Multiplex PCR (Polymerase Chain Reaction) primer group for rapidly detecting oxazolidinone drug-resistant gene and detection method

Abstract

The invention discloses a multiplex PCR primer group for rapidly detecting oxazolidinone drug resistance genes and a detection method, and belongs to the technical field of molecular biology. The invention designs a multiplex PCR primer group for rapidly detecting the oxazolidinone drug-resistant gene, and the primer sequence is shown as SEQ ID NO.1-8. The multiplex PCR method for detecting the oxazolidinone drug-resistant gene comprises the following steps: firstly, extracting total DNA (deoxyribonucleic acid) of a strain, amplifying the oxazolidinone drug-resistant genes cfr, cfr (D), optrA and poxtA by adopting the multiplex PCR primer group through multiplex PCR reaction, and detecting the oxazolidinone drug-resistant gene by adopting the multiplex PCR primer group to obtain the oxazolidinone drug-resistant gene cfr, cfr (D), optrA and poxtA. And carrying out agarose gel electrophoresis separation on the obtained PCR product, and comparing the obtained PCR product with agarose gel electrophoretograms of cfr, cfr (D), optrA and poxtA genes so as to identify the drug-resistant gene type of the to-be-detected bacteria. Four common oxazolidinone drug-resistant genes (cfr, cfr (D), optrA and poxtA) can be conveniently and rapidly detected from a bacterial sample, compared with single gene PCR, the accuracy, specificity and sensitivity can reach 100%, and the detection efficiency and accuracy are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a multiplex PCR primer set and a detection method for simultaneously and rapidly detecting oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA in bacteria. