Primer and probe set for detecting carbapenemase type through multiple digital PCR (polymerase chain reaction) and application of primer and probe set

Abstract

The invention provides a primer and a probe set for detecting carbapenemase by multiple digital PCR (polymerase chain reaction) and application of the primer and the probe set, and belongs to the technical field of biological detection. The primer and the probe group provided by the invention are as shown in SEQ ID NO: 1-15, the carbapenemase type can be quickly and accurately identified by utilizing the primer and the probe group provided by the invention, and the detection sensitivity reaches 25 copies / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a primer and probe group for multiple digital PCR detection of carbapenemase type and application thereof. BACKGROUND

[0002] Carbapenems are a class of antibiotics widely used in the treatment of multiple drug-resistant gram-negative bacterial infections. However, with the increasing frequency of use, bacteria have gradually evolved mechanisms of resistance to this class of antibiotics, the most critical of which is the production of carbapenemases. Carbapenemases are a class of beta-lactamases that can hydrolyze carbapenems, including KPC, NDM, VIM, IMP and OXA-48. The genes of these enzymes are usually located on mobile genetic elements such as plasmids or transposons, so they can quickly spread among different strains, exacerbating the spread of drug resistance.

[0003] Detecting carbapenemase genes is of great significance for clinical diagnosis, infection control and epidemiological surveillance. Currently, the techniques for detecting CRE from anal swabs are mainly: 1. CIM and eCIM experiments, which use anal swabs to culture bacteria, and then use the bacteria to perform CIM experiments or eCIM experiments to roughly determine the CRE enzyme type. However, this method can only determine whether the CRE is a metalloenzyme or a serine enzyme, and cannot accurately determine the subtype, and the culture time is relatively long; 2. Carbapenem detection kit (colloidal gold method): this method uses anal swabs to culture bacteria, and then uses the bacteria to detect the colloidal gold reagent strip to determine the five enzyme types of CRE. However, the culture time is long, and the sensitivity is low. The detection technology based on digital PCR technology for detecting carbapenemase type collects patient blood or anal swabs for bacterial detection in the target, and can also detect the CRE enzyme type, but the coverage of the subtype range is small, and the use of universal primers has low sensitivity.

[0004] Therefore, the development of a carbapenemase type detection kit that is efficient, accurate and covers a wide range of subtypes is of great significance in curbing the spread of drug-resistant bacteria, guiding rational drug use, and formulating public health policies. SUMMARY

[0005] The purpose of the present application is to provide a primer and probe group for multiple digital PCR detection of carbapenemase type, which can quickly and accurately detect carbapenemase type with high sensitivity and specificity.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a primer and probe group for multiple digital PCR detection of carbapenemase type: The nucleotide sequence of the primer for detecting the KPC gene is shown as SEQ ID NO: 1-2, and the nucleotide sequence of the probe is shown as SEQ ID NO: 3; The nucleotide sequence of the primer for detecting the IMP gene is shown as SEQ ID NO: 4-5, and the nucleotide sequence of the probe is shown as SEQ ID NO: 6; The nucleotide sequence of the primer for detecting the OXA48 gene is shown as SEQ ID NO: 7-8, and the nucleotide sequence of the probe is shown as SEQ ID NO: 9; The nucleotide sequence of the primer for detecting the NDM gene is shown as SEQ ID NO: 10-11, and the nucleotide sequence of the probe is shown as SEQ ID NO: 12; The nucleotide sequence of the primer for detecting the VIM gene is shown as SEQ ID NO: 13-14, and the nucleotide sequence of the probe is shown as SEQ ID NO: 15.

[0007] The application also provides a use of the above-mentioned primer and probe set in the preparation of a carbapenamase detection kit.

[0008] The application also provides a kit containing the above-mentioned primer and probe set.

[0009] Beneficial effects: The primer and probe set provided by the application can detect five enzyme types of KPC, IMP, OXA-48, NDM and VIM of CRE, and covers known subtypes, avoiding missed detection. The quantitative detection is performed by a multiplex digital PCR method, the sensitivity is as low as 25 copies / mL, the whole process only takes 4 hours, the detection time is significantly shortened, the enzyme type can be quickly and clearly determined, and the doctor is assisted in treatment. DETAILED DESCRIPTION

[0010] The technical solutions provided by the application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the application.

