Detection Method for KPC-Type Carbapenemase Resistance Genes

Abstract

The present invention provides a method for detecting KPC-type carbapenemase resistance genes and a specific primer pair and probe used therefor. Specifically, the specific primer pair and probe of the present invention can efficiently and specifically amplify the KPC gene, with a sensitivity reaching 0.01 ng / μL and no cross-reaction with genomic DNA of NDM, VIM, OXA, and IMP. The detection method avoids aerosol contamination, is simple to operate, can rapidly, conveniently, and highly sensitively detect the KPC gene under isothermal conditions, and can be used for rapid screening and detection of carbapenem-resistant bacteria producing KPC.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a method for detecting 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 bacterial species faced clinically, the most important one is the carbapenem-resistant Enterobacteriaceae (CRE) that has rapidly increased in recent years. CRE infections mostly occur in patients with severe underlying diseases, immune deficiencies, and / or long-term repeated use of broad-spectrum antibacterial drugs, and the prognosis is poor. Especially for patients with CRE bloodstream infections, the fatality rate is as high as over 50%. In 2019, the US Centers for Disease Control and Prevention classified CRE as a pathogen of "urgent threat" level that endangers human health due to drug resistance.

[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 class A enzymes (serine carbapenemases) and is the most widely prevalent carbapenemase among global Enterobacteriaceae, especially Klebsiella pneumoniae. Studies have found that the blaKPC gene is often located on conjugative plasmids, which can be conjugatively transferred to other bacteria to form multi-drug resistant bacteria, enabling the widespread transmission 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 such super bacteria as early as possible, and adopt effective treatment plans to avoid the occurrence of further drug resistance.

[0004] Currently, the methods for detecting KPC are divided into phenotypic detection and genotypic detection. Phenotypic detection methods include the Carba NP test, mCIM and eCIM, carbapenemase inhibitor enhancement test, and time-of-flight mass spectrometry technology, etc. These methods mostly have disadvantages such as long time consumption and cumbersome operation. Genotypic detection methods include enzyme immunoassay chromatography technology and molecular detection technology. Traditional gene identification methods are the PCR method and DNA sequencing method. Among them, the traditional PCR method requires a thermocycling instrument with high cost, complex operation, and long time consumption; the Sanger DNA sequencing method has high requirements for primer specificity, high price, and complex data analysis in the later stage, and is not suitable for clinical promotion.

[0005] Therefore, there is an urgent need to develop a KPC gene detection method with simple operation, short time consumption, high sensitivity, strong specificity, and suitable for on-site detection at room temperature. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for detecting KPC gene with simple operation, short time consumption, high sensitivity, strong specificity, and suitable for on-site detection at room temperature.

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

[0008] In another preferred example, the lowest detectable concentration of KPC in the method is 0.005 - 0.015 ng / μL.

[0009] In another preferred example, the lowest detectable concentration of KPC in the method is 0.01 ng / μL.

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

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

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

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

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

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

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

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

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

[0019] In another preferred embodiment, the primer pair comprises the sequence shown in SEQ ID NO: 4 or its complementary strand, and the sequence shown in SEQ ID NO: 9 or its complementary strand.

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

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

[0022] In another preferred embodiment, the reaction system further comprises amplification components such as DNA polymerase, single-strand binding protein, endonuclease IV, recombinase, and accessory proteins.

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

[0024] In another preferred embodiment, the method is an enzyme-mediated isothermal amplification (ERA).

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

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

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

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

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

[0030] (b) performing 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 using 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 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 example, the positive control group includes Klebsiella pneumoniae resistant strains producing KPC.

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

[0037] In another preferred example, the lowest detectable concentration of KPC in the method is 0.005 - 0.015 ng / μL.

[0038] In another preferred example, the lowest detectable concentration of the method is approximately 0.01 ng / μL.

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

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

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

[0042] In the third aspect of the present invention, there is provided a detection kit for rapidly detecting the KPC gene, and the detection kit includes:

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

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

[0045] In another preferred example, the detection kit further includes sample processing reagents, and the sample processing reagents include cell lysate, nucleic acid extraction solution, washing solution, etc.

[0046] In another preferred example, the detection kit further includes ERA reaction reagents, and the ERA reaction reagents include one or more of a general solvent, an activator, a fluorescence amplification reagent, and ddH2O.

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

[0048] In another preferred example, the detection kit further includes an instruction manual for guiding the detection of the KPC gene.

