Detection method and kit for drug-resistant genes in bloodstream infection pathogens and application

Abstract

The invention discloses a method for detecting drug-resistant genes in blood flow infection pathogens, a kit and application. Specifically, the invention provides a primer probe combination for detecting a drug-resistant gene in a bloodstream infection pathogen, and the primer probe combination comprises: (a) a primer and a probe for detecting mecA / C genes as shown in SEQ ID NO: 1-3; (b) a primer and a probe which are shown as SEQ ID NO: 4-6 and are used for detecting the OXA48 gene; (c) a primer and a probe which are shown as SEQ ID NO: 7-9 and are used for detecting the KPC gene; (d) a primer and a probe which are shown as SEQ ID NO: 10-12 and are used for detecting the NDM gene; (e) a primer and a probe which are shown as SEQ ID NO: 13-15 and are used for detecting the IMP gene; (f) a primer and a probe which are shown as SEQ ID NO: 16-19 and are used for detecting the MCR gene; and (g) a primer and a probe which are shown as SEQ ID NO: 20-23 and are used for detecting the vanA / B gene. The primer probe combination provided by the invention can be used for realizing rapid, high-sensitivity and high-specificity detection of drug-resistant genes in blood flow infection pathogens.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular relates to a method for detecting drug-resistant genes in blood stream infection pathogens, a kit and application thereof. BACKGROUND

[0002] The massive use of antibiotics leads to bacteria to obtain genes or chromosomal mutations to enable them to tolerate antibacterial drugs, thereby producing bacterial drug resistance, so that many bacterial infectious diseases may have no effective antibiotic treatment, which poses a huge risk to human health. Antibiotic resistance is one of the "ten threats to global health in 2019" published by WHO, and its severity and harm are gradually increasing. In 2017, the World Health Organization (WHO) released 12 "superbugs" that pose the greatest threat to human health, indicating that bacterial antibiotic resistance has become a global public health problem.

[0003] As the problem of bacterial resistance becomes more and more serious, it is also more and more important to detect pathogens and drug-resistant genes. The current conventional detection method has the disadvantages of low detection rate, long time consumption, and especially cannot accurately identify multiple pathogens at the same time, etc., and cannot provide timely, comprehensive and accurate pathogen diagnosis basis for the clinic, resulting in the fact that the clinic generally uses broad-spectrum antibacterial drugs for empirical treatment, which causes low efficacy, induces high drug resistance rate of strains and increases the medical burden of patients, etc.

[0004] Therefore, in order to determine the types of drug-resistant genes of common pathogens as soon as possible, continuously do a good job in drug resistance monitoring, fully understand the changes of bacterial species and drug resistance rate to antibacterial drugs, and better guide the rational use of drugs in the clinic, the development of a method for detecting drug-resistant genes with rapidity, high specificity, high sensitivity, and strong anti-interference ability is urgently needed in the field. SUMMARY

[0005] The present application provides a method for detecting drug-resistant genes with rapidity, high specificity, high sensitivity, and strong anti-interference ability.

[0006] In a first aspect of the present application, a primer probe combination for detecting drug-resistant genes in blood stream infection pathogens is provided, and the primer probe combination is selected from the following group:

[0007] (a) the upstream and downstream primers and the probe for detecting mecA and mecC (or mecA / C) genes as shown in SEQ ID NO: 1-3;

[0008] (b) the upstream and downstream primers and the probe for detecting OXA48 gene as shown in SEQ ID NO: 4-6;

[0009] (c) the upper and lower primers and probes for detecting KPC gene shown in SEQ ID NO: 7-9;

[0010] (d) the upper and lower primers and probes for detecting NDM gene shown in SEQ ID NO: 10-12;

[0011] (e) the upper and lower primers and probes for detecting IMP gene shown in SEQ ID NO: 13-15;

[0012] (f) the upper and lower primers and probes for detecting MCR gene shown in SEQ ID NO: 16-19;

[0013] (g) the upper and lower primers and probes for detecting vanA and vanB (or vanA / B) gene shown in SEQ ID NO: 20-23;

[0014] or a combination thereof.

[0015] In another preferred embodiment, the primer probe combination further comprises primers and probes for detecting other drug-resistant genes in blood stream infectious pathogens.

[0016] In another preferred embodiment, the primer probe combination is the same as, and can be used interchangeably with, primer probe combination.

[0017] In another preferred embodiment, the primer probe combination further comprises:

[0018] (h) the upper and lower primers and probes for detecting internal reference gene shown in SEQ ID NO: 24-26.

[0019] In another preferred embodiment, the primers and probes for detecting internal reference are designed for specific conserved regions of human PIK3C gene.

[0020] In another preferred embodiment, the fluorescence reporter group of the mecA and mecC gene probes is FAM; the fluorescence reporter group of the OXA48 gene probe is HEX; the fluorescence reporter group of the KPC gene probe is ROX; the fluorescence reporter group of the NDM gene probe is CY5; the fluorescence reporter group of the IMP gene probe is CY5; the fluorescence reporter groups of the MCR gene probes are FAM and ROX respectively; the fluorescence reporter groups of the vanA and vanB gene probes are ROX and CY5 respectively.

[0021] In another preferred embodiment, the fluorescence reporter group of the internal reference gene probe is Q705.

[0022] In another preferred embodiment, the 3' end of the probe further has a fluorescence quenching group.

[0023] In another preferred embodiment, the fluorescence quenching group is selected from the group consisting of BHQ1, BHQ2, and BHQ3.

[0024] In another preferred embodiment, the fluorescence quenching group of the mecA and mecC gene probes is BHQ1; the fluorescence quenching group of the OXA48 gene probe is BHQ1; the fluorescence quenching group of the KPC gene probe is BHQ2; the fluorescence quenching group of the NDM gene probe is BHQ2; the fluorescence quenching group of the IMP gene probe is BHQ2; the fluorescence quenching groups of the MCR gene probes are BHQ1 and BHQ2, respectively; the fluorescence quenching groups of the vanA and vanB gene probes are BHQ2 and BHQ2, respectively.