[0011] Example 1: Detection process

[0012] After sampling with an anal swab, insert the transport medium (composition: composed of a disposable sterile test tube, transport medium dry powder, sterile purified water, and a sterile cotton swab. The composition of the transport medium dry powder (g / L) is: potassium dihydrogen phosphate 0.2 g, sodium phosphate dibasic 1.15 g, potassium chloride 0.2 g, sodium chloride 3.0 g, magnesium chloride 0.1 g, sodium thio glycolate 1.0 g, calcium chloride 0.1 g, and agar 7.5 g). After being sent to the laboratory, the swab is taken out for digital PCR detection.

[0013] Detection method: take clean centrifuge tube, add 2 ml pure water, put the anal swab inserted into the transport medium into the pure water and mix well, then add the anal swab-stirred pure water into the Easy-CF2 reagent strip hole for nucleic acid extraction (machine extraction). System configuration: 5 μL nucleic acid extract + 4 μL primer probe + 3 μL reaction solution + 3 μL PCR water. After configuration, add to the sample cup of digital PCR microdroplet chip, and after 4 hours of testing, it can be known whether CRE exists in the anal swab of the patient, and the corresponding enzyme type is known. The primer probe sequence is shown in Table 1.

[0014] Table 1 Primer probe sequence

[0015] Example 2 Simulation experiment

[0016] Five strains carrying IMP, KPC, NDM, VIM and OXA-48 respectively were subcultured for two generations, and were prepared into bacterial liquid with a concentration of 1.5×10 8 and 1.5×10 6 , and then the anal swab was used to pick up the bacterial liquid and then inserted into the transport medium.

[0017] Nucleic acid extraction: 1. Take the anal swab out of the transport medium and put it into 2 ml pure water and mix well.

[0018] 2. The eluent is subjected to nucleic acid extraction on the automatic nucleic acid extractor.

[0019] System detection: 1. Preparation of premix solution Take 4 μL primer probe mixture (the volume ratio of primer and probe is 4:1) and 3 μL reaction solution, add 3 μL PCR water and mix well to obtain the premix solution required for testing 1 sample; 2. Droplet generation 3. After determining the required number of tests N (sample number + 2), take out the corresponding amount of premix solution.

[0020] 4. Add 5 μL of nucleic acid extraction product of the sample to be tested and negative quality control (ultra-pure water) or corresponding positive control (containing all target gene fragments in CRE) to each tube, respectively, vortex well, avoid air bubbles, and centrifuge.

[0021] 5. Take 14 μL of the reaction solution after adding sample, and add it to each channel of the digital PCR microdroplet chip.

[0022] 6. Use droplet generator DG32 to generate droplets, and the operation steps are according to the instructions of droplet generator DG32.

[0023] 7. PCR amplification

[0024] After the droplet generation, the chip was placed into a PCR amplifier TC1, and the reaction was performed according to the following PCR parameters.

[0025] 95℃ 5 min; [95℃ 15 s, 60℃ 30 s] 40 cycles; 25℃ 1 min.

[0026] 8. Chip scanning

[0027] After the PCR, the chip was placed into a biochip reader, FAM, VIC, ROX, CY5, CY5.5, and A425 fluorescence channels were selected, A425 channel was set as a positioning channel, and the chip hole was set. Chip scanning and analysis were performed. The CRE enzyme types corresponding to the fluorescence channels are shown in Table 2.

[0028] Table 2: CRE enzyme types corresponding to the fluorescence channels

[0029] 9. The detection results are shown in Table 3

[0030] Table 3: Sample detection results

[0031] As shown in Table 3, this test simulates the method of directly detecting five carbapenemase types of anal swabs using digital PCR, which proves that the kit can directly detect anal swabs, and the five enzyme types of CRE can be directly detected.

