Detection method of carbapenem drug-resistant genes

Abstract

The invention relates to a detection method of carbapenem drug-resistant genes, and belongs to the technical field of biological detection. Comprising a specific RPA amplification primer pair, an RPA amplification reaction system and a test strip, the specific RPA amplification primer pair comprises a primer pair aiming at an OXA-48 gene and a primer pair aiming at an IPM gene, the RPA amplification reaction system comprises recombinase, single-chain binding protein, DNA polymerase, dNTPs and a buffer solution, and the test strip comprises a detection line, a quality control line, a sample pad and a water absorption pad. A probe specifically hybridized with OXA-48 and IPM gene amplification products is fixed on the detection line, a probe specifically combined with a reference gene amplification product is fixed on the quality control line, the sample pad and the water absorption pad are used for sample flow and reaction control, and rapid and high-sensitivity detection of OXA-48 and IPM genes is realized by optimizing the design of primers, a reaction system and a test strip. The method is suitable for clinical and field application.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and particularly relates to a method for detecting carbapenem-resistant genes, which is used for detecting carbapenem-resistant genes OXA-48 and IPM. Background Art

[0002] Carbapenem antibiotics are key drugs for treating severe bacterial infections, but the spread of resistant genes (such as OXA-48 and IPM) has significantly increased the difficulty of clinical treatment. Existing detection methods (such as PCR) rely on complex instruments and professional personnel, and it is difficult to meet the requirements of rapid and on-site detection. Recombinase polymerase amplification (RPA), as an isothermal nucleic acid amplification technology, has advantages such as rapidity and no need for thermal cycling, but its application in the detection of resistant genes still needs to be optimized. Summary of the Invention

[0003] Provide a method for detecting carbapenem-resistant genes, which is used for rapidly, highly sensitively and highly specifically detecting OXA-48 and IPM genes, and is applicable to primary medical institutions and on-site detection.

[0004] The object of the present invention is achieved through the following technical solutions, including specific RPA amplification primer pairs, an RPA amplification reaction system and a test strip: The specific RPA amplification primer pairs include primer pairs for the OXA-48 gene and primer pairs for the IPM gene; For the primer pair for the OXA-48 gene, its sequences are as follows: Forward primer: 5'-ACACCAAGTCTTIAAGTGGGATG-3', Reverse primer: 5'-CCCGAAATGTCCTCATIACC-3'; For the primer pair for the IPM gene, its sequences are as follows: Forward primer: 5'-GGAATAGAGTGGCTTAAYTCTC-3', Reverse primer: 5'-GGTTTAAYAAAACAACCACC-3'; The RPA amplification reaction system includes recombinase, single-stranded binding protein, DNA polymerase, dNTPs and buffer; The test strip includes a detection line, a quality control line, a sample pad and a water absorption pad; The detection line is fixed with a probe that specifically hybridizes with the amplification products of the OXA-48 and IPM genes; The quality control line is fixed with a probe that specifically binds to the amplification products of the internal reference gene; The sample pad and the water absorption pad are used for sample flow and reaction control.

[0005] Furthermore, the reaction temperature of the RPA amplification reaction system is 37-42°C, and the reaction time is 15-20 minutes.

[0006] Furthermore, the internal reference gene is the 16S rRNA gene.

[0007] Furthermore, the following steps are also included: S1: Extract the genomic DNA of the sample to be tested; S2: Add the genomic DNA into the RPA amplification reaction system, and perform an isothermal amplification reaction at 37-42°C for 15-20 minutes to obtain an amplification product; S3: Drop the amplification product onto the sample pad of the test strip and let it flow through capillary action; S4: Observe the test line and the control line. If a band appears on the test line, the sample contains the OXA-48 or IPM gene. If only a band appears on the control line, the target gene is not detected in the sample.

[0008] Furthermore, the genomic DNA is extracted by the boiling method, specifically by suspending the colony in sterile deionized water, performing a water bath at 100°C for 10 minutes, and centrifuging to take the supernatant as the template.

[0009] Furthermore, the test line probe and the control line probe of the test strip are labeled with fluorescein and biotin respectively.

[0010] Furthermore, the test line and the control line of the test strip are fixed on the nitrocellulose membrane by the gold spraying technique.

[0011] Furthermore, in the RPA amplification system, the primer concentration is 0.4-0.5 μM, and the dNTPs concentration is 0.2 mM.

[0012] Furthermore, the detection sensitivity of the method is 2.0×10 for the OXA-48 gene 3 CFU / ml, and 2.0×10 for the IPM gene 2 CFU / ml.