[0025] In another preferred embodiment, the fluorescence quenching group of the internal reference gene probe is BHQ3.

[0026] In another preferred embodiment, each component of the primer probe combination is present in a separate package.

[0027] In another preferred embodiment, each component of the primer probe combination is present in the same package.

[0028] In another preferred embodiment, each component of the combination is present in a mixed form.

[0029] In another preferred embodiment, the 5' end of the probe sequence is linked to a fluorescence reporter group, and the 3' end is linked to a fluorescence quenching group.

[0030] In a second aspect of the present application, there is provided use of the primer probe combination as described in the first aspect of the present application for preparing a kit for detecting drug-resistant genes in blood stream infection pathogens.

[0031] The drug-resistant genes are selected from the group consisting of mecA gene, mecC gene, OXA48 gene, KPC gene, NDM gene, IMP gene, MCR gene, vanA gene, vanB gene, or a combination thereof.

[0032] In a third aspect of the present application, there is provided a kit for detecting drug-resistant genes in blood stream infection pathogens, the kit comprising the primer probe combination as described in the first aspect of the present application.

[0033] In another preferred embodiment, the kit further comprises a negative control and a positive control.

[0034] In another preferred embodiment, the negative control is at least one of DEPC-H2O, physiological saline, internal reference gene pseudovirus, and normal human leukocyte genomic DNA.

[0035] In another preferred embodiment, the negative quality control is normal human genomic DNA.

[0036] In another preferred embodiment, the copy number concentration of the human genomic DNA is 1000 copies / μL.

[0037] In another preferred embodiment, the kit further comprises:

[0038] (h) the upstream and downstream primers and probes for detecting the reference genes as shown in SEQ ID NO: 24-26.

[0039] In another preferred embodiment, the positive quality control is a mixture of pathogenic bacteria DNA containing KPC, NDM, IMP, OXA48, mecA, mecC, vanA, vanB, MCR genes.

[0040] In another preferred embodiment, the copy number of all target sites in the positive quality control is 50 copies / μL.

[0041] In another preferred embodiment, the kit further comprises a PCR reaction solution.

[0042] In another preferred embodiment, the PCR reaction solution comprises at least one of dNTP, PCR buffer, Mg 2+ and DNA polymerase.

[0043] In another preferred embodiment, the DNA polymerase is 5×HS Taq Buffer with Mg 2+ reaction system.

[0044] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used for detecting drug-resistant genes in blood stream infection pathogens.

[0045] In another preferred embodiment, the drug-resistant genes are selected from the group consisting of mecA / C gene, OXA48 gene, KPC gene, NDM gene, IMP gene, MCR gene, vanA / B gene, or a combination thereof.

[0046] In another preferred embodiment, the kit further comprises a blank control, which is ultrapure water.

[0047] In a fourth aspect of the present application, there is provided a use of the primer probe combination according to the first aspect of the present application or the kit according to the third aspect of the present application for preparing a detection reagent for drug-resistant genes in blood stream infection pathogens.

[0048] The drug resistance gene is selected from the group consisting of mecA gene, mecC gene, OXA48 gene, KPC gene, NDM gene, IMP gene, MCR gene, vanA gene, vanB gene, or a combination thereof.

[0049] In another preferred embodiment, the detecting comprises the following steps:

[0050] (i) extracting free DNA in the sample to be tested;

[0051] (ii) performing fluorescent quantitative PCR on the free DNA obtained in step (i) using the primer probe combination of the first aspect of the present application or the kit of the third aspect of the present application;

[0052] (iii) reading the fluorescence data and calculating the copy concentration of the sequence of interest, thereby obtaining the detection result.

[0053] In another preferred embodiment, in step (iii), the determination of the detection result is as follows:

[0054] (1) positive droplet ≥ 3 or positive signal ≥ 5 copies / reaction, and clustering and positive control are consistent: positive result, directly report the drug resistance gene and copy number.

[0055] (2) no positive signal, and the clustering of the internal reference and the negative control are consistent: negative result, report not detected.

[0056] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or a combination thereof.

[0057] In the fifth aspect of the present application, a method for detecting drug resistance genes in blood stream infection pathogens is provided, comprising the following steps:

[0058] (i) extracting free DNA in the sample to be tested;

[0059] (ii) performing fluorescent quantitative PCR on the free DNA obtained in step (i) using the primer probe combination of the first aspect of the present application or the kit of the third aspect of the present application;

[0060] (iii) reading the fluorescence data and calculating the copy concentration of the sequence of interest, thereby obtaining the detection result.

[0061] In another preferred embodiment, the drug resistance gene is selected from the group consisting of mecA gene, mecC gene, OXA48 gene, KPC gene, NDM gene, IMP gene, MCR gene, vanA gene, vanB gene, or a combination thereof.

[0062] In another preferred embodiment, in step (iii), the determination of the detection result is as follows:

[0063] (1) Positive droplet > 3 or positive signal > 5 copies / reaction, and cluster with positive control: positive result, report drug resistance gene and copy number directly.

[0064] (2) No positive signal, and cluster with internal control and negative control: negative result, report not detected.

[0065] In another preferred embodiment, the sample to be tested is selected from the group consisting of whole blood, plasma, serum, or a combination thereof.

[0066] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0067] In another preferred embodiment, the method is an in vitro method.

[0068] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 Screening results of IMP primer probe are shown.

[0070] Figure 2 Screening results of NDM primer probe are shown.

[0071] Figure 3 Screening results of KPC primer probe are shown.

[0072] Figure 4 Screening results of OXA48 primer probe are shown.

[0073] Figure 5 Screening results of mecA / mecC primer probe are shown.

[0074] Figure 6 Screening results of vanA / vanB primer probe are shown.

[0075] Figure 7 Screening results of MCR primer probe are shown.

[0076] Figure 8 Screening results of internal control gene are shown.

[0077] Figure 9 Detection results of vanA / B gene are shown.

[0078] Figure 10 Detection results of KPC gene are shown.

[0079] Figure 11Results of detection of MCR genes are shown.

[0080] Figure 12 Results of detection of mecA / C genes are shown.

[0081] Figure 13 Results of detection of IMP genes are shown.