[0032] Example 3

[0033] There are five types of CRE enzymes, namely KPC, IMP, OXA-48, NDM, and VIM, and more subtypes are covered (as shown in Table 4). The detection method of the present application can detect the above-mentioned subtypes. And 34 strains of different subtypes were found out from the CRE strain library of our hospital, and the results were all detected.

[0034] Table 4: CRE subtypes

[0035] The detection process is as follows: 1. After two generations of subculture of the 34 strains of bacteria, bacterial liquid was prepared, and the concentration of the bacterial liquid was 1.5 x 10 8 2. Take 2 ml of bacterial liquid and extract nucleic acid on a full-automatic nucleic acid extractor.

[0036] ​The system detection part is the same as that in Example 2.

[0037] The detection results are shown in Table 5 below

[0038] Table 5: 34 strains of different subtypes and detection results

[0039] As can be seen from Table 5, the detection method of the present application can accurately identify different subtypes of CRE.

[0040] Example 4: Sensitivity verification

[0041] The detection was performed according to the detection process of Example 1.

[0042] The KPC and NDM reference stock solutions were cfDNA containing KPC and NDM genes, respectively. The process was as follows: bacterial genomic DNA containing KPC and NDM genes was extracted, broken by ultrasonication, and then diluted into KPC reference stock solution and NDM reference stock solution (final concentration of KPC and NDM genes was 500 copies / μL) using DNA preservation solution. The detection limit reference stock solution of KPC and NDM was diluted to the target nucleic acid concentration gradient of 5 working solutions using negative anal swab matrix, i.e., working solutions with final concentrations of 400 copies / mL, 200 copies / mL, 100 copies / mL, 50 copies / mL, and 25 copies / mL. Each concentration sample was repeatedly detected 20 times (after nucleic acid extraction), and the positive detection rate was calculated. The concentration with a positive detection rate of more than 95% was determined as the detection limit.

[0043] The dilution process of the KPC detection limit reference stock solution and the NDM detection limit reference stock solution with a final concentration of 500 copies / μL to the working solution concentration is shown in Table 6 below: Table 6: KPC / NDM stock solution dilution process

[0044] The KPC / NDM working solution with a final concentration of 400 copies / mL, 200 copies / mL, 100 copies / mL, 50 copies / mL, and 25 copies / mL was repeatedly extracted 20 times for each concentration, and the positive detection rate of each target gene was calculated separately. The concentration with a positive detection rate of more than 95% was determined as the detection limit. The detection results are shown in Tables 7-9 below: Table 7: KPC resistance gene detection rate test raw data for different working solution concentrations

[0045] Table 8 Test original data of detection rate of NDM drug-resistant gene at different working solution concentrations

[0046] Table 9 Statistical table of establishment results of minimum detection limit

[0047] From the above table 9, it can be seen that when the working solution concentration is 25 copies / mL, 50 copies / mL, 100 copies / mL, 200 copies / mL and 400 copies / mL, KPC and NDM can be detected at 95%. The minimum detection limit of KPC and NDM is 25 copies / mL.

[0048] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A primer and probe set for multiplex digital PCR detection of carbapenemase types, characterized by: The nucleotide sequences of the primers used to detect the KPC gene are shown in SEQ ID NOs: 1-2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 3; The nucleotide sequences of the primers used to detect the IMP gene are shown in SEQ ID NOs: 4-5, and the nucleotide sequence of the probe is shown in SEQ ID NO: 6; The nucleotide sequences of the primers used to detect the OXA48 gene are shown in SEQ ID NOs: 7-8, and the nucleotide sequence of the probe is shown in SEQ ID NO: 9; The nucleotide sequences of the primers used to detect the NDM gene are shown in SEQ ID NOs: 10-11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12; The nucleotide sequences of the primers used to detect the VIM gene are shown in SEQ ID NOs: 13-14, and the nucleotide sequence of the probe is shown in SEQ ID NO:

15.

2. Use of the primer and probe set according to claim 1 in preparing a kit for detecting carbapenemase types.

3. A kit comprising the primer and probe set according to claim 1.

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