059] 5'-GGATCTTCAGAACCACTTCTATT-3' (SEQ ID NO:8)

[0060] 5'-AGCTCAGCAAATGCATCACAAACAGAT-3' (SEQ ID NO:9)

[0061] SEQ ID NOs: 10 to 13 are the first round pre-amplification outer primer pair, the second round multiplex fluorescent PCR amplification inner primer pair and the probe set for detecting Escherichia coli;

[0062] 5'-ACCTTTGCTGAAACGGCAAC-3' (SEQ ID NO:10)

[0063] 5'-TCGAGATCGACCGTCTCG-3' (SEQ ID NO:11)

[0064] 5'-CGCTTCCGTCACCGAATT-3' (SEQ ID NO:12)

[0065] 5'-CGCCTGTGCCTGTTCACGCAGC-3' (SEQ ID NO: 13)

[0066] SEQ ID NOs: 14 to 17 are the first round pre-amplification outer primer pair, the second round multiplex fluorescent PCR amplification inner primer pair and the probe set for detecting Enterococcus faecium;

[0067] 5'-GGTTCTTTGCTTTATCCGATG-3' (SEQ ID NO: 14)

[0068] 5'-GCGCTTCAATTCCTTGTTCAA-3' (SEQ ID NO:15)

[0069] 5'-CCGGCGAATATGGGTTCTA-3' (SEQ ID NO:16)

[0070] 5'-CGGCATTACAAAAGCAGAAAACCGAGAAGAGC-3' (SEQ ID NO: 17)

[0071] SEQ ID NOs: 18 to 22 are the first round pre-amplification outer primer pair, the second round multiplex fluorescent PCR amplification inner primer pair and the probe set for detecting Enterococcus faecalis;

[0072] 5'-TGTCGGAATTAGCAAAGCG-3' (SEQ ID NO:18)

[0073] 5'-GCGACATCTTTCACCACTT-3' (SEQ ID NO:19)

[0074] 5'-TGTCGGAATTAGCAAAGCGGAA-3' (SEQ ID NO: 20)

[0075] 5'-ACGTGCTTCAATCCCTTGTTCAA-3' (SEQ ID NO: 21)

[0076] 5'-TGCTCGGGCATCATAACGGAAAGCTTCTTCCA-3' (SEQ ID NO: 22)

[0077] SEQ ID NOs: 23 to 27 are the first round pre-amplification outer primer pair, the second round multiplex fluorescence PCR amplification inner primer pair and the probe set for detecting Acinetobacter baumannii;

[0078] 5'-TAATGCTTTGATCGGCCTTG-3' (SEQ ID NO: 23)

[0079] 5'-TGGATTGCACTTCATCTTGG-3' (SEQ ID NO: 24)

[0080] 5'-TGGCAATGCAGATATCGGTAC-3' (SEQ ID NO: 25)

[0081] 5'-TGGAAGCGTTTTATTAGCTAGCTTG-3' (SEQ ID NO: 26)

[0082] 5'-ACTCCTCAGCAAGAGGCACAGTTTGC-3' (SEQ ID NO: 27)

[0083] SEQ ID NOs: 28 to 32 are the first round pre-amplification outer primer pair, the second round multiplex fluorescent PCR amplification inner primer pair and the probe set for detecting Pseudomonas aeruginosa;

[0084] 5'-GTAGTGCTGGAAGGCCACAC-3' (SEQ ID NO: 28)

[0085] 5'-AACGTCAGGACACGCAGGT-3' (SEQ ID NO: 29)

[0086] 5'-GAACTGGTTTCCTATGGTAAAGAG-3' (SEQ ID NO:30)

[0087] 5'-CTTGGGCATAACGACTTCTTAC-3' (SEQ ID NO:31)

[0088] 5'-CCGGCCACGACGAGCAGTCC-3' (SEQ ID NO:32)

[0089] SEQ ID NOs: 33-36 are the first round pre-amplification outer primer pair, the second round multiplex fluorescent PCR amplification inner primer pair and the probe set for detecting Stenotrophomonas maltophilia.

