Detection method of KPC type carbapenemase drug-resistant gene

Abstract

The invention provides a detection method of a KPC type carbapenemase drug-resistant gene, and a specific primer pair and a probe used in the detection method. Specifically, the specific primer pair and the probe can efficiently and specifically amplify the KPC gene, the sensitivity of the specific primer pair and the probe reaches 0.01 ng / mu L, and the specific primer pair and the probe have no cross reaction with NDM genome DNA, VIM genome DNA, OXA genome DNA and IMP genome DNA. The detection method avoids aerosol pollution, is simple to operate, can rapidly, conveniently and highly sensitively detect the KPC gene under isothermal conditions, and can be used for rapidly screening and detecting carbapenem antibiotic drug-resistant bacteria producing KPC.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular, relates to a method for detecting a KPC gene based on ERA technology. Background Art

[0002] Bacterial resistance has become a major challenge in the field of global public health. Among the many drug-resistant strains faced in clinical practice, the most important is the rapidly increasing number of carbapenem-resistant Enterobacteriaceae (CRE) in recent years. CRE infections often occur in patients with serious underlying diseases, immunodeficiency and / or long-term repeated use of broad-spectrum antibiotics, with a poor prognosis, especially in patients with CRE bloodstream infections, with a mortality rate of more than 50%. In 2019, the US Centers for Disease Control and Prevention listed CRE as an "emergency threat" level pathogen that threatens human health.

[0003] One of the main reasons for the resistance of Enterobacteriaceae to carbapenem antibiotics is the production of carbapenemases. Carbapenemases are divided into three categories: A, B, and D. KPC belongs to the A-class enzyme (serine carbapenemases), which is the most widespread carbapenemases in Enterobacteriaceae, especially Klebsiella pneumoniae, in the world. Studies have found that the blaKPC gene is often located on a conjugative plasmid, which can be transferred to other bacteria by conjugation to form multidrug-resistant bacteria, causing widespread spread of drug resistance. Therefore, in the face of KPC-producing bacterial infections, it is particularly important to establish a rapid, accurate, and highly sensitive detection method to screen and diagnose this type of superbug as early as possible, adopt effective treatment plans, and avoid the occurrence of further drug resistance.

[0004] At present, the methods for detecting KPC are divided into phenotypic detection and genotypic detection. Phenotypic detection methods include Carba NP test, mCIM and eCIM, carbapenemase inhibitor enhancement test and time-of-flight mass spectrometry, etc. These methods have the disadvantages of being time-consuming and cumbersome to operate. Genotypic detection methods include enzyme immunochromatography and molecular detection technology. Traditional gene identification methods are PCR and DNA sequencing. Among them, the traditional PCR method requires a high-cost thermal cycler, which is complicated and time-consuming to operate. The Sanger DNA sequencing method has high requirements for primer specificity and is expensive. The subsequent data analysis is complicated and not suitable for clinical promotion.

[0005] Therefore, there is an urgent need to develop a KPC gene detection method that is simple to operate, time-saving, highly sensitive, and specific and suitable for on-site detection at room temperature. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting the KPC gene which is simple to operate, short in time, highly sensitive, highly specific and suitable for on-site detection at room temperature.

[0007] In a first aspect of the present invention, a non-diagnostic and non-therapeutic KPC gene amplification method is provided, the amplification method comprising the steps of: performing amplification in a reaction system, the reaction system containing a specific primer pair for amplifying the KPC gene, the primer pair comprising: a sequence as shown in SEQ ID NO: 1, and a sequence as shown in SEQ ID NO: 6.

[0008] In another preferred embodiment, the minimum detection concentration of KPC in the method is 0.005-0.015 ng / μL.

[0009] In another preferred embodiment, the minimum detection concentration of KPC in the method is 0.01 ng / μL.

[0010] In another preferred example, the reaction system further comprises a probe, and the probe comprises a sequence as shown in SEQ ID NO:19.

[0011] In another preferred embodiment, the probe is labeled with a fluorescent group and a quencher group.

[0012] In another preferred embodiment, the 5' end of the probe is labeled with a fluorescent gene FAM, and the 3' end is labeled with a quenching group BHQ.