[0082] Figure 14 Results of detection of NDM genes are shown.

[0083] Figure 15 Results of detection of OXA48 genes are shown.

[0084] Figure 16 Results of detection of internal reference genes are shown.

[0085] Figure 17 (A) Concentration results of amplification of vanA genes using multiplex PCR combination are shown; (B) Concentration results of single amplification of vanA genes are shown.

[0086] Figure 18 (A) Concentration results of amplification of vanB genes using multiplex PCR combination are shown; (B) Concentration results of single amplification of vanB genes are shown.

[0087] Figure 19 (A) Concentration results of amplification of KPC genes using multiplex PCR combination are shown; (B) Concentration results of single amplification of KPC genes are shown.

[0088] Figure 20 (A) Concentration results of amplification of MCR genes using multiplex PCR combination are shown; (B) Concentration results of single amplification of MCR genes are shown.

[0089] Figure 21 (A) Concentration results of amplification of mecA genes using multiplex PCR combination are shown; (B) Concentration results of single amplification of mecA genes are shown.

[0090] Figure 22 (A) Concentration results of amplification of mecC genes using multiplex PCR combination are shown; (B) Concentration results of single amplification of mecC genes are shown.

[0091] Figure 23 (A) Concentration results of amplification of IMP genes using multiplex PCR combination are shown; (B) Concentration results of single amplification of IMP genes are shown.

[0092] Figure 24(A) Concentration results showing amplification of NDM genes using multiplex PCR combination; (B) Concentration results showing single amplification of NDM genes.

[0093] Figure 25 (A) Concentration results showing amplification of OXA48 genes using multiplex PCR combination; (B) Concentration results showing single amplification of OXA48 genes.

[0094] Figure 26 Results of dual color fluorescently labeled screening for vanA / B.

[0095] Figure 27 Results of dual color fluorescently labeled screening for KPC.

[0096] Figure 28 Results of dual color fluorescently labeled screening for MCR.

[0097] Figure 29 Results of dual color fluorescently labeled screening for mecA.

[0098] Figure 30 Results of dual color fluorescently labeled screening for mecC.

[0099] Figure 31 Results of dual color fluorescently labeled screening for IMP.

[0100] Figure 32 Results of dual color fluorescently labeled screening for NDM.

[0101] Figure 33 Results of dual color fluorescently labeled screening for OXA48.

[0102] Figure 34 Detection sensitivity of mecA.

[0103] Figure 35 Detection sensitivity of mecC.

[0104] Figure 36 Detection sensitivity of MCR.

[0105] Figure 37 Detection sensitivity of vanA.

[0106] Figure 38 Detection sensitivity of vanB.

[0107] Figure 39 Detection sensitivity of IMP.

[0108] Figure 40 Detection sensitivity of NDM.

[0109] Figure 41 The detection sensitivity of OXA48 is shown.

[0110] Figure 42 The detection sensitivity of KPC is shown. DETAILED DESCRIPTION

[0111] The present inventors, through extensive and in-depth research, unexpectedly developed primers and probes that can be used to rapidly and highly specifically and sensitively detect drug-resistant genes in blood stream infection pathogens through a large amount of screening. The drug-resistant genes include: mecA / C genes, OXA48 genes, KPC genes, NDM genes, IMP genes, MCR genes, and vanA / B genes. The experiments of the present invention show that the digital PCR method of the present invention has a minimum detection sensitivity of 5 copies / reaction, and is specific. On this basis, the present invention is completed.

[0112] TERMS

[0113] In order that the disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically stated otherwise, each of the following terms has the meaning given below. Additional definitions are set forth throughout the application.

[0114] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101, and all values in between.

[0115] As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0116] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the entire range of values as well as any value within that range (e.g., one-tenth and one-hundredth of an integer) as appropriate.

[0117] As used herein, the term "and / or" refers to any and all possible combinations of one or more of the associated listed items.

[0118] As used herein, "mecA / C" refers to mecA and mecC.

[0119] As used herein, "vanA / B" refers to vanA and vanB.

[0120] Drug-resistant genes of blood stream infection pathogens

[0121] The 2023 National Bacterial Drug Resistance Monitoring Report shows that among the 13 key drug resistance indicators detected nationwide in 2023, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative Staphylococcus (MRCNS), erythromycin-resistant Streptococcus pneumoniae (ERSP), vancomycin-resistant Enterococcus faecium (VREM), carbapenem-resistant Klebsiella pneumoniae (CR-KPN), carbapenem-resistant Acinetobacter baumannii (CR-ABA), carbapenem-resistant Escherichia coli (CR-ECO), third-generation cephalosporin-resistant Escherichia coli (CTX / CRO-R-ECO), and quinolone-resistant Escherichia coli (QNR-ECO) have increased to varying degrees, cefotaxime or ceftriaxone-resistant Klebsiella pneumoniae (CTX / CRO-R KPN) and vancomycin-resistant Enterococcus faecium (VREA) have remained the same, carbapenem-resistant Pseudomonas aeruginosa (CR-PAE) and penicillin-resistant Streptococcus pneumoniae (PRSP) have decreased, especially CTX / CRO-R-ECO, which has rebounded for the first time after years of continuous decline, and further efforts are needed to strengthen drug resistance prevention and control.

[0122] It has been reported that Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis have become increasingly resistant to commonly used antibiotics such as β-lactams and carbapenems; Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis producing extended-spectrum β-lactamases (ESBLs) have gradually increased, and multiple reports have pointed out that the average incidence of ESBLs-producing Escherichia coli and Klebsiella pneumoniae is close to 50%. In the past decade, carbapenem-resistant Enterobacteriaceae (CRE) such as Escherichia coli and Klebsiella pneumoniae have emerged and rapidly spread globally, causing great distress for clinicians in terms of drug use, and how to quickly and accurately detect CRE strains has become a hot topic in antimicrobial therapy.