[0090] 5'-GCGGAATACTTCGGTCTG-3' (SEQ ID NO:33)

[0091] 5'-AACAAGGGCAAGCTGATCA-3' (SEQ ID NO:34)

[0092] 5'-AAGGCGATGGTCTCGGTTT-3' (SEQ ID NO:35)

[0093] 5'-CGTTCTCCAAGCTGAAGTTCATGGTCAGCATGG-3' (SEQ ID NO:36)

[0094] The primer-probe composition for multi-target joint detection of nucleic acid of bloodstream infection pathogens provided by the present invention adopts a specially designed nested primer-probe group scheme to carry out the first round of single-tube nested multiple oil-in-water emulsion PCR (≥8-fold) pre-amplification reaction, obtain the effective amplification product of the trace DNA starting template, and directly carry out the second round of multiple fluorescent quantitative PCR reaction with the obtained aqueous phase first round amplification product as a template to realize the multi-target joint detection of pathogens. Through the special design method of the present invention, high-sensitivity and high-specificity rapid detection of bacteria in the blood, including but not limited to Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, is realized, and the target detection sensitivity reaches 60 copies / mL or 10 CFU / mL.

[0095] Furthermore, the primer-probe composition also includes an upstream primer and a downstream primer for detecting an internal standard, one of which is a modified primer, which can be used for melting curve analysis and judgment, thereby increasing the number of single-channel target detections. In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is GAPDH, and its upstream and downstream primer sequences are: 5'-GATCATCAGCAATGCCTCCT' (SEQ ID NO: 37) and 5'-TGAGTCCTTCCACGATACCA-3' (SEQID NO: 38), respectively.

[0096] In some specific embodiments, the fluorescent reporter group of the Klebsiella pneumoniae and Stenotrophomonas maltophilia probes is FAM; the fluorescent reporter group of the Enterococcus faecalis and Enterococcus faecium probes is HEX; the fluorescent reporter group of the Staphylococcus aureus and Acinetobacter baumannii probes is ROX; and the fluorescent reporter group of the Pseudomonas aeruginosa and Escherichia coli probes is CY5.

[0097] Further, in some embodiments, the primer-probe combination of the present invention may include one or more of the above primer and probe sets. In the present invention, "group" refers to a set of "nested" primer-probe pairs for each detection target, through which the specific first round of nested pre-amplification and the second round of fluorescent PCR amplification are achieved.

[0098] The primer-probe combination of the present invention can be arbitrarily combined into any combination form corresponding to the detection of 8 targets. Those skilled in the art can combine as needed to detect which targets, that is, combine the first and second round primers and probe sets corresponding to the targets. These combinations are all included in the present invention.

[0099] For example, it may include any 7 of the above 8 sets of primers and probes, it may include any 6 of the above 8 sets of primers and probes, it may include any 5 of the above 8 sets of primers and probes, it may include any 4 of the above 8 sets of primers and probes, it may include any 3 of the above 8 sets of primers and probes, it may include any 2 of the above 8 sets of primers and probes, and it may also include any 1 of the above 8 sets of primers and probes.

[0100] Wherein, the fluorescent groups of the composite probes do not interfere with each other.

[0101] In some specific embodiments, the primer-probe combination of the present invention is ultimately used for real-time fluorescent PCR.

[0102] In the present invention, the components of the primer-probe composition are present in a mixed form.

[0103] The present invention also provides a reagent / kit / product for the joint detection and differentiation of multiple targets of nucleic acid of bloodstream infection pathogens, wherein the reagent / kit / product comprises the primer-probe combination as described above.

[0104] Furthermore, the reagent / kit / product also includes negative quality control products and positive quality control products.

[0105] In a specific embodiment, the negative control is at least one of 1×TE, ultrapure water, and human cells. The positive control is at least one of a plasmid or DNA fragment of Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

[0106] Furthermore, the reagent / kit / product also includes dNTP, PCR buffer and Mg 2+ At least one of .

[0107] Furthermore, the reagent / kit / product also includes: a nucleic acid extraction reagent, and at least one of multiple hot-start DNA polymerases.

[0108] Furthermore, the reagent / kit / product also includes a nucleic acid extraction reagent, a multiple hot start DNA polymerase, dNTP, dUTP, uracil glycosylase (UDG), a PCR buffer, and Mg 2+ At least one of .

[0109] In a specific embodiment, the reagent / kit / product of the present invention comprises a multiple hot start DNA polymerase, Mg 2+ , dNTP(U)s, primers, probes and PCR reaction buffer.