[0013] In summary, compared with the prior art, the present invention has the following advantages: In the present invention, rapid detection is completed within 30 minutes, the detection limit of OXA-48 is 2.0×10 3 CFU / ml, the detection limit of IPM is 2.0×10 2 CFU / ml, the detection sensitivity is high, no complex equipment is required, the test strip interpretation is intuitive, the operation is simple, the reagent cost is low, and it is suitable for areas with limited resources. Detailed implementation mode

[0014] The technical solution of the present invention will be further specifically described below through specific embodiments, but the present invention is not limited to these embodiments.

[0015] Example 1 Primer screening and verification: (1) Primer design: Using bioinformatics software, a large number of OXA-48 and IPM gene sequences in the GenBank database were compared and analyzed, and multiple groups of primers were designed for highly conserved regions. Among them, the downstream primers were labeled with biotin, and the probes were labeled with fluorescein.

[0016] (2) Primer synthesis: The designed primer sequences were handed over to a professional biological company for synthesis.

[0017] (3) Primer specificity verification: Using the genomic DNA of the standard strain containing OXA-48 and IPM genes as a template, and the genomic DNA of other strains without these two genes as a negative control, the synthesized primers were used for RPA amplification reactions respectively. The amplification products were detected by agarose gel electrophoresis. The results showed that the primers designed for the OXA-48 gene amplified specific bands only in the template containing the OXA-48 gene, and the primers designed for the IPM gene amplified specific bands only in the template containing the IPM gene. There were no amplification bands in the negative controls, proving that the primers had good specificity.

[0018] (4) Primer sensitivity verification: The genomic DNA of the standard strain containing OXA-48 and IPM genes was diluted in a 10-fold gradient, from 2.0×10 7 CFU / ml diluted to 2.0×10 CFU / ml, and the screened primers were used for RPA amplification reactions.

[0019] The results showed that: the sensitivity of OXA-48 was 2.0×10 3 CFU / ml, while the sensitivity of IMP was 2.0×10 2 CFU / ml, and the sensitivity met the detection requirements.

[0020] Example 2 Optimization of the RPA amplification system (1)Optimization of reaction components: Single-factor optimization experiments were carried out on primer concentration, enzyme concentration, dNTPs concentration, buffer components, etc. in the RPA amplification system. RPA amplification reactions were carried out with different concentrations of primers, enzymes, dNTPs, etc. respectively, and the brightness, clarity and other indicators of the amplified bands were detected by agarose gel electrophoresis to determine the optimal reaction concentration. For example, after multiple experiments, the PCR amplification system was 25 uL. It was determined that the optimal primer concentration for OXA-48 gene amplification was 0.4 μM (0.5 uL for each of the upstream and downstream primers), and the optimal primer concentration for IPM gene amplification was 0.5 μM (0.5 uL for each of the upstream and downstream primers); the optimal addition amount of RPA reaction enzyme was 5 μL; the optimal concentration of dNTPs was 0.2 mM (12.5 uL), and the template DNA was 2 uL; ddH2O was added to make up to 25 μL. The PCR products were analyzed by 1.2% agarose gel electrophoresis, the gel was cut and recovered, and then sequenced.

[0021] (2)Optimization of reaction temperature and time: Different isothermal reaction temperatures (37 °C, 39 °C, 41 °C, 42 °C) and reaction times (10 minutes, 15 minutes, 20 minutes, 25 minutes) were set, and RPA amplification reactions were carried out on the genomic DNA of standard strains containing OXA-48 and IPM genes. The results showed that when the reaction was carried out at 39 °C for 15 - 20 minutes, the amplification effect was the best, and the amplified bands were clear and bright.

[0022] Example 3 Preparation of test strip (1)Probe synthesis and labeling: Specific oligonucleotide probes were designed for the RPA amplification products of OXA-48 and IPM genes. The probes were labeled with fluorescein for subsequent fixation on the test line of the test strip.

[0023] (2)Assembly of test strip: The treated sample pad, conjugate pad (used to adsorb the conjugate of the fluorescein-labeled probe and the amplification product), nitrocellulose membrane (with a test line and a control line drawn on it), and absorbent pad were sequentially pasted on the PVC bottom plate to assemble a complete test strip.

[0024] On the test line of the nitrocellulose membrane, specific probes for the amplification products of OXA-48 and IPM genes were fixed by techniques such as gold spraying; on the control line, probes specifically binding to the internal reference amplification products were fixed (the internal reference gene can be a gene that is commonly present and stably expressed in the sample, such as the 16S rRNA gene, which is used to monitor whether the detection process is normal).