[0049] In the 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 test sample contains KPC-type carbapenemase-resistant bacteria.

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

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

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

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

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

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

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

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

[0058] In another preferred embodiment, the primer pair comprises the sequence shown in SEQ ID NO:2 or its complementary strand, and the sequence shown in SEQ ID NO:6 or its complementary strand.

[0059] In another preferred embodiment, the primer pair comprises the sequence shown in SEQ ID NO:4 or its complementary strand, and the sequence shown in SEQ ID NO:6 or its complementary strand.

[0060] In another preferred embodiment, the primer pair comprises the sequence shown in SEQ ID NO:4 or its complementary strand, and the sequence shown in SEQ ID NO:9 or its complementary strand.

[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 in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0063] Figure 2 Shows the further screening test results of the optimal primer pair for detecting the KPC resistance gene of Klebsiella pneumoniae to carbapenem antibiotics by the ERA method.

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

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

[0066] Figure 5 Shows the cross-reaction specificity test results of the optimal primer pair in different genomes. DETAILED DESCRIPTION OF THE INVENTION

[0067] The present inventors have conducted extensive and in-depth research and a large number of screenings, and developed a detection method for rapidly detecting the KPC gene based on the enzyme-mediated recombinase amplification (ERA) technology. Experiments show that its sensitivity reaches 0.01 ng / μL, the detection specificity is high, and there is no cross-reaction with genomic DNA of NDM, VIM, OXA, and IMP, and it can be used for rapid screening and detection of carbapenem-resistant bacteria producing KPC. Based on this, the present invention has been completed.

[0068] Terminology

[0069] For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, 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 pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0070] As used herein, when referring to a specifically 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 "comprising", "including", "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the said terms include "consisting of", "consisting essentially of".

[0072] Primers and probes

[0073] As used herein, the term "specific primer for the KPC gene" refers to such a primer (pair) whose amplified product has a complementary strand sequence of the KPC gene. Preferred primer sequences include the 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 primers, one or more probes, such as KPC fluorescent probes, may also be included in the reaction system for detecting the amplification effect of the KPC gene. The said probe carries 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 the nucleotide sequence shown in SEQ ID NO:19.

[0075] In the present invention, a number of primer sequences are designed, and 32 primer pairs are formed by pairwise combination. 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 is 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 XianDa Gene Technology Co., Ltd. in Suzhou, China. Through genetic engineering, a multi-enzyme system such as recombinase, exonuclease, and polymerase is modified to establish a special amplification reaction system. At room temperature, specific segments of trace DNA / RNA can be amplified billions of times within a few minutes, with the advantages of being fast, simple, easy to operate, and having reliable results.

[0078] In the present invention, ERA is used to detect the KPC gene. In addition to specific primers and probes, it also includes general reaction reagents for ERA, including one or more of a general solvent, activator, fluorescent amplification reagent, and ddH2O. When using other methods to detect KPC, the specific primers and probes of the present invention can also be used in combination with reaction reagents applicable to other methods for KPC detection.

[0079] KPC

[0080] Carbapenemases are divided into three categories: A, B, and D. KPC belongs to class A enzymes (serine carbapenemases) and is the most widespread carbapenemase in Enterobacteriaceae bacteria globally, 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-resistant bacteria" and "carbapenemase-resistant bacteria" can be used interchangeably to represent bacteria that express carbapenemase and are resistant to carbapenem antibiotics.

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

[0083] The 18 primers designed for the KPC gene in the present invention include: 6 upstream primers (SEQ ID NO: 1-4, 11-12) and 12 downstream primers (SEQ ID NO: 5-10, 13-18), as well as 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) herein represents that the probe consists of an oligonucleotide backbone composed of a flanking FAM-dT fluorophore, a deoxynucleotide analogue (tetrahydrofuran [THF] residue), and a BHQ1-dT quencher group.

[0087] (C3-SPACER) herein represents a 3'-blocking group to block the polymerase extension of the probe.

[0088] Detection method

[0089] The present invention provides a method for detecting the KPC gene, including amplifying a test sample with the specific primers of the present invention; and

[0090] During or after the amplification reaction, detecting the amplification product of the KPC gene, for example, by detecting the fluorescence signal emitted by a specific probe.

[0091] The method may also optionally include setting 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 the genomic DNA of Klebsiella pneumoniae resistant bacteria expressing KPC.