[0123] The research shows that the drug resistance mechanisms of CRE mainly include the following three aspects: production of carbapenemases, including KPC enzyme in class A enzymes, VIM and IMP in class B enzymes, and NDM-1 type enzyme in superbugs; deletion of membrane pore proteins and / or high expression of cephalosporinases; hyperfunction of active efflux pumps; and change of the target site of carbapenem antibiotics on bacteria. Among these drug resistance mechanisms, the most important one is the production of carbapenemases, and the most important ones are class A enzymes and class B enzymes. Carbapenemases are a class of beta-lactamases that can hydrolyze carbapenem antibiotics. According to carbapenemases, they can be divided into A, B and D, among which A and D belong to serine enzymes (including KPC, IMI, NMC-A, SME, etc.), and B belongs to metalloenzymes (including NDM, IMP, VIM, SIM, etc.). At present, KPC is the most popular carbapenemase type in the world. CRE has become an important challenge in clinical practice, spreads quickly and has a high mortality rate. KPC and NDM are the main drug resistance mechanisms and can be transmitted through plasmids. Early detection and monitoring are important measures to prevent the spread of CRE. Since KPC-producing Klebsiella pneumoniae was first reported in the United States, this pathogen has been widely detected as a multidrug-resistant bacteria around the world, posing a serious challenge to global anti-infective work. Once KPC-producing Klebsiella pneumoniae hydrolyzes carbapenem antibiotics, carbapenem antibiotics are the last line of defense in controlling gram-negative bacterial infections. It has been reported that the final mortality rate due to the lack of effective drugs to control KPC-producing Klebsiella pneumoniae infections can be as high as 75%. Therefore, it is of great significance to quickly determine whether bacteria carry carbapenemase-producing genes through molecular detection for monitoring bacterial drug resistance, preventing the spread and transmission of drug-resistant bacteria.

[0124] In addition, there are certain differences in the types of carbapenemases used by different types of carbapenemases, and rapid determination of the genotype of carbapenemases can help clinicians provide certain medication guidance.

[0125] The drug resistance genes studied in the present application and their basic information are shown in Table A.

[0126] Table A Drug resistance genes and their effects

[0127]

[0128]

[0129] The detection method of the drug resistance gene of the present application

[0130] The present application provides a detection method of a drug resistance gene, comprising the following steps:

[0131] 1) According to the gene sequence of drug resistance gene, design specific primer probe. The above-mentioned primer and probe are used for digital PCR detection, and the optimal detection condition of PCR system is optimized. The drug resistance genes include KPC, NDM, IMP, OXA48, mecA, mecC, vanA, vanB, MCR gene.

[0132] 2) Extraction of nucleic acid of sample. The nucleic acid is extracted by selecting a method suitable for the type of sample, including blood culture, peripheral blood, peripheral blood plasma / serum. In particular, for the extraction of free nucleic acid of blood sample, the steps are as follows:

[0133] First, the selection of blood collection tube. If a common blood collection tube is used, it cannot contain heparin, and the plasma needs to be separated within 4h. If the blood cannot be processed in time, a blood collection tube containing blood cell stabilizing technology needs to be selected.

[0134] Second, the separation of plasma. The separation of plasma: the first step is to remove cells by low-speed centrifugation, 4℃, 1600g, 10min.

[0135] Third, extraction. Use the special extraction kit for plasma free nucleic acid, and extract according to the requirements of the instruction manual.

[0136] 3) Digital PCR.

[0137] First, the configuration of PCR system. In the reagent preparation area, configure the PCR system according to the following proportion:

[0138] 5×HS Taq Buffer with Mg 2+6 μL, dNTPs (10 mM each) 0.75 μL, HotStart Taq DNA Polymerase 0.2 μL, SEQ ID NO: 1 (50 μM) 0.2 μL, SEQ ID NO: 2 (50 μM) 0.2 μL, SEQ ID NO: 3 (50 μM) 0.2 μL, SEQ ID NO: 4 (50 μM) 0.1 μL, SEQ ID NO: 5 (50 μM) 0.2 μL, SEQ ID NO: 6 (50 μM) 0.1 μL, SEQ ID NO: 7 (50 μM) 0.2 μL, SEQ ID NO: 8 (50 μM) 0.2 μL, SEQ ID NO: 9 (50 μM) 0.1 μL, SEQ ID NO: 10 (50 μM) 0.2 μL, SEQ ID NO: 11 (50 μM) 0.2 μL, SEQ ID NO: 12 (50 μM) 0.1 μL, SEQ ID NO: 13 (50 μM) 0.2 μL, SEQ ID NO: 14 (50 μM) 0.2 μL, SEQ ID NO: 15 (50 μM) 0.1 μL, SEQ ID NO: 16 (50 μM) 0.2 μL, SEQ ID NO: 17 (50 μM) 0.2 μL, SEQ ID NO: 18 (50 μM) 0.1 μL, SEQ ID NO: 19 (50 μM) 0.1 μL, SEQ ID NO: 20 (50 μM) 0.2 μL, SEQ ID NO: 21 (50 μM) 0.2 μL, SEQ ID NO: 22 (50 μM) 0.1 μL, SEQ ID NO: 23 (50 μM) 0.1 μL, SEQ ID NO: 24 (50 μM) 0.2 μL, SEQ ID NO: 25 (50 μM) 0.2 μL, SEQ ID NO: 26 (50 μM) 0.1 μL, template 5 μL, add water to 30 μL.

[0139] Second, add templates. Add templates in the following order: sample to be tested, blank control, negative control, positive control.

[0140] Third, droplet generation. Perform droplet generation according to the requirements of the instrument.

[0141] Fourth, PCR. After annealing temperature optimization experiment, it is found that the digital PCR effect is optimal at 56 °C annealing for 15 s. Perform PCR according to the following program: 95 °C for 10 min, 40 cycles (95 °C for 30 s, 56 °C for 15 s, 72 °C for 15 s), temperature rising and falling rate 2 °C / s.

[0142] Fifth, scanning. According to the scanning results, judge whether the sample is positive or negative.