[0013] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 11 or its complementary chain, and a sequence as shown in SEQ ID NO: 17 or its complementary chain.

[0014] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 3 or its complementary chain, and a sequence as shown in SEQ ID NO: 6 or its complementary chain.

[0015] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 3 or its complementary chain, and a sequence as shown in SEQ ID NO: 8 or its complementary chain.

[0016] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 1 or its complementary chain, and a sequence as shown in SEQ ID NO: 9 or its complementary chain.

[0017] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 2 or its complementary chain, and a sequence as shown in SEQ ID NO: 6 or its complementary chain.

[0018] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 4 or its complementary chain, and a sequence as shown in SEQ ID NO: 6 or its complementary chain.

[0019] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 4 or its complementary chain, and a sequence as shown in SEQ ID NO: 9 or its complementary chain.

[0020] In another preferred embodiment, the method further comprises: performing an amplification reaction in another positive (control) reaction system or a negative (control) reaction system.

[0021] In another preferred embodiment, the positive reaction system contains genomic DNA of drug-resistant Klebsiella pneumoniae expressing KPC.

[0022] In another preferred embodiment, the reaction system further comprises amplification elements such as DNA polymerase, single-stranded binding protein, endonuclease IV, recombinase and auxiliary protein.

[0023] In another preferred embodiment, the method further comprises the step of detecting the fluorescent signal emitted by the specific probe during or after the amplification reaction.

[0024] In another preferred embodiment, the method is enzymatic isothermal amplification (ERA).

[0025] In another preferred embodiment, the reaction reagents of the ERA further include: one or more of a universal solvent for the ERA reaction system, an activator, a fluorescent amplification reagent, and ddH2O.

[0026] In another preferred embodiment, the reaction temperature of the ERA isothermal amplification is 36°C to 42°C, preferably 37°C to 39°C.

[0027] In another preferred embodiment, the reaction time of the ERA isothermal amplification is 15-60 minutes, preferably 15-20 minutes.

[0028] In a second aspect of the present invention, a non-diagnostic KPC gene detection method is provided, the method comprising the steps of:

[0029] (a) amplifying the sample to be tested using the method described in the first aspect of the present invention; and

[0030] (b) Perform signal detection.

[0031] In another preferred embodiment, the method further comprises processing the sample to be tested to obtain genomic DNA, and then amplifying the target DNA by the method described in the first aspect of the present invention.

[0032] In another preferred embodiment, the sample to be tested is the nucleic acid of a human excrement sample.

[0033] In another preferred embodiment, the method further comprises setting up one or more control groups.

[0034] In another preferred example, the control group includes a negative control group and a positive control group.

[0035] In another preferred embodiment, the positive control group includes a drug-resistant strain of Klebsiella pneumoniae that produces KPC.

[0036] In another preferred embodiment, the method includes qualitative detection and quantitative detection.

[0037] In another preferred embodiment, the minimum detection concentration of KPC in the method is 0.005-0.015 ng / μL.

[0038] In another preferred embodiment, the minimum detection concentration of the method is about 0.01 ng / μL.

[0039] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0040] In another preferred example, the detection includes fluorescence signal detection.

[0041] In another preferred embodiment, the fluorescence detection includes using a fluorescence quantitative PCR instrument or using a handheld fluorescence detector for real-time detection.

[0042] In a third aspect of the present invention, a detection kit for rapid detection of KPC gene is provided, the detection kit comprising:

[0043] A container, and a specific primer pair and a probe for the KPC gene in the container, wherein the specific primer pair comprises a sequence as shown in SEQ ID NO: 1 and a sequence as shown in SEQ ID NO: 6; and the probe comprises a sequence as shown in SEQ ID NO: 19.

[0044] In another preferred embodiment, the detection kit further comprises elements such as DNA polymerase, single-strand binding protein, endonuclease IV, recombinase and auxiliary protein.

[0045] In another preferred example, the detection kit further comprises a sample processing reagent, and the sample processing reagent comprises a cell lysis solution, a nucleic acid extraction solution, a washing solution and the like.

[0046] In another preferred example, the detection kit further includes an ERA reaction reagent, and the ERA reaction reagent includes one or more of a universal dissolving agent, an activating agent, a fluorescent amplification reagent, and ddH2O.