[0025] Example 4 Detection of clinical samples (1)Sample collection: 30 specimens such as sputum, urine, and blood were collected from clinical patients suspected of being infected with carbapenem-resistant bacteria.

[0026] (2) Sample processing: The genomic DNA of the collected specimens was extracted using a conventional nucleic acid extraction kit.

[0027] (3) RPA amplification and strip detection: The extracted genomic DNA was added to the optimized RPA amplification system and amplified at 39 °C for 15 minutes. After the amplification was completed, 10 μL of the amplification product was dropped onto the prepared sample pad of the test strip, and after waiting for 5 - 10 minutes, the test line and the control line of the test strip were observed.

[0028] Result interpretation and verification: The results showed that among the 30 samples, after strip detection, 12 samples had bands on the test line, indicating that they might contain OXA - 48 and IPM resistance genes; 18 samples had bands only on the control line and no bands on the test line, indicating that the corresponding resistance genes were not detected. The samples with positive test results were sent to a third - party testing agency and verified using real - time fluorescence quantitative PCR technology. The verification results showed that the coincidence rate of the detection results of the two methods reached 96%, proving that the RPA + strip detection method of the present invention has high accuracy and reliability.

[0029] The embodiments of the present invention are not limited to those described in the above examples. Without departing from the spirit and scope of the present invention, those of ordinary skill in the art can make various changes and improvements in form and details, and all of these are considered to fall within the protection scope of the present invention.

Claims

1. A method for detecting carbapenem-resistant genes, characterized in that, It includes a specific RPA amplification primer pair, an RPA amplification reaction system and a test strip: The specific RPA amplification primer pair includes a primer pair targeting the OXA-48 gene and a primer pair targeting the IPM gene; For the primer pair targeting the OXA-48 gene, its sequences are as follows: Forward primer: 5'-ACACCAAGTCTTIAAGTGGGATG-3', Reverse primer: 5'-CCCGAAATGTCCTCATIACC-3'; For the primer pair targeting the IPM gene, its sequences are as follows: Forward primer: 5'-GGAATAGAGTGGCTTAAYTCTC-3', Reverse primer: 5'-GGTTTAAYAAAACAACCACC-3'; The RPA amplification reaction system includes recombinase, single-stranded binding protein, DNA polymerase, dNTPs and buffer; The test strip includes a test line, a quality control line, a sample pad and a water absorption pad; The test line is immobilized with a probe that specifically hybridizes with the amplification products of the OXA-48 and IPM genes; The quality control line is immobilized with a probe that specifically binds to the amplification product of the internal reference gene; The sample pad and the water absorption pad are used for sample flow and reaction control.

2. The detection method of a carbapenem resistance gene according to claim 1, characterized in that: The reaction temperature of the RPA amplification reaction system is 37 - 42 °C, and the reaction time is 15 - 20 minutes.

3. The detection method of a carbapenem resistance gene according to claim 1, characterized in that: The internal reference gene is the 16S rRNA gene.

4. The detection method of a carbapenem-resistant gene according to claim 1, characterized in that, It also includes the following steps: S1: Extract the genomic DNA of the sample to be tested; S2: Add the genomic DNA into the RPA amplification reaction system, and carry out an isothermal amplification reaction at 37 - 42 °C for 15 - 20 minutes to obtain amplification products; S3: Drop the amplification products onto the sample pad of the test strip and let them flow through capillary action; S4: Observe the test line and the quality control line. If a band appears on the test line, the sample contains the OXA-48 or IPM gene. If only a band appears on the quality control line, the target gene is not detected in the sample.

5. The detection method of a carbapenem-resistant gene according to claim 4, characterized in that: The genomic DNA is extracted by the boiling method, specifically by suspending the colony in sterile deionized water, heating in a water bath at 100 °C for 10 minutes, and centrifuging to take the supernatant as the template.

6. The detection method of a carbapenem resistance gene according to claim 4, characterized in that: The test line probe and the quality control line probe of the test strip are labeled with fluorescein and biotin respectively.

7. The detection method of a carbapenem-resistant gene according to claim 1, wherein: The test line and the quality control line of the test strip are immobilized on the nitrocellulose membrane by the gold spraying technique.

8. The detection method of a carbapenem resistance gene according to claim 4, wherein: In the RPA amplification system, the primer concentration is 0.4 - 0.5 μM, and the dNTPs concentration is 0.2 mM.

9. The detection method of a carbapenem-resistant gene according to claim 4, characterized in that: The detection sensitivity of the method is 2.0×10 3 CFU / ml for the OXA-48 gene and 2.0×10 2 CFU / ml for the IPM gene.