[0143] Detection kit

[0144] The present application provides a kit for detecting KPC, NDM, IMP, OXA48, mecA, mecC, vanA, vanB, MCR genes. The kit comprises PCR primer probe, reaction system, reaction condition, negative control DNA sample, positive control DNA sample. The following content:

[0145] Primer and probe. The following oligonucleotide fragments and modified oligonucleotide fragments are synthesized and dissolved in Tris-EDTA Buffer (10 μM). See the foregoing part for details

[0146] PCR reagent. DNA polymerase uses 5×HS Taq Buffer with Mg 2+ Reaction system, see the foregoing part.

[0147] Reaction condition, see the foregoing part.

[0148] Control: blank control is water, negative control is human genome, copy concentration is 1000 copies / μL. Positive control is pathogenic bacteria DNA mixture containing KPC, NDM, IMP, OXA48, mecA, mecC, vanA, vanB, MCR genes. The copy number of all target sites of the positive control solution is 50 copies / μL.

[0149] Drug guide

[0150] Since the NDM and IMP drug resistance range and sensitive antibiotics are basically the same, the clinical drug is also the same, so in the actual detection, the two genes are not distinguished, only detection is needed.

[0151] As shown in Table A above, KPC, NDM, IMP and OXA-48 all belong to carbapenamase, but KPC is resistant to carbapenems, third-generation cephalosporins and amikacin, NDM and IMP are resistant to carbapenems, but sensitive to amikacin, and OXA-48 is low-level resistant to carbapenems, and sensitive to third-generation cephalosporins. Therefore, because the drug resistance spectrum of NDM and IMP is the same, they can be detected together, and the drug resistance spectrum of other resistant genes is different, and they need to be detected separately.

[0152] Since the detection device of the present application is 5 fluorescence channels, but the detection target (including internal reference) is more than 5, and a tube reaction contains all the target points, one or two target points must be double fluorescence labeled. Therefore, the present application finally determines that the probe of the MCR gene is double labeled with FAM and ROX, and vanA / B is double labeled with ROX and CY5 based on the results of the screening based on double-color fluorescence labeling, and in this combination, the positive and negative points of each target point are well distinguished (for details of the screening process, see Example 4 of the present application).

[0153] The main advantages of the present application include:

[0154] (a) Compared with the prior art blood culture, MALD-TOF MS, NGS, etc., the present application uses Taqman probe and combines digital PCR method, which can solve the problems of long detection time, low sensitivity, poor specificity, high requirements for sample type and quality, complex positive interpretation method, etc.

[0155] (b) The method of the present application has high sensitivity: since the present method uses a digital PCR platform, the reaction system can be divided into about 20000 small reactions, which can theoretically detect a single copy of the mutation, and has a sensitivity advantage that other technologies cannot match. The detection method of the present application can achieve a minimum detection limit of 5 copies / reaction through verification.

[0156] (c) The method of the present application has strong specificity: the designed specific primers and probes are designed for specific positions of KPC, NDM, IMP, OXA48, mecA, mecC, vanA, vanB and MCR genes, and can specifically amplify the target positions.

[0157] (d) The method of the present application has a wide range of requirements for sample type and quality, and strong anti-interference ability. Due to the high sensitivity of the present application, the applicable sample type is peripheral blood sample; and due to the uniqueness of the digital PCR platform, the reaction system can be divided into about 20000 small systems, and at the same time, the interfering substances can also be divided into about 20000 parts, which can greatly reduce the influence of interfering substances on the reaction, and of course, more complex background samples can be detected. This is something that other platforms cannot do.

[0158] (e) The positive interpretation method of the method of the present application is simple: since the present application uses an absolute quantitative method, there is no need to set a standard curve, and the result can be determined according to the two-dimensional fluorescence chart, that is, whether the target mutation template is contained. The specific interpretation method is as follows:

[0159] (1) Positive droplet ≥ 3 or positive signal ≥ 5 copies / reaction, and clustering and positive control are consistent: positive result, directly report the drug resistance gene and copy number.

[0160] (2) No positive signal, and the internal reference clusters are consistent with the negative control: negative result, report not detected.

[0161] (f) The method of the present application can detect 7 drug-resistant genes at one time, reducing repeated operations and detection costs

[0162] (g) The method of the present application can comprehensively guide the use of antibiotics, covering the drug-resistant genes of common gram-negative bacteria (KPC, NDM, IMP, OXA-48) and common gram-positive bacteria (mecA / C), and polymyxin (MCR-1) is the last line of defense for gram-negative bacterial infections, and vancomycin (vanA / B) is the last line of defense for gram-positive bacteria.

[0163] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, and the conditions are generally according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0164] Example 1 Primer / probe screening

[0165] 1.1 Design primers / probes for detecting target points KPC, NDM, IMP, OXA48, mecA / C, vanA / B, and MCR genes. Design multiple sets of primers / probes for each target point until the appropriate primer / probe combination is screened.

[0166] 1.2 PCR system

[0167] 5×HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNA Polymerase 0.2μL, primer / probe concentration 50μM, add proportion according to 0.2μL:0.2μL:0.1μL, template 5μL, add water to 30μL.

[0168] 1.3 PCR program: 95℃ 10min, 40 cycles (95℃ 30s, 56℃ 15s, 72℃ 15s).

[0169] 1.4 Chip scanning.

[0170] The primer / probe combination of IMP is shown in Table 1 below, and the digital PCR results are as followsFigure 1 and Table 2. Combination 1 and combination 5 can distinguish the positive and negative droplets well, in which combination 1 has higher intensity ratio of positive and negative droplets, slightly better distinguishability and slightly higher concentration, thus combination 1 is selected as the subsequent primer probe combination.

[0171] Table 1

[0172]

[0173] Note: + represents locked nucleic acid; M represents A / C degenerate base; R represents A / G degenerate base; Y represents C / T degenerate base; W represents A / T degenerate base;

[0174] Table 2. IMP primer screening concentration table

[0175] Combination name Concentration (copies / μL) 1 1219.440 2 1194.284 3 1156.931 4 1110.905 5 1166.882

[0176] The primer probe combination of NDM is shown in Table 3 below, and the digital PCR results are shown in Figure 2 and Table 4. Combination 1 and combination 2 can distinguish the positive and negative droplets well, in which combination 1 has slightly higher concentration, thus combination 1 is selected as the subsequent primer probe combination.