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

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

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

[0096] S1. Extracting DNA from a fecal sample by thermal lysis as a template;

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

[0098] S3. Detecting signals through the fluorescence signal carried by the specific probe.

[0099] Among them, the ERA reaction system further includes other ERA reaction reagents, such as a universal solubilizer, an activator, a fluorescent amplification reagent, ddH2O, etc.

[0100] Detection kit

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

[0103] The detection reagent of the present invention further includes a container and an instruction manual. The container is used to hold the above-mentioned detection reagents, and the instruction manual is used to guide the use of the above-mentioned detection reagents.

[0104] Based on this kit, the present invention can further provide the antibacterial effect of drugs against KPC-type carbapenemase-resistant bacteria. After detecting the content Z0 of the KPC gene in the sample after treating with drugs against KPC-type carbapenemase-resistant bacteria, and the content Z1 of the KPC gene in the sample without using the above-mentioned drugs; if the ratio of Z0 / Z1 ≤ 2 / 3, preferably ≤ 1 / 2, 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 the enzymatic amplification reaction, uses a specific primer and probe combination, and can complete the detection within 20 minutes through the isothermal amplification technology. Moreover, it does not require a complex 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, which does not require particularly strict temperature control, does not need to set a variety of parameters, and has low energy consumption. Therefore, it is particularly suitable for on-site operation.

[0108] 3. The technical solution of the present invention determines the result by reading the fluorescence value, omits the process of verifying the PCR product by electrophoresis, and avoids aerosol contamination.

[0109] 4. The specific primer pair and probe for the KPC gene of the present invention have extremely high sensitivity, and its lowest detection concentration is about 0.01 ng / μL. At the same time, it also has high specificity and has 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 is used for scientific research and clinical applications.

[0111] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out 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 according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight percentage and weight parts.

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

[0113] Table 2 Primer pairs for detecting specific amplification of the KPC gene

[0114]

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

[0116] DNA sample preparation: The template DNA was genomic DNA extracted after culturing the above-mentioned strains. 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 serially diluted 10-fold, and the working concentration was 10.0 ng / μl.

[0117] ERA isothermal amplification: Amplification was carried out using the ERA isothermal amplification reaction system, and a 50 μl amplification reaction system was constructed as follows:

[0118]

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

[0120] Fluorescence detection: During the reaction, the change value of fluorescence was detected with a GS8 fluorescence constant temperature amplifier, and the fluorescence was read every 30 s until the reaction ended. On the fluorescence detector, the positive group showed an "S" curve with an exponential amplification trend; the negative group was a flat horizontal line with no amplification trend; the TT (time threshold) value was 4 - 8 min. Parallel experiments were carried out with different primer pairs to detect the fluorescence results.

[0121] The detection results show 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 have good sensitivity and specificity (Figure 1).

[0122] Example 2. Further screening of the optimal primer pair

[0123] To screen the optimal primer pair, the Klebsiella pneumoniae resistant strain producing KPC 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 Klebsiella pneumoniae resistant strain producing KPC described in Example 1 was extracted as a template for detection, and a negative control group without a DNA template was set up simultaneously. The reaction system was as described in Example 1. The amplification temperature was 37°C, and the reaction was carried out for 20 min. The change value of fluorescence was detected using a GS8 fluorescence constant temperature amplifier, and the fluorescence was read every 30 s. The positive showed an "S" curve with an exponential amplification trend, while the negative control was a flat horizontal line with no amplification trend.

[0125] Parallel experiments were carried out with different concentrations of the template to detect the fluorescence results.

[0126] The results show that the optimal primer pair is SEQ ID NO:1 + SEQ ID NO:6, see Figure 2 。

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

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

[0129] Example 3. The lowest detection concentration of the KPC gene

[0130] The following optimal primer pair and probe sequence were selected:

[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 lower limit of detection of ERA. The pure genomic DNA of Klebsiella pneumoniae was serially diluted 10-fold, with the content ranging from 0.01 - 10 ng / μL. The reaction system was as described in Example 1, and the primers and probes were as described in Example 3. The amplification temperature was 37°C, and the reaction was carried out for 20 min. The GS8 fluorescence constant temperature amplifier was used to detect the change value of fluorescence, and the fluorescence was read every 30 s. Parallel experiments were carried out with templates of different concentrations to detect the fluorescence results.

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

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

[0138] Use a sterile spatula to collect 1 g of fecal sample, melt it on ice, and add 10 ml of ice-cold phosphate buffered saline (PBS). Transfer 900 μL of the fecal suspension into a new Eppendorf tube, and inoculate 100 μL of the overnight cultured Klebsiella pneumoniae resistant strain producing KPC to make the level reach 10 8 CFU / 1.0 g of feces. Take 15 μL of the suspension and resuspend it in 150 μL of extraction buffer, gently vortex, and boil at 95°C for 5 minutes to release DNA. The obtained sample was serially diluted 10-fold to obtain DNA sample products corresponding to 10 8 ~10 5 CFU / 1.0 g. Take 2 μL of the lysate as the template DNA in this example. The reaction system was as described in Example 1, and the primer pair and probe sequence were as described in Example 3. Detect the fluorescence results.

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

[0140] Example 5. Specificity of the Optimal Primer Pair and Probe for the Carbapenemase KPC Gene

[0141] Select the optimal primer pair and probe sequences as described in Example 2, and use genomic DNA samples containing different subtypes of carbapenemase (blaNDM, blaKPC, blaKPC, blaOXA, blaKPC) at a concentration of 20 ng to evaluate the specificity of this primer pair and probe combination for detecting the KPC gene. The reaction system is as described in Example 1. ddH2O is used as a negative control to ensure that amplification does not occur in the absence of target DNA. The amplification temperature is 37 °C, and the reaction is carried out for 20 min. The change value of fluorescence is detected using a GS8 fluorescence constant temperature amplifier, and the fluorescence is read every 30 s.

[0142] The results show that: the amplification curve appears only in the KPC-positive DNA samples, and there is no cross-reaction with other subtypes of carbapenemase genes blaNDM, blaIMP, blaOXA, 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. Detection Rate of the KPC Gene by Comparing the Method of the Present Invention with a Commercially Available Kit

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

[0145] The results show that: as shown in Table 3, the detection rate of KPC+ by the method of the present invention can reach 100%, which is more accurate than that of the commercially available kit (Xpert).

[0146] Table 3. Detection Rate of KPC+

[0147]

[0148] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. 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 appended claims of the present application.

Claims

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

6. The sequence of the probe is: 5’-TCGGAGACAAAACCGGAACCTGCGGAGTG / i6FAMdT / iTHF / / IBHQ1dT / GGCACGGCAAATGA-3’. Wherein, the reaction system further includes a DNA polymerase, a single-stranded binding protein, an endonuclease IV, a recombinase, and an auxiliary protein. The method further includes the step of detecting the fluorescence signal emitted by the specific probe during or after the amplification reaction. And, the amplification method is an isothermal enzymatic amplification method.

2. A non-diagnostic detection method for KPC gene, characterized in that, The method includes the steps of: (a) amplifying a test sample by the method described in claim 1; and (b) performing signal detection, wherein, the method further includes setting a KPC negative control and / or a KPC positive control.

3. The method according to claim 2, wherein The detection includes fluorescence signal detection, and the fluorescence signal detection is real-time detection using a handheld fluorescence detector.

4. The method according to claim 2, wherein The minimum detection concentration of the method is 0.005 - 0.015 ng / μL.

5. A detection kit for rapidly detecting the KPC gene for the method according to claim 2, characterized in that, The detection kit includes: a container, and a specific primer pair and a probe for the KPC gene in the container, wherein, the primer pair is the sequence shown in SEQ ID NO:1 and the sequence shown in SEQ ID NO:6; the probe is: 5’-TCGGAGACAAAACCGGAACCTGCGGAGTG / i6FAMdT / iTHF / / IBHQ1dT / GGCACGGCAAATGA-3’. The kit further includes a DNA polymerase, a single-stranded binding protein, an endonuclease IV, a recombinase, an auxiliary protein, a KPC negative control, and / or a KPC positive control.

6. Use of a detection kit as described in claim 5, characterized in that, For non-diagnostic and non-therapeutic detection of whether a test sample contains KPC-type carbapenemase-resistant bacteria.

7. A reagent combination for detecting KPC-type carbapenemase-resistant bacteria in a sample to be tested, characterized in that, The reagent combination includes: a specific primer pair and a probe combination for amplifying the KPC gene. The primer pair is the sequence shown in SEQ ID NO:1 and the sequence shown in SEQ ID NO:

6. The probe is: 5’-TCGGAGACAAAACCGGAACCTGCGGAGTG / i6FAMdT / iTHF / / IBHQ1dT / GGCACGGCAAATGA-3’.

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