[0047] In another preferred embodiment, the detection kit further includes a KPC positive control and a KPC negative control.

[0048] In another preferred embodiment, the detection kit further comprises an instruction manual, wherein the instruction manual is used to guide the detection of the KPC gene.

[0049] In a fourth aspect of the present invention, there is provided a use of the detection kit as described in the third aspect, for non-diagnostic and non-therapeutic detection of whether a sample to be tested contains KPC-type carbapenemase-resistant bacteria.

[0050] In another preferred embodiment, the sample to be tested is the nucleic acid of a human excrement sample.

[0051] In another preferred embodiment, the kit is used to identify whether the sample to be tested contains KPC-type carbapenemase-resistant bacteria. By detecting the KPC gene content C0 of the sample to be tested and the KPC gene content C1 in the negative control sample, if C0>C1 or C0 / C1>1.5, preferably>2, and more preferably>3, it indicates that the sample to be tested may contain KPC-type carbapenemase-resistant bacteria.

[0052] In another preferred embodiment, the kit is also used to evaluate the antibacterial effect of drugs against KPC-type carbapenemase-resistant bacteria. The KPC gene content Z0 in the sample after the drug treatment of KPC-type carbapenemase-resistant bacteria and the KPC gene content Z1 in the sample without the above-mentioned drug are detected; if the ratio of Z0 / Z1 is ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 3, it indicates that the drug has the effect of inhibiting KPC-type carbapenemase-resistant bacteria.

[0053] In a fifth aspect of the present invention, a specific primer pair for amplifying the KPC gene is provided, the primer pair comprising a sequence as shown in SEQ ID NO:1 and a sequence as shown in SEQ ID NO:6.

[0054] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 11 or its complementary chain, and a sequence as shown in SEQ ID NO: 17 or its complementary chain.

[0055] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 3 or its complementary chain, and a sequence as shown in SEQ ID NO: 6 or its complementary chain.

[0056] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 3 or its complementary chain, and a sequence as shown in SEQ ID NO: 8 or its complementary chain.

[0057] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 1 or its complementary chain, and a sequence as shown in SEQ ID NO: 9 or its complementary chain.

[0058] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 2 or its complementary chain, and a sequence as shown in SEQ ID NO: 6 or its complementary chain.

[0059] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 4 or its complementary chain, and a sequence as shown in SEQ ID NO: 6 or its complementary chain.

[0060] In another preferred example, the primer pair includes a sequence as shown in SEQ ID NO: 4 or its complementary chain, and a sequence as shown in SEQ ID NO: 9 or its complementary chain.

[0061] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 shows the results of the ERA method for detecting the KPC resistance gene of Klebsiella pneumoniae using 32 primer pairs: A. Primer pairs A1 to A8; B. Primer pairs B1 to B8; C. Primer pairs C1 to C8; D. Primer pairs D1 to D8. The primer pair information is shown in Table 2.

[0063] Figure 2 The results of further screening and testing of the optimal primer pairs for detecting carbapenem antibiotic KPC resistance genes in Klebsiella pneumoniae by ERA method are shown.

[0064] Figure 3 Shown are the test results of the optimal primer pair for detecting different concentrations of Klebsiella pneumoniae genomic DNA.

[0065] Figure 4 Shown are the test results of the optimal primer pair for detecting different concentrations of Klebsiella pneumoniae genomic DNA in human feces.

[0066] Figure 5 The results of cross-reactivity specificity testing of the optimal primer pairs on different genomes are shown. DETAILED DESCRIPTION

[0067] After extensive and in-depth research and a large number of screenings, the inventors have developed a method for rapid detection of KPC genes based on enzymatic recombinant isothermal amplification (ERA). Experiments have shown that the method has a sensitivity of 0.01 ng / μL, high detection specificity, and no cross-reaction with NDM, VIM, OXA, and IMP genomic DNA, and can be used for rapid screening and detection of KPC-producing carbapenem-resistant bacteria. The present invention was completed on this basis.

[0068] the term

[0069] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0070] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0071] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0072] Primers and probes

[0073] As used herein, the term "primers specific for the KPC gene" refers to primers (pairs) that amplify products having complementary sequences of the KPC gene. Preferred primer sequences include a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 6.