[0110] In a specific embodiment, the reagent / kit / product of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0111] The present invention also provides a multi-target joint detection system for nucleic acid of bloodstream infection pathogens, the system comprising the primer-probe combination as described above, wherein the target-specific amplification inner primer probe group is "nested" or designed inside the specific product of the first round of nested multiple emulsion PCR reaction; in the target-specific inner primer pair involved, its upstream or downstream primer is shared with the upstream or downstream primer of its corresponding outer primer, further improving the amplification efficiency and system stability, and can minimize the complexity of the first round single-tube 8-plex PCR reaction system.

[0112] The present invention also provides the use of the primer-probe combination in preparing reagents / kits / products.

[0113] The present invention also provides application of the primer-probe combination in multi-target joint detection of trace amounts of pathogen nucleic acids.

[0114] The present invention also provides the use of the primer-probe combination in the joint detection and differentiation of multiple targets of nucleic acid of bloodstream infection pathogens.

[0115] The present invention also provides the use of the primer-probe combination in preparing a reagent for multi-target joint detection and differentiation of bloodstream infection pathogens.

[0116] The present invention also provides the use of the reagent / kit / product in the multi-target joint detection of trace amounts of pathogen nucleic acids.

[0117] The present invention also provides the use of the reagent / kit / product in preparing a reagent for multi-target joint detection and differentiation of bloodstream infection pathogens.

[0118] The present invention also provides the application of the bloodstream infection pathogen nucleic acid multi-target joint detection system in the multi-target joint detection of trace pathogen nucleic acids.

[0119] The present invention also provides a use of the bloodstream infection pathogen nucleic acid multi-target joint detection system in preparing a reagent for multi-target joint detection and differentiation of bloodstream infection pathogens.

[0120] The present invention also provides a method for joint detection and differentiation of pathogens, the method comprising the following steps:

[0121] 1) Extract nucleic acid from the sample to be tested;

[0122] 2) using the primer-probe combination or the reagent / kit / product as described above to perform a first round of single-tube nested multiplex emulsion PCR pre-amplification and a second round of multiplex fluorescence quantitative PCR on the nucleic acid obtained in step 1);

[0123] 3) Obtain and analyze the results.

[0124] In the present invention, the sample used for detection may be blood, plasma, etc., but is not limited thereto. It may also be joint puncture fluid, joint cavity effusion, cerebrospinal fluid, etc.

[0125] Furthermore, the reaction conditions of the first round of single-tube nested multiple emulsion PCR pre-amplification are 95° C. for 30 s; 94° C. for 10 s, 53° C. for 30 s, and 15 cycles.

[0126] The reaction conditions of the second round of multiplex fluorescence PCR were 37°C for 2 min, 95°C for 1 min; 95°C for 15 s, 63-55°C for 30 s, 10 cycles; 95°C for 15 s, 55°C for 30 s, 30 cycles; and fluorescence collection at 68°C→90°C.

[0127] The beneficial effects of the present invention are as follows: the latex droplets provided by the present invention are combined with a specially designed nested PCR technology, and a trace DNA multiplex detection method combined with a multiplex fluorescence PCR amplification technology, which realizes a single-tube ≥8-fold fluorescence PCR multiplex detection when the template concentration is as low as dozens of copies, has high specificity and high sensitivity, and the target gene detection sensitivity is as low as 60 copies / mL or 10 CFU / mL of target bacteria, providing a highly sensitive, effective and rapid molecular detection solution for the detection of pathogens such as bloodstream infection and bone and joint infection.

[0128] In addition, the final detection of the method and kit developed by the present invention can be achieved through a conventional four-channel fluorescent quantitative PCR instrument, and such instruments are equipped in clinical testing departments. Compared with other molecular detection methods currently available for sterile body fluid samples such as bloodstream infections, the kit developed by the method of the present invention has the characteristics of simple operation, lower detection cost, and a wide range of applications. It can realize multi-target joint detection of trace pathogen nucleic acids without the need for additional expensive instruments. It is economical and practical, and more suitable for general clinical inspections. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 This is a flow chart of the two-step nested emulsion multiplex fluorescence PCR detection method of the present invention;

[0130] Figure 2 is a graph showing the test results of the composition of the present invention (8 pathogens); wherein, Figure 2A This is the test result diagram of Klebsiella pneumoniae / Acinetobacter baumannii / Escherichia coli / Enterococcus faecalis; Figure 2B This is the test result diagram of Stenotrophomonas maltophilia / Staphylococcus aureus / Pseudomonas aeruginosa / Enterococcus faecium;

