A primer, probe set for detecting carbapenamase type by multiplex digital PCR and application thereof

Abstract

The application provides a primer, a probe set and an application thereof for multiple digital PCR detection of carbapenemase, and belongs to the technical field of biological detection.The primer and the probe set provided by the application are shown in SEQ ID NO:1-15, and the primer and the probe set can be used for quickly and accurately identifying carbapenemase, and the detection sensitivity reaches 25 copies / mL.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a primer and probe set for multiplex digital PCR detection of carbapenem enzyme types and their applications. Background Technology

[0002] Carbapenem antibiotics are important drugs widely used to treat multidrug-resistant Gram-negative bacterial infections. However, with increased frequency of use, bacteria have gradually evolved resistance mechanisms to these antibiotics, the most critical of which is the production of carbapenemases. Carbapenemases are a class of β-lactamases that hydrolyze carbapenem antibiotics, and they are divided into several molecular types, including KPC, NDM, VIM, IMP, and OXA-48. The genes for these enzymes are usually located on mobile genetic elements, such as plasmids or transposons, and therefore can spread rapidly between different strains, exacerbating the spread of resistance.

[0003] Detection of carbapenemase genes is of great significance for clinical diagnosis, infection control, and epidemiological surveillance. Currently, the main techniques for detecting CRE in anal swabs are: 1. CIM and eCIM assays: Bacteria are cultured from anal swabs, and the CIM or eCIM assays are performed to roughly determine the CRE enzyme type. However, this method can only determine whether the CRE belongs to a metalloenzyme or serine enzyme, not the specific subtype, and the culture time is long; 2. Carbapenem detection kits (colloidal gold method): This method uses bacteria cultured from anal swabs, and then uses these bacteria to detect the CRE using colloidal gold reagent strips. It can identify five CRE enzyme types, but the culture time is long and the sensitivity is low. Digital PCR technology for detecting carbapenemase types involves collecting patient blood or anal swabs for target bacterial detection, which can also detect CRE enzyme types. However, it has a limited subtype coverage and uses universal primers, resulting in low sensitivity.

[0004] Therefore, developing efficient, accurate, and comprehensive carbapenem enzyme detection kits is of profound significance for curbing the spread of drug-resistant bacteria, guiding rational drug use, and formulating public health policies. Summary of the Invention

[0005] The purpose of this invention is to provide a primer and probe set for multiplex digital PCR detection of carbapenem enzyme types, which can rapidly and accurately detect carbapenem enzyme types with high sensitivity and specificity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a primer and probe set for multiplex digital PCR detection of carbapenemase types: The nucleotide sequences of the primers used to detect the KPC gene are shown in SEQ ID NO: 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 NO: 4~5, and the nucleotide sequences of the probes are shown in SEQ ID NO: 6. The nucleotide sequences of the primers used to detect the OXA48 gene are shown in SEQ ID NO: 7-8, and the nucleotide sequences of the probes are shown in SEQ ID NO: 9. The nucleotide sequences of the primers used to detect the NDM gene are shown in SEQ ID NO: 10-11, and the nucleotide sequences of the probes are shown in SEQ ID NO: 12. The nucleotide sequences of the primers used to detect the VIM gene are shown in SEQ ID NO: 13-14, and the nucleotide sequences of the probes are shown in SEQ ID NO: 15.

[0007] The present invention also provides the application of the above-mentioned primer and probe set in the preparation of a kit for detecting carbapenem enzyme types.

[0008] The present invention also provides a kit containing the above-described primer and probe set.

[0009] Beneficial effects: Using the primer and probe set provided by this invention, five enzyme types of CRE—KPC, IMP, OXA-48, NDM, and VIM—can be detected, covering known subtypes and avoiding missed detections. Quantitative detection is performed using multiplex digital PCR, with a sensitivity as low as 25 copies / mL, and the entire process takes only 4 hours, significantly shortening the detection time and enabling rapid identification of enzyme types to assist doctors in treatment. Detailed Implementation

[0010] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0011] Example 1: Detection Process

[0012] After collecting samples using anal swabs, the samples were inserted into transport culture medium (composition: disposable sterile test tubes, transport culture medium powder, sterile purified water, and sterile cotton swabs. The transport culture medium powder composition (g / L) is: potassium dihydrogen phosphate 0.2 g, disodium hydrogen phosphate 1.15 g, potassium chloride 0.2 g, sodium chloride 3.0 g, magnesium chloride 0.1 g, sodium thioglycolate 1.0 g, calcium chloride 0.1 g, and agar 7.5 g). The swabs were then removed from the laboratory for digital PCR testing.

[0013] Detection method: Take a clean centrifuge tube, add 2 ml of pure water, place the anal swab containing the transport culture medium into the pure water and mix well. Then add the pure water mixed with the anal swab into the well of the Easy-CF2 reagent strip for nucleic acid extraction (machine extraction). System preparation: 5 μL nucleic acid extract + 4 μL primer probe + 3 μL reaction solution + 3 μL PCR water. After preparation, add it to the sample cup of the digital PCR microdroplet chip. After testing for 4 hours, it can be determined whether CRE is present in the patient's anal swab and the corresponding enzyme type. The primer probe sequences are shown in Table 1 below.