[0074] In the amplification method of the present invention, in addition to the primers, the reaction system may also include one or more probes, such as a KPC fluorescent probe, for detecting the amplification effect of the KPC gene. The probe has a FAM fluorescent group and a BHQ quenching group, and has a blocking group at the 3'-end to block the polymerase extension of the probe. A preferred detection probe of the present invention has a nucleotide sequence as shown in SEQ ID NO: 19.

[0075] In the present invention, many primer sequences were designed, and 32 primer pairs were formed by combining two of them. After a large number of experimental screenings, the combination of SEQ ID NO: 1 and SEQ ID NO: 6 with high specificity and high sensitivity was finally selected, and its sensitivity can reach 2.664 copies / μL.

[0076] ERA

[0077] Enzymatic Recombinase Amplification (ERA) is an isothermal amplification technology developed by Suzhou Xinda Gene Technology Co., Ltd. in China. Through genetic engineering, the multi-enzyme system of recombinase, exonuclease, polymerase, etc. is modified to establish a special amplification reaction system. At room temperature, the specific segment of trace DNA / RNA can be amplified billions of times within a few minutes. It has the advantages of being fast and simple, easy to operate, and having reliable results.

[0078] The present invention uses ERA to detect KPC gene, and in addition to specific primers and probes, also includes universal reaction reagents of ERA, including one or more of universal solvent, activator, fluorescent amplification reagent, and ddH2O. When other methods are used to detect KPC, the specific primers and probes of the present invention can also be used to cooperate with reaction reagents applicable to other methods to perform KPC detection.

[0079] KPC

[0080] Carbapenemases are divided into three categories: A, B, and D. KPC belongs to the A category (serine carbapenemases), and is the most prevalent carbapenemases in Enterobacteriaceae, especially Klebsiella pneumoniae. KPC is one of the subtypes of carbapenemases. Carbapenemases also include OXA type, IMP type, NDM type, and VIM type.

[0081] In the present invention, "carbapenem antibiotic-resistant bacteria" and "carbapenemase-resistant bacteria" are used interchangeably to refer to bacteria that express carbapenemase and are resistant to carbapenem antibiotics.

[0082] The KPC-producing Klebsiella pneumoniae resistant strains involved in the embodiments of the present invention were donated by the First Affiliated Hospital of Nanchang University. The fecal samples of healthy volunteers were obtained from the Shanghai Institute of Biomedical Technology.

[0083] The 18 primers designed for the KPC gene of the present invention include: 6 upstream primers (SEQ ID NOs: 1-4, 11-12), 12 downstream primers (SEQ ID NOs: 5-10, 13-18), and 1 fluorescent probe (SEQ ID NO: 19), as shown in Table 1.

[0084] Table 1 Primer and probe sequences

[0085]

[0086] (FAM-dT)(THF)(BHQ1-dT) indicates that the probe consists of an oligonucleotide backbone consisting of a flanking FAM-dT fluorophore, a deoxynucleotide analog (tetrahydrofuran [THF] residue), and a BHQ1-dT quencher.

[0087] (C3-SPACER) here stands for: 3'-blocking group to block polymerase extension of the probe.

[0088] Detection Methods

[0089] The present invention provides a method for detecting KPC gene, comprising amplifying a sample to be detected using the specific primers of the present invention; and

[0090] During or after the amplification reaction, the amplified product of the KPC gene is detected, for example, by detecting the fluorescent signal emitted by the specific probe.

[0091] The method may also optionally include setting up one or more control groups, such as a KPC positive control group, a KPC negative control group, etc.

[0092] Preferably, the KPC positive control group is genomic DNA of drug-resistant Klebsiella pneumoniae expressing KPC.

[0093] Preferably, the KPC negative control group is PCR water.

[0094] In the present invention, the detection method can be a conventional PCR method, or a different method such as real-time fluorescence PCR. Preferably, the present invention uses the ERA method.

[0095] Typically, the ERA method of the present invention comprises the following steps:

[0096] S1. DNA from stool samples was extracted using thermal lysis method as a template;

[0097] S2. Amplifying DNA in an ERA reaction system comprising specific primers and specific probes of the present invention;

[0098] S3. Signal detection is performed through the fluorescent signal carried by the specific probe.