[0131] FIG3 is a graph showing the sensitivity results of the composition of the present invention (8 pathogens); wherein, Figure 3A This is the sensitivity result diagram of Escherichia coli; Figure 3B This is the sensitivity result diagram of Stenotrophomonas maltophilia; Figure 3C This is the sensitivity result diagram of Acinetobacter baumannii; Figure 3D This is the sensitivity result diagram of Enterococcus faecalis; Figure 3E This is the sensitivity result diagram of Klebsiella pneumoniae; Figure 3F This is the sensitivity result diagram of Staphylococcus aureus; Figure 3G This is the sensitivity result diagram of Enterococcus faecium; Figure 3H This is the sensitivity result diagram of Pseudomonas aeruginosa;

[0132] FIG4 is a sensitivity result diagram of the composition of the present invention (3 targets); wherein, Figure 4A The 50CFU / mL test results for Klebsiella pneumoniae / Enterococcus faecium / Staphylococcus aureus; Figure 4BThe test results are 20CFU / mL for Klebsiella pneumoniae / Enterococcus faecium / Staphylococcus aureus; Figure 4C The results are 10 CFU / mL for Klebsiella pneumoniae / Enterococcus faecium / Staphylococcus aureus;

[0133] Figure 5 It is a specific result diagram of the composition of the present invention;

[0134] FIG6 is a comparative analysis diagram of target specificity between the emulsion microdroplet nested multiplex fluorescence PCR detection method of the present invention and the conventional fluorescence multiplex PCR detection method, wherein: Fig. 6A The target detection result of Klebsiella pneumoniae using the method of the present invention (Ct24.61); Figure 6B This is the result of the Klebsiella pneumoniae target detection using a common fluorescent multiplex method (Ct32.33); Figure 6C The target detection result of Pseudomonas aeruginosa using the method of the present invention (Ct28.24); Fig.6D This is the result of Pseudomonas aeruginosa target detection using a common fluorescent multiplex method (Ct32.24). DETAILED DESCRIPTION

[0135] The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may be subject to various modifications and changes.

[0136] Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0137] The invention provides a method, a kit and use for multi-target joint detection of trace pathogen nucleic acids. The method comprises the following steps: 1) emulsion microdroplet nested multiplex PCR pre-amplification, comprising multiple primer sets required for pre-amplification, template DNA, dNTPs, PCR buffer and DNA polymerase as an aqueous phase, microdroplet generated oil as an oil phase, preparing the aqueous phase and the oil phase in a certain ratio to generate a microdroplet system, and then performing 10-20 cycles of the first round of nested emulsion pre-amplification on the prepared microdroplet system; after the microdroplet pre-amplification PCR, demulsification is performed to obtain a pre-amplified PCR product of the aqueous phase; 2) multiplex nested real-time fluorescent PCR amplification, using a group of "nested" primer probe pairs for each detection target, and realizing the specific first round of nested pre-amplification and the second round of fluorescent PCR amplification multiplex detection through the group of "nested" primer probe groups. The final detection of the method developed by the present invention can be achieved by a conventional fluorescent quantitative PCR instrument, and this instrument is equipped in most scientific research institutes and clinical testing departments. Therefore, the kit developed based on the method described in the present invention has simple operation, low detection cost, and a wide range of applications. It can realize multi-target joint detection of trace pathogen nucleic acids without the need for additional expensive instruments, and is economical and practical. The latex droplets provided by the present invention are combined with a specially designed nested PCR technology, and a micro-DNA multiple detection method combined with multiple fluorescent PCR amplification technology, which realizes a single tube ≥ 8-fold fluorescent PCR multiple detection when the template concentration is as low as dozens of copies, has high specificity and high sensitivity, and the target gene detection sensitivity is as low as 60 copies / mL or 10 CFU / mL of target bacteria, providing a highly sensitive, effective and rapid molecular detection solution for the detection of pathogens such as blood influenza and bone and joint infections.

[0138] Example 1: The primers and probes used in the present invention are shown in Table 1 below.