[0014] Table 1 Primer and probe sequences

[0015] Example 2 Simulation Experiment

[0016] Five bacterial strains carrying IMP, KPC, NDM, VIM, and OXA-48, respectively, were passaged twice to prepare a solution with a concentration of 1.5 × 10⁻⁶. 8 and 1.5×10 6 The bacterial solution was collected using an anal swab and then inserted into the transport medium.

[0017] Nucleic acid extraction: 1. Remove the anal swab from the transport culture medium and place it in 2ml of pure water and stir well.

[0018] 2. Extract nucleic acids from the eluent using a fully automated nucleic acid extractor.

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

[0020] 4. Add 5 μL of nucleic acid extract of the sample to be tested and the negative control (ultrapure water) or the corresponding positive control (containing all target gene fragments in CRE) to each tube, vortex to mix, avoid the generation of air bubbles, and centrifuge briefly.

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

[0022] 6. Use the DG32 droplet generator to generate droplets. Follow the instructions in the DG32 droplet generator manual for operation.

[0023] 7. PCR amplification

[0024] Place the chip after droplet generation into the PCR amplification instrument TC1 and perform the reaction 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 PCR, the chip was placed in a biochip reader, and the FAM, VIC, ROX, CY5, CY5.5, and A425 fluorescence channels were selected. Channel A425 was used as the positioning channel to set the chip well positions, and chip scanning and analysis were performed. The CRE enzyme types corresponding to the fluorescence channels are shown in Table 2 below.

[0028] Table 2. CRE enzyme types corresponding to fluorescent channels

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

[0030] Table 3 Sample Detection Results

[0031] As shown in Table 3, this experiment simulated the direct detection of five carbapenem enzyme types in anal swabs using digital PCR, proving that this kit can directly detect anal swabs and that all five CRE enzyme types can be directly detected.

[0032] Example 3

[0033] There are five enzyme types of CRE: KPC, IMP, OXA-48, NDM, and VIM, covering a wider range of subtypes (as shown in Table 4). The detection method in this application can detect all of these subtypes. Validation was performed using 34 strains of different subtypes identified from our institute's CRE strain library, and all were detected.

[0034] Table 4 CRE subtypes

[0035] The testing process is as follows: 1. After subculturing the 34 bacterial strains twice, bacterial culture was prepared to a concentration of 1.5 × 10⁻⁶. 8 bacterial solution 2. Take 2ml of bacterial culture and extract nucleic acid using a fully automated nucleic acid extractor.

[0036] The system testing section is the same as in Example 2.

[0037] The test results are shown in Table 5 below.

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

[0039] As shown in Table 5, the detection method of this application can accurately identify different subtypes of CRE.

[0040] Example 4 Sensitivity Verification

[0041] The testing was performed according to the testing procedure in Example 1.

[0042] The KPC and NDM reference stock solutions were cfDNA containing the KPC and NDM genes, respectively. The procedure involved extracting bacterial genomic DNA containing the KPC and NDM genes, fragmenting it using sonication, determining its composition by digital PCR, and then diluting it with DNA preservation solution to prepare KPC and NDM reference stock solutions (both with a final concentration of 500 copies / μL). Using negative rectal swab matrix, the KPC and NDM detection limit reference stock solutions were diluted to five working solutions at five concentration gradients of the target nucleic acid: final concentrations of 400 copies / mL, 200 copies / mL, 100 copies / mL, 50 copies / mL, and 25 copies / mL. Each concentration sample was tested 20 times (after nucleic acid extraction), and the positive detection rate for each sample was calculated. The concentration with a positive detection rate of 95% or higher was used as the definitive detection limit concentration.

[0043] The dilution process for the KPC detection limit reference stock solution and the NDM detection limit reference stock solution to their respective working solution concentrations at a final concentration of 500 copies / μL is shown in Table 6 below: Table 6. Dilution process of KPC / NDM mother liquor

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

[0045] Table 8. Raw data for NDM resistance gene detection rate test at different working solution concentrations.

[0046] Table 9. Statistical Table of the Establishment Results of the Limit of Detection

[0047] As shown in Table 9 above, KPC and NDM can be detected with 95% accuracy at working solution concentrations of 25 copies / mL, 50 copies / mL, 100 copies / mL, 200 copies / mL, and 400 copies / mL. The minimum detection limit for KPC and NDM is 25 copies / mL.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer and probe set for multiplex digital PCR detection of carbapenemase types, characterized in that, The primer and probe set includes: The nucleotide sequences of the primers used to detect the KPC gene are shown in SEQ ID NO: 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 NO: 4~5, and the nucleotide sequences of the probes are shown in SEQ ID NO:

6. The nucleotide sequences of the primers used to detect the OXA48 gene are shown in SEQ ID NO: 7-8, and the nucleotide sequences of the probes are shown in SEQ ID NO:

9. The nucleotide sequences of the primers used to detect the NDM gene are shown in SEQ ID NO: 10-11, and the nucleotide sequences of the probes are shown in SEQ ID NO:

12. The nucleotide sequences of the primers used to detect the VIM gene are shown in SEQ ID NO: 13-14, and the nucleotide sequences of the probes are shown in SEQ ID NO:

15.

2. A kit for multiplex digital PCR detection of carbapenemase types, characterized in that, The kit contains the primer and probe set as described in claim 1.

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