[0099] Among them, the ERA reaction system also includes other ERA reaction reagents, such as universal solvent, activator, fluorescence amplification reagent, ddH2O, etc.

[0100] Detection Kits

[0101] The present invention also provides a KPC gene detection kit, which contains a specific detection reagent and can more accurately identify whether the sample to be tested contains carbapenemase-resistant bacteria, especially KPC-type carbapenemase-resistant bacteria. By detecting the KPC gene content C0 of the sample to be tested and the KPC gene content C1 in the negative control, if C0>C1 or C0 / C1>1.5, preferably>2, and more preferably>3, it indicates that the sample to be tested may contain KPC-type carbapenemase-resistant bacteria.

[0102] Preferably, the detection reagents of the detection kit of the present invention include specific primers, specific probes, reagents for preparing the ERA reaction system, and the like.

[0103] The detection reagent of the present invention further comprises a container and an instruction manual, wherein the container is used to contain the detection reagent, and the instruction manual is used to guide the use of the detection reagent.

[0104] Based on this kit, the present invention can further provide the antibacterial effect of the drug against KPC-type carbapenemase-resistant bacteria. The KPC gene content Z0 in the sample after the drug treatment of KPC-type carbapenemase-resistant bacteria and the KPC gene content Z1 in the sample without the above-mentioned drug are detected; if the ratio of Z0 / Z1 is ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 3, it indicates that the drug has the effect of inhibiting KPC-type carbapenemase-resistant bacteria.

[0105] Compared with the prior art, the advantages of the present invention are as follows:

[0106] 1. The technical solution provided by the present invention takes the KPC gene as the detection target, relies on enzymatic amplification reaction, uses a specific primer and probe combination, and completes the detection within 20 minutes through constant temperature amplification technology. It does not require a complicated sample DNA pretreatment process, does not require thermal deformation of the template, is easy to operate, and takes a short time.

[0107] 2. The reaction conditions of the technical solution of the present invention are 37-42°C, and the temperature control does not need to be particularly strict, nor does it need to set multiple parameters, and the energy consumption is low. Therefore, it is particularly suitable for on-site operation.

[0108] 3. The technical solution of the present invention uses fluorescence numerical interpretation results, eliminating the process of electrophoresis verification of PCR products and avoiding aerosol contamination.

[0109] 4. The specific primer pair and probe of the KPC gene of the present invention have extremely high sensitivity, with a minimum detection concentration of about 0.01 ng / μL. They also have high specificity and have no cross-reaction with other subtype genes of carbapenemase.

[0110] 5. The KPC gene detection kit of the present invention can be used as a reagent for detecting the KPC gene in clinical specimens (excreta, etc.) or clinical isolates, and can be used for scientific research and clinical applications.

[0111] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0112] Example 1. Screening for the optimal primer pair for KPC gene specific amplification

[0113] Table 2 Primer pairs used to detect KPC gene specific amplification

[0114]

[0115] The upstream and downstream primers described in Table 1 were paired in pairs to form 32 primer pairs, as shown in Table 2. The drug-resistant Klebsiella pneumoniae strains producing KPC were used as the detection object to detect the effects of different primer pairs in the specific amplification of the KPC gene, and SEQ ID NO: 19 was the probe sequence.

[0116] DNA sample preparation: The template DNA is the genomic DNA extracted from the above strains after culture. Specifically, the strains were cultured in brain heart infusion broth (BHI) medium containing ampicillin (50 μg / mL) at 37°C according to the standard protocol, and then Plasmid DNA Mini Kit I (D6942-01) was used to extract genomic DNA according to the instructions. The template DNA was diluted 10 times in a row, and the working concentration was 10.0 ng / μl.

[0117] ERA isothermal amplification: Amplification was performed using the ERA isothermal amplification reaction system. The 50ul amplification reaction system was constructed as follows:

[0118]

[0119] The amplification temperature was 37°C and the reaction lasted for 20 min.