[0139] Table 1: Primer and probe information

[0140]

[0141] Among them, the fluorescent reporter group of the Klebsiella pneumoniae target gene probe is FAM; the fluorescent reporter group of the Staphylococcus aureus target gene probe is ROX; the fluorescent reporter group of the Escherichia coli target gene probe is CY5 or FAM; the fluorescent reporter group of the Enterococcus faecium and Enterococcus faecalis target gene probe is HEX; the fluorescent reporter group of the Acinetobacter baumannii target gene probe is ROX; the fluorescent reporter group of the Pseudomonas aeruginosa target gene probe is CY5 or ROX; the fluorescent reporter group of the Stenotrophomonas maltophilia target gene probe is FAM. It should be noted that the multi-selectivity of the second round of fluorescent PCR amplification management scheme in the two-step nested emulsion multiplex fluorescent PCR detection method provided by the present invention can greatly improve the freedom of target channel selection, and flexibly select and use channels with relatively weak fluorescence signals or channels with relatively strong fluorescence interference. For example, Enterococcus faecalis and Enterococcus faecium can both select the HEX fluorescent channel without affecting the result judgment, and the fluorescent reporter groups of Pseudomonas aeruginosa and Escherichia coli target gene probes can be CY5 or ROX or FAM.

[0142] Example 2: Method for multiple pathogen detection

[0143] The first round of single tube 8-fold nested emulsion PCR pre-amplification reaction solution: contains PCR reaction buffer, emulsion PCR multiplex enzyme, Mg2 + , dNTP, primers, etc. The specific reaction system is shown in Table 2.

[0144] Table 2:

[0145] Components Volume / concentration in each reaction PCR Buffer 5μL DNA polymerase 1.25μL Primer (nM) 3μL Template DNA 10μL <![CDATA[ddH 2 The]]> Make up to 30 μL

[0146] The reaction procedure of the first round single tube 8-plex nested emulsion PCR amplification is shown in Table 3.

[0147] Table 3:

[0148]

[0149] The second round of nested fluorescent PCR multiplex amplification reaction solution: PCR reaction buffer, Taq enzyme, Mg2 + , dNTP, UDG, primer probe, etc. The specific reaction system is shown in Table 4.

[0150] Table 4:

[0151]

[0152] The reaction procedure of the second round of nested fluorescent PCR multiplex detection amplification is shown in Table 5.

[0153] Table 5:

[0154]

[0155] Result analysis and judgment:

[0156] After the reaction is completed, the results are automatically saved, and the amplification curve and melting curve of the detection target in each tube are analyzed separately. For the amplification curve, adjust the Start value, End value and Threshold value of the Baseline according to the image after analysis (the user can adjust it according to the actual situation, the Start value can be set between 3 and 15, the End value can be set between 5 and 20, and adjust the amplification curve of the negative control to make it flat or below the threshold line), click Analyze for analysis, make all parameters meet the requirements of the following "Quality Control", and then record the qualitative results in the Plate window.

[0157] Quality Control:

[0158] Negative control: no Ct value is displayed in the curves of the four channels of FAM, HEX, ROX, and CY5;

[0159] Positive control: The curves of the three channels of FAM, ROX, and HEX are Ct≤36;

[0160] The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be invalid and must be repeated.

[0161] In addition, during the first round of emulsion droplet generation and amplification, the reaction system should be in a stable emulsified state, and there should be no obvious droplet breakage or no emulsification state when observed with the naked eye. If the above-mentioned emulsion droplet abnormalities occur, droplet generation and pre-amplification need to be repeated.

[0162] Positive judgment value:

[0163] The Ct reference value of the target gene detected by this kit was determined to be 38 through reference value research.

[0164] According to the above test results, the judgment results are as follows:

[0165]

[0166] Example 3: Detection results of the nested primer-probe combination test sample of the present invention

[0167] The primers and probes of the two-round nested multiplex fluorescent PCR multiplex detection shown in Example 1 of the present invention were used to detect Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Stenotrophomonas maltophilia, Staphylococcus aureus, Enterococcus faecium and Pseudomonas aeruginosa on a SLAN96 fluorescent PCR instrument after two rounds of multiple nested amplification. The detection results are shown in Figure 2. It can be seen from Figure 2 that the composition of the present invention can perform good detection on various pathogens.

[0168] Example 4: Sensitivity of the nested primer-probe combination of the present invention

[0169] Using the primer-probe combination in Example 1 of the present invention, LOD (minimum limit of detection) detection was performed on each target to simulate clinical samples, and after 2 rounds of nested multiplex fluorescence PCR amplification, detection was performed on a SLAN96 fluorescence PCR instrument. The results are shown in FIG3 , indicating that each channel can still accurately detect samples as low as 60 copies / mL, indicating that the minimum detection limit of the primer-probe combination of the present invention is 60 copies / mL.