[0177] Table 3

[0178]

[0179] Table 4. NDM primer screening concentration table

[0180] Combination name Concentration (copies / μL) 1 159.367 2 146.513 3 25.283

[0181] The primer probe combination of KPC is shown in Table 5 below, and the digital PCR results are shown in Figure 3 and Table 6. Three combinations can distinguish the positive and negative droplets well, in which combination 3 has slightly higher concentration, thus combination 3 is selected as the subsequent primer probe combination.

[0182] Table 5

[0183]

[0184]

[0185] Table 6. KPC primer screening concentration table

[0186] Combination name Concentration (copies / μL) 1 85.085 2 87.635 3 91.803

[0187] The primer probe combination of OXA48 is shown in Table 7 below, and the digital PCR results are shown in Figure 4and Table 8. All four combinations can well distinguish between positive and negative droplets, and combination 2 and combination 3 have similar concentrations, and the separation degree of positive and negative droplets of combination 2 is slightly better, so combination 2 is selected as the subsequent primer probe combination.

[0188] Table 7

[0189] Note: M represents A / C degenerate base; R represents A / G degenerate base; Y represents C / T degenerate base; W represents A / T degenerate base; K represents G / T degenerate base.

[0190] Table 8 OXA48 primer screening concentration table

[0191] Combination name Concentration (copies / μL) 1 307.659 2 345.276 3 356.243 4 308.009

[0192] The primer probe combination of mecA / C is shown in the following Table 9. The digital PCR results are as shown in Figure 5 and Table 10. All four combinations can well distinguish between positive and negative droplets, and combination 1 has a higher concentration, so combination 1 is selected as the subsequent primer probe combination.

[0193] Table 9

[0194] Note: R represents A / G degenerate base; Y represents C / T degenerate base.

[0195] Table 10 mecA / mecC primer screening concentration table

[0196] Combination name Concentration (copies / μL) 1 24.523 2 20.979 3 19.208 4 17.690

[0197] The primer probe combination of vanA / B is shown in the following Table 11. The digital PCR results are as shown in Figure 6 and Table 12. All three combinations can well distinguish between positive and negative droplets, and combination 3 has a higher concentration, so combination 3 is selected as the subsequent primer probe combination.

[0198] Table 11

[0199] Note: M represents A / C degenerate base; R represents A / G degenerate base; Y represents C / T degenerate base; W represents A / T degenerate base; K represents G / T degenerate base.

[0200] Table 12 vanA / B primer screening concentration table

[0201]

[0202]

[0203] The primer probe combination of MCR is shown in the following Table 13, and the digital PCR results are as shown in Figure 7and Table 14. Four combinations can well distinguish between positive and negative droplets, in which combination 4 has higher concentration and better separation between positive and negative droplets, so combination 4 is selected as the subsequent primer probe combination.

[0204] Table 13

[0205]

[0206] Table 14 MCR primer screening concentration table

[0207]

[0208] Example 2 internal reference primer screening

[0209] 2.1 Primer design

[0210] The primer probe is designed according to the specific segment of EGFR L858, CEP-17, EGFR T790, PIK3CA N345 site, and the sequence is shown in Table 15 as follows.

[0211] Table 15

[0212]

[0213] Note: + represents locked nucleic acid.

[0214] 2.2 PCR system

[0215] 5×HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNA Polymerase 0.2μL, SEQ ID NO: 27 (50μM) 0.2μL, SEQ ID NO: 28 (50μM) 0.2μL, SEQ ID NO: 29 (50μM) 0.1μL, template 5μL, add water to 30μL.

[0216] 5×HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNA Polymerase 0.2μL, SEQ ID NO: 30 (50μM) 0.2μL, SEQ ID NO: 31 (50μM) 0.2μL, SEQ ID NO: 32 (50μM) 0.1μL, template 5μL, add water to 30μL.

[0217] 5×HS Taq Buffer with Mg 2+6 μL, dNTPs (10 mM each) 0.75 μL, HotStart Taq DNA Polymerase 0.2 μL, SEQ ID NO: 33 (50 μM) 0.2 μL, SEQ ID NO: 34 (50 μM) 0.2 μL, SEQ ID NO: 35 (50 μM) 0.1 μL, template 5 μL, add water to 30 μL.

[0218] 5x HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart Taq DNA Polymerase 0.2 μL, SEQ ID NO: 24 (50 μM) 0.2 μL, SEQ ID NO: 25 (50 μM) 0.2 μL, SEQ ID NO: 26 (50 μM) 0.1 μL, template 5 μL, add water to 30 μL.

[0219] 2.3 PCR procedure: 95 °C 10 min, 40 cycles (95 °C 30 s, 56 °C 15 s, 72 °C 15 s).

[0220] 2.4 Chip scanning

[0221] The results of digital PCR are shown in Figure 8 and Table 16 below. The primers were screened in combination with signal intensity and amplification concentration. From the ddPCR amplification results of the four groups of candidate internal reference genes, PIK3CA N345 had the highest amplification concentration and higher signal intensity. Therefore, PIK3CA N345 was selected as the internal reference.

[0222] Table 16

[0223] Target Concentration EGFR L858 3091.513 CEP17 3893.708 EGFR T790 2083.29 PIK3CA N345 4739.915

[0224] Example 3 Detection of KPC, NDM, IMP, OXA48, mecA / C, vanA / B and MCR genes by digital PCR

[0225] 3.1 Primer combination

[0226] The optimal primer probe combination of each target selected according to Example 1 was combined into a multiplex PCR reaction, wherein the probe for detecting MCR gene was labeled with FAM and ROX double color, and the probe for detecting vanA / B was labeled with ROX and CY5 double color, and the sequences are shown in Table 17 below.

[0227] Table 17

[0228] Note: + represents locked nucleic acid; M represents A / C degenerate base; R represents A / G degenerate base; Y represents C / T degenerate base; W represents A / T degenerate base; K represents G / T degenerate base.