[0120] Fluorescence detection: During the reaction, the GS8 fluorescence thermostatic amplification instrument was used to detect the change in fluorescence value, and the fluorescence was read every 30 seconds until the reaction was completed. On the fluorescence detector, the positive group showed an "S" curve with an exponential amplification trend; the negative group showed a flat horizontal straight line with no amplification trend; the TT (time threshold) value was 4 to 8 minutes. Parallel experiments were performed with different primer pairs to detect the fluorescence results.

[0121] The test results showed that after a large number of screenings, among the 32 primer pair combinations, the following combinations were screened out: SEQ ID NO: 1 + SEQ ID NO: 6, SEQ ID NO: 1 + SEQ ID NO: 9 and SEQ ID NO: 2 + SEQ ID NO: 6 had good sensitivity and specificity (Figure 1).

[0122] Example 2. Further screening of optimal primer pairs

[0123] In order to screen the optimal primer pair, the KPC-producing Klebsiella pneumoniae resistant strain was used as the detection object, and the primer pairs SEQ ID NO: 1 + SEQ ID NO: 6, SEQ ID NO: 1 + SEQ ID NO: 9 and SEQ ID NO: 2 + SEQ ID NO: 6 described in Example 1 were further screened.

[0124] The genomic DNA of the KPC-producing Klebsiella pneumoniae resistant strain described in Example 1 was extracted as a template for detection, and a negative control group without a DNA template was set up. The reaction system is as described in Example 1. The amplification temperature is 37°C, the reaction is 20 minutes, and the change value of fluorescence is detected by GS8 fluorescence constant temperature amplification instrument, and the fluorescence is read every 30 seconds. The positive shows an "S" curve with an exponential amplification trend, while the negative control is a flat horizontal straight line with no amplification trend.

[0125] Parallel experiments were performed with templates of different concentrations, and the fluorescence results were detected.

[0126] The results showed that the optimal primer pair was SEQ ID NO:1+SEQ ID NO:6. Figure 2 .

[0127] SEQ ID NO: 1: 5'-GCAGTTTGTTGATTGGCTAAAGGGAAACACGA-3'

[0128] SEQ ID NO:6: 5'-ACTTGTCATCCTTGTTAGGCGCCCGGGTGTAG-3'

[0129] Example 3. Minimum detection concentration of KPC gene

[0130] Select the following optimal primer pairs and probe sequences:

[0131] SEQ ID NO: 1: 5'-GCAGTTTGTTGATTGGCTAAAGGGAAACACGA-3'

[0132] SEQ ID NO:6: 5'-ACTTGTCATCCTTGTTAGGCGCCCGGGTGTAG-3'

[0133] SEQ ID NO:19(probe):5'-TCGGAGACAAAACCGGAACCTGCG

[0134] GAGTG / i6FAMdT / iTHF / / IBHQ1dT / GGCACGGCAAATGA-3'

[0135] The genomic DNA of Klebsiella pneumoniae expressing KPC was used as a positive template, and PCR water was used as a negative control to perform ERA isothermal amplification to detect the detection limit of ERA. The pure genomic DNA of Klebsiella pneumoniae was diluted 10 times continuously, and the content range was 0.01-10ng / μL. The reaction system is as described in Example 1, and the primers and probes are as described in Example 3. The amplification temperature is 37°C, the reaction is 20min, and the change in fluorescence is detected using a GS8 fluorescence constant temperature amplification instrument, and the fluorescence is read every 30s. Parallel experiments were performed with templates of different concentrations to detect the fluorescence results.

[0136] The results show that the KPC gene detection method of the present invention has an extremely low detection concentration of 0.01 ng / μL (i.e. 2.664 copies / μL). Figure 3 .

[0137] Example 4. KPC gene detection in human fecal samples

[0138] A 1 g stool sample was collected using a sterile spatula, thawed on ice, and added to 10 ml of ice-cold phosphate-buffered saline (PBS). 900 μL of the stool suspension was transferred to a new Eppendorf tube and inoculated with 100 μL of an overnight culture of a KPC-producing Klebsiella pneumoniae resistant strain to a level of 10 8 CFU / 1.0g feces. Take 15μL of the suspension and resuspend it in 150μL of extraction buffer, vortex gently, and boil at 95℃ for 5 minutes to release DNA. Perform 10-fold gradient dilution of the obtained sample to obtain 10 8 ~10 5 CFU / 1.0g corresponding DNA sample. Take 2 μL of the lysate as the template DNA in this example. The reaction system is as described in Example 1, and the primer pair and probe sequence are as described in Example 3. Detect the fluorescence result.

[0139] The results showed that the detection method of the present invention is effective for human fecal samples (10 5 CFU / 1.0g, corresponding to 12.5 CFU Klebsiella pneumoniae) is also effective, see Figure 4 .

[0140] Example 5. Optimal primer pairs and probe specificity for carbapenemase KPC gene

[0141] The optimal primer pair and probe sequence as described in Example 2 were selected, and genomic DNA samples containing different subtypes of carbapenemase (blaNDM, blaKPC, blaKPC, blaOXA, blaKPC) at a concentration of 20 ng were used to evaluate the specificity of the primer pair and probe combination in detecting the KPC gene. The reaction system is as described in Example 1. ddH2O was used for negative control to ensure that amplification would not occur in the absence of target DNA. The amplification temperature was 37°C, the reaction was 20 minutes, and the change in fluorescence was detected using a GS8 fluorescence thermostatic amplification instrument, and the fluorescence was read every 30 seconds.

[0142] The results showed that the amplification curve only appeared in KPC-positive DNA samples, and there was no cross-reaction with other carbapenemase subtype genes blaNDM, blaIMP, blaOXA, and blaVIM ( Figure 5 ), indicating that the primer pair and probe for the KPC gene screened by the present invention have extremely high specificity.

[0143] Example 6. Comparison of the detection rate of KPC gene by the method of the present invention and commercially available kits

[0144] In order to test the effectiveness and specificity of the technology of the present invention in detecting KPC+ in clinical samples, stool samples of 8 KPC+ patients were obtained from the First Affiliated People's Hospital of Nanchang. The fecal swab was soaked in 200 μL of lysis solution, vortexed and homogenized, and boiled in a 95°C water bath for 10 minutes to release DNA. Subsequently, 2 μL of lysis solution was used to perform ERA reaction according to the reaction system of Example 1. At the same time, the test results were compared with the test results of the commercial kit (Cepe).

[0145] The results show that: as shown in Table 3, the KPC+ detection rate of the method of the present invention can reach 100%, which is higher than the commercial kit (Cepheid).

[0146] Table 3. KPC+ detection rate

[0147]

[0148] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A non-diagnostic and non-therapeutic method for amplifying the KPC gene, characterized in that: The amplification method comprises the steps of: performing amplification in a reaction system, wherein the reaction system contains a specific primer pair for amplifying the KPC gene, and the primer pair comprises: a sequence as shown in SEQ ID NO:1, and a sequence as shown in SEQ ID NO:

6.

2. The method according to claim 1, characterized in that The method further comprises a probe comprising the sequence shown in SEQ ID NO:

19.

3. A non-diagnostic detection method for KPC gene, characterized in that: The method comprises the steps of: (a) amplifying the sample to be tested using the method according to claim 1; and (b) Perform signal detection.

4. The method according to claim 3, characterized in that The method further comprises setting a KPC negative control and / or a KPC positive control.

5. The method according to claim 3, characterized in that The sample to be tested is a human excrement sample.

6. The method according to claim 3, characterized in that The minimum detection concentration of the method is 0.005-0.015 ng / μL.

7. A detection kit for rapid detection of KPC gene, characterized in that: The detection kit comprises: a container, and a primer pair and a probe specific to the KPC gene in the container, Wherein, the primer pair includes the sequence shown as SEQ ID NO:1 and the sequence shown as SEQ ID NO:6; and the probe includes the sequence shown as SEQ ID NO:

19.

8. A use of the detection kit according to claim 7, characterized in that: Used for non-diagnostic and non-therapeutic detection of whether the test sample contains KPC-type carbapenemase-resistant bacteria.

9. The use according to claim 8, characterized in that The kit is also used to evaluate the antibacterial effect of drugs against KPC-type carbapenemase-resistant bacteria.

10. A specific primer pair for amplifying the KPC gene, characterized in that: The primer pair includes a sequence as shown in SEQ ID NO:1 and a sequence as shown in SEQ ID NO:6.

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