[0170] The prepared whole blood samples with different bacterial concentrations (final concentrations of 10 CFU / mL, 20 CFU / mL, and 50 CFU / mL) were subjected to nucleic acid extraction, and the primer-probe combination in Example 1 of the present invention was used to perform LOD (limit of detection) detection on Klebsiella pneumoniae, Enterococcus faecium, and Staphylococcus aureus targets. The results are shown in FIG4 , indicating that the target gene of the sample as low as 10 CFU / mL can still be accurately detected, that is, the minimum detection limit can reach 10 CFU / mL.

[0171] Example 5: Specificity of the nested primer-probe combination of the present invention

[0172] In order to test the blank specificity of the primer-probe combination in Example 1 of the present invention, the negative control was used as a sample and the detection was carried out according to the method of Example 2. The results are as follows Figure 5 It was shown that there was no nonspecific amplification in each target channel and the blank specificity of the kit was good.

[0173] Comparative Example 1: Difference in sensitivity between emulsion microdroplet nested multiplex fluorescence PCR and conventional fluorescence multiplex PCR

[0174] Using the primer-probe combination in Example 1 of the present invention, 10 CFU of Klebsiella pneumoniae and Pseudomonas aeruginosa were added as simulated clinical samples. The emulsion microdroplet nested multiplex fluorescence PCR detection method of the present invention showed better sensitivity and smaller Ct value than the conventional fluorescence multiplex PCR detection. The specific comparison test results are as follows: Figure 6A-6B (Klebsiella pneumoniae target) and Figure 6C-6D (Pseudomonas aeruginosa target) as shown.

[0175] Example 6: Clinical sample detection test

[0176] Clinical culture positive and negative samples were selected for testing, a total of 12 samples.

[0177] 1) Sample pretreatment and nucleic acid extraction:

[0178] 2) The primers and probes of the two-round nested multiplex fluorescent PCR multiplex detection shown in Example 1 of the present invention were used to detect Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Stenotrophomonas maltophilia, Staphylococcus aureus, Enterococcus faecium and Pseudomonas aeruginosa on a SLAN96 fluorescent PCR instrument after two rounds of multiple nested amplification. The first-generation Sanger sequencing was used to verify the results that were inconsistent with the culture results. The specific test results are shown in Table 6:

[0179] Table 6: Comparison with clinical results

[0180] Sample No. Culture results The test results of the present invention Sanger Verification Results Consistency of the present invention with Sanger S1 Negative Pseudomonas aeruginosa Pseudomonas aeruginosa Consistency S2 Negative Negative / / S3 Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Consistency S4 Negative Acinetobacter baumannii Acinetobacter baumannii Consistency S5 Negative Stenotrophomonas maltophilia Stenotrophomonas maltophilia Consistency S6 Staphylococcus aureus, Enterococcus faecalis Staphylococcus aureus, Enterococcus faecalis Staphylococcus aureus, Enterococcus faecalis Consistency S7 Negative Acinetobacter baumannii Acinetobacter baumannii Consistency S8 Negative Klebsiella pneumoniae Klebsiella pneumoniae Consistency S9 Negative Acinetobacter baumannii Acinetobacter baumannii Consistency S10 Negative Klebsiella pneumoniae Klebsiella pneumoniae Consistency S11 Negative Negative / / S12 Negative Negative / /