[0229] 3.2 PCR system

[0230] 5x HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart Taq DNA Polymerase 0.2 μL, SEQ ID NO: 1 (50 μM) 0.2 μL, SEQ ID NO: 2 (50 μM) 0.2 μL, SEQ ID NO: 3 (50 μM) 0.1 μL, SEQ ID NO: 4 (50 μM) 0.2 μL, SEQ ID NO: 5 (50 μM) 0.2 μL, SEQ ID NO: 6 (50 μM) 0.1 μL, SEQ ID NO: 7 (50 μM) 0.2 μL, SEQ ID NO: 8 (50 μM) 0.2 μL, SEQ ID NO: 9 (50 μM) 0.1 μL, SEQ ID NO: 10 (50 μM) 0.2 μL, SEQ ID NO: 11 (50 μM) 0.2 μL, SEQ ID NO: 12 (50 μM) 0.1 μL, SEQ ID NO: 13 (50 μM) 0.2 μL, SEQ ID NO: 14 (50 μM) 0.2 μL, SEQ ID NO: 15 (50 μM) 0.1 μL, SEQ ID NO: 16 (50 μM) 0.2 μL, SEQ ID NO: 17 (50 μM) 0.2 μL, SEQ ID NO: 18 (50 μM) 0.1 μL, SEQ ID NO: 19 (50 μM) 0.1 μL, SEQ ID NO: 20 (50 μM) 0.2 μL, SEQ ID NO: 21 (50 μM) 0.2 μL, SEQ ID NO: 22 (50 μM) 0.1 μL, SEQ ID NO: 23 (50 μM) 0.1 μL, SEQ ID NO: 24 (50 μM) 0.2 μL, SEQ ID NO: 25 (50 μM) 0.2 μL, SEQ ID NO: 26 (50 μM) 0.1 μL, template 5 μL, add water to 30 μL.

[0231] 3.3 PCR program: 95 °C 10 min, 40 cycles (95 °C 30 s, 56 °C 15 s, 72 °C 15 s).

[0232] 3.4 Chip scanning

[0233] The test can effectively detect vanA / B, KPC, MCR, mecA / C, IMP, NDM, OXA48 genes and human reference genes, and the detection results are shown in Table 6 and Table 7. Figure 9-16

[0234] By comparing the single and multiple results of detecting each target point, it can be seen that there is no difference in the amplification concentration results of single and multiple. Therefore, there is no mutual influence between primer probes that leads to the reduction of PCR amplification efficiency, and the results are shown in Table 8 and Table 9. Figure 17-25

[0235] Table 9

[0236]

[0237] Example 4 Dual-color fluorescence screening

[0238] 4.1 Fluorescence channel

[0239] Because the separation degrees of positive and negative points of primer probes of different genes in different fluorescence channels are different, in order to ensure good separation effect of positive and negative points, according to the primer sequences of the target points in Example 1, the probes of KPC, NDM, IMP, OXA48, mecA, mecC, vanA, vanB and MCR genes are marked with different single or dual colors for combination, and the scheme is shown in Table 19.

[0240] Among them, the purpose of setting dual-color labels for some genes in the application is that: since the detection equipment is a 5-channel equipment, the reagent kit has 8 target points, so 3 target points need to be labeled with dual colors to detect 8 target points at a time; and the labeling scheme is determined after screening, and the main index of screening is the discrimination degree of target points.

[0241] Table 19

[0242]

[0243] 4.2 PCR system

[0244] 5×HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNA Polymerase 0.2μL, the concentration of primer probe is 50μM, and the addition ratio is 0.2μL:0.2μL:0.1μL, the dual-color probe is added according to the primer probe ratio of 0.2μL:0.2μL:0.1μL:0.1μL, the template is 5μL, and water is added to 30μL.

[0245] ​​4.3 PCR procedure: 95°C 10 min, 40 cycles (95°C 30 s, 56°C 15 s, 72°C 15 s).

[0246] 4.4 Chip scanning

[0247] The results of digital PCR are shown in Table 1. Figure 26-33 According to the results of the figures, the positive point signal intensity of each target in scheme 1 is better than others, so scheme 1 is selected as the subsequent probe fluorescence labeling scheme.

[0248] Example 5 Sensitivity verification of digital PCR method for detecting KPC, NDM, IMP, OXA48, mecA / C, vanA / B and MCR genes

[0249] According to Example 1, the copy number of each pathogen template (copies / μL) was calculated, and each pathogen template was diluted to 8 copies / μL, 4 copies / μL, 2 copies / μL and 1 copies / μL with Tris-EDTA Buffer to make gradient dilution templates.

[0250] Verification of digital PCR system: 5x HS Taq Buffer with Mg 2+6 μL, dNTPs (10 mM each) 0.75 μL, HotStart Taq DNA Polymerase 0.2 μL, SEQ ID NO: 1 (50 μM) 0.2 μL, SEQ ID NO: 2 (50 μM) 0.2 μL, SEQ ID NO: 3 (50 μM) 0.2 μL, SEQ ID NO: 4 (50 μM) 0.1 μL, SEQ ID NO: 5 (50 μM) 0.2 μL, SEQ ID NO: 6 (50 μM) 0.1 μL, SEQ ID NO: 7 (50 μM) 0.2 μL, SEQ ID NO: 8 (50 μM) 0.2 μL, SEQ ID NO: 9 (50 μM) 0.1 μL, SEQ ID NO: 10 (50 μM) 0.2 μL, SEQ ID NO: 11 (50 μM) 0.2 μL, SEQ ID NO: 12 (50 μM) 0.1 μL, SEQ ID NO: 13 (50 μM) 0.2 μL, SEQ ID NO: 14 (50 μM) 0.2 μL, SEQ ID NO: 15 (50 μM) 0.1 μL, SEQ ID NO: 16 (50 μM) 0.2 μL, SEQ ID NO: 17 (50 μM) 0.2 μL, SEQ ID NO: 18 (50 μM) 0.1 μL, SEQ ID NO: 19 (50 μM) 0.1 μL, SEQ ID NO: 20 (50 μM) 0.2 μL, SEQ ID NO: 21 (50 μM) 0.2 μL, SEQ ID NO: 22 (50 μM) 0.1 μL, SEQ ID NO: 23 (50 μM) 0.1 μL, SEQ ID NO: 24 (50 μM) 0.2 μL, SEQ ID NO: 25 (50 μM) 0.2 μL, SEQ ID NO: 26 (50 μM) 0.1 μL, Template 5 μL, add water to 30 μL.

[0251] The templates were added in the following order in the sample preparation area: blank control, negative control, gradient dilution templates. The blank control was water, the negative control was human genomic DNA, and the gradient dilution templates were 8 copies / μL, 4 copies / μL, 2 copies / μL, 1 copies / μL of each pathogen genome.

[0252] The PCR reaction system was generated into droplets according to the same method of Example 1. PCR was performed according to the PCR program: 95 °C for 10 min, 40 cycles (95 °C for 30 s, 56 °C for 15 s, 72 °C for 15 s). The plate was read according to the instrument requirements.

[0253] The digital PCR results are shown in Table 1. Figure 34-42As shown, the digital PCR method of the application uses water or human genomic DNA as a template, and has clean background and no pollution.

[0254] In addition, the digital PCR method of the application can effectively detect positive points when detecting 1 copies / µL template, that is, the minimum detection sensitivity of the digital PCR method of the application is 5 copies / reaction.

[0255] Discussion

[0256] The application discloses a method for detecting drug-resistant genes in blood stream infection pathogens and application, and relates to primers and probes.

[0257] The application provides a primer probe composition, which mainly utilizes a multiplex fluorescence PCR analysis method to detect and distinguish different pathogens by designing specific primers and probes on different drug-resistant genes.

[0258] The 5' end of the probe sequence of the mecA gene and the mecC gene is provided with a FAM fluorescent group, and the 3' end is provided with a BHQ1 group; the 5' end of the probe sequence of the OXA48 gene is provided with a HEX fluorescent group, and the 3' end is provided with a BHQ1 group; the 5' end of the probe sequence of the KPC gene is provided with a ROX fluorescent group, and the 3' end is provided with a BHQ2 group; the 5' end of the probe sequence of the NDM gene and the IMP gene is provided with a CY5 fluorescent group, and the 3' end is provided with a BHQ2 group; the 5' end of the probe sequence of the MCR gene is respectively provided with a FAM fluorescent group and a ROX fluorescent group, and the 3' end is respectively provided with a BHQ1 group and a BHQ2 group; the 5' end of the probe sequence of the vanA gene and the vanB gene is respectively provided with a ROX fluorescent group and a cy5 fluorescent group, and the 3' end is provided with a BHQ2 group.

[0259] The digital PCR detection kit for drug-resistant genes can detect nucleic acid fragments of specific drug-resistant genes, make up for the shortcomings of low throughput and long time consumption of conventional drug sensitivity analysis methods, provide comprehensive, accurate and low-cost drug-resistant gene diagnosis for the clinic in the first time, and provide an important reference for individualized drug use and precision medicine.

[0260] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the appended claims.

Claims

1. A primer probe combination for detecting drug resistant genes in blood stream infection pathogens, characterized by, The primer probe combination comprises: (a) the upper and lower primers and probes for detecting the mecA and mecC genes as shown in SEQ ID NO: 1-3; (b) the upper and lower primers and probes for detecting the OXA48 gene as shown in SEQ ID NO: 4-6; (c) the upper and lower primers and probes for detecting the KPC gene as shown in SEQ ID NO: 7-9; (d) the upper and lower primers and probes for detecting the NDM gene as shown in SEQ ID NO: 10-12; (e) the upper and lower primers and probes for detecting the IMP gene as shown in SEQ ID NO: 13-15; (f) the upper and lower primers and probes for detecting the MCR gene as shown in SEQ ID NO: 16-19; (g) the upper and lower primers and probes for detecting the vanA and vanB genes as shown in SEQ ID NO: 20-23.

2. The primer probe combination of claim 1, wherein, The primer probe combination further comprises primers and probes for detecting other drug-resistant genes in blood stream infection pathogens.

3. The primer probe combination of claim 1, wherein The primer probe combination further comprises: (h) the upper and lower primers and probes for detecting the internal reference gene as shown in SEQ ID NO: 24-26.

4. The primer probe combination of claim 1, wherein The fluorescence reporter group of the mecA and mecC gene probe is FAM; the fluorescence reporter group of the OXA48 gene probe is HEX; the fluorescence reporter group of the KPC gene probe is ROX; the fluorescence reporter group of the NDM gene probe is CY5; the fluorescence reporter group of the IMP gene probe is CY5; the fluorescence reporter groups of the MCR gene probe are FAM and ROX, respectively; the fluorescence reporter groups of the vanA and vanB gene probes are ROX and CY5, respectively.

5. The primer probe combination of claim 3, wherein The fluorescence reporter group of the internal reference gene probe is Q705.

6. The use of a primer probe combination according to claim 1, characterized in that, A kit for preparing a reagent for detecting drug-resistant genes in blood stream infection pathogens; The drug-resistant genes are selected from the group consisting of the mecA gene, the mecC gene, the OXA48 gene, the KPC gene, the NDM gene, the IMP gene, the MCR gene, the vanA gene, the vanB gene, or a combination thereof.

7. A kit for detecting drug resistance genes in blood stream infection pathogens, characterized by, The kit comprises the primer probe combination of claim 1.

8. The kit of claim 7, wherein The kit further comprises negative and positive quality controls.

9. Use of a primer probe combination according to claim 1 or a kit according to claim 7, characterized in that, A reagent for detecting drug-resistant genes in blood stream infection pathogens; The drug-resistant genes are selected from the group consisting of the mecA gene, the mecC gene, the OXA48 gene, the KPC gene, the NDM gene, the IMP gene, the MCR gene, the vanA gene, the vanB gene, or a combination thereof.

10. Use according to claim 9, characterized in that, The detection comprises the following steps: (i) extracting free DNA in a sample to be tested; (ii) performing fluorescent quantitative PCR on the free DNA obtained in step (i) using the primer probe combination of claim 1 or the kit of claim 7; (iii) reading the fluorescence data and calculating the copy concentration of the target sequence to obtain a detection result.

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