[0181] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0182] As used in the present invention, the terms "include" and "comprising" are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0183] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0184] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A method for emQPCR multi-target joint detection of trace DNA not for the purpose of disease diagnosis, characterized in that: The method comprises the following steps: (1) The first round of single-tube nested multiple emulsion PCR pre-amplification based on the emulsion droplet reaction system: the water phase and the oil phase are mixed to prepare droplets to generate no less than 40,000 water-in-oil emulsion droplets, and then the prepared emulsion droplets are collected into eight PCR tubes with sealed rubber caps, and then the first round of single-tube nested multiple emulsion PCR pre-amplification is performed for 10 to 20 cycles; The final concentration of the forward and reverse primers in the aqueous phase is 0.1-0.9 μM, and the fragment size of the amplified product of the forward and reverse primers is 150-450 bp; the program of the first round of nested multiple emulsion PCR pre-amplification is 95° C. for 10-100 s; 94° C. for 5-30 s, 52-62° C. for 10-60 s, and 10-20 cycles; (2) Demulsification and separation of water phase and oil phase: After the first round of single-tube nested multiple emulsion PCR pre-amplification in step (1), the product is demulsified and separated into water phase and oil phase, thereby obtaining the first round of nested multiple emulsion PCR pre-amplification product of the water phase; (3) Second round of multiplex real-time fluorescence PCR multiplex detection: Using the first round pre-amplification product of the aqueous nested multiplex emulsion PCR obtained in step 2) as a template, add 8 detection target and internal standard gene specific modified primer pairs and probe sets, perform the second round of multiplex real-time fluorescence PCR amplification, and perform multi-target fluorescence amplification analysis and detection of trace pathogen nucleic acids; The final concentrations of the specific modified inner primers and modified probe sets for the second round of multiplex real-time fluorescent PCR for the eight detection target genes were 0.05-0.5 μM and 0.05-0.3 μM, respectively; the program for the second round of multiplex real-time fluorescent PCR amplification was 37°C for 2-5 min, 95°C for 1-5 min, 95°C for 10-30 s, 63-55°C for 30-90 s, 30-45 cycles, and fluorescence was collected at 60°C→90°C; The target detection requires the use of a primer probe set, which is a nested primer probe set; the sequence of the nested primer probe set includes: An outer primer pair, an inner primer pair and a probe for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 1 to 5; An outer primer pair, an inner primer and a probe for detecting Staphylococcus aureus as shown in SEQ ID NOs: 6 to 9; An outer primer pair, an inner primer and a probe for detecting Escherichia coli as shown in SEQ ID NOs: 10 to 13; An outer primer pair, an inner primer and a probe for detecting Enterococcus faecium as shown in SEQ ID NOs: 14 to 17; An outer primer pair, an inner primer pair and a probe for detecting Enterococcus faecalis as shown in SEQ ID NOs: 18 to 22; An outer primer pair, an inner primer pair and a probe for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 23 to 27; An outer primer pair, an inner primer pair and a probe for detecting Pseudomonas aeruginosa as shown in SEQ ID NOs: 28 to 32; and, The outer primer pair, inner primer pair and probe for detecting Stenotrophomonas maltophilia are shown in SEQ ID NOs: 33-36.

2. The method according to claim 1, characterized in that The primer set for the first round of single-tube nested multiple emulsion PCR pre-amplification is an outer primer for target-specific gene amplification, and the modified primer probe set for the second round of multiple real-time fluorescence PCR multiplex detection is an inner primer; wherein the upstream or downstream end of the outer primer can be shared with the upstream or downstream end of the inner primer, and no additional cross or non-specific amplification can be generated, which will further improve the amplification efficiency and specificity of the micro-template; And / or, the Mix of the first round of single-tube nested multiple emulsion PCR pre-amplification reaction has different components from the Mix of the second round of multiple real-time fluorescence PCR multiple detection reaction. The Mix of the first round of single-tube nested multiple PCR pre-amplification reaction should not only meet the specificity and efficiency of PCR amplification, but also ensure the stable generation of the emulsion micro-droplet system and the stability of subsequent micro-droplet PCR amplification.

3. The method according to claim 1, characterized in that In step (2), the volume ratio of the water phase to the oil phase is 1:5 to 1:10; And / or, the amount of the microdroplet demulsifier after amplification is at least 1 to 3 times the reaction volume before droplet amplification, so as to ensure that the oil phase and the water phase are completely separated after demulsification and a stable recovery rate is achieved.

4. The method according to claim 1, characterized in that: In step (1), the final concentration of the forward and reverse outer primers in the aqueous phase is 0.15-0.75 μM, and the fragment size of the amplified product of the forward and reverse primer pairs is 190-410 bp; the first round of nested multiple emulsion PCR pre-amplification program is 95°C 30s; 94°C 10s, 53°C 30s, 15 cycles.

5. The method according to claim 1, characterized in that In step (3), the final concentrations of the eight specific modified inner primers and modified probe sets for detecting target genes are 0.15-0.3 μM and 0.06-0.15 μM, respectively; And / or, the second round of multiplex real-time fluorescence PCR amplification program is 37°C for 2 min, 95°C for 1 min; 95°C for 15 s, 63-55°C for 30 s, 10 cycles of TD amplification; 95°C for 15 s, 55°C for 30 s, 30 cycles; 68°C→90°C to collect fluorescence.

6. The method according to claim 1, characterized in that The detected pathogens include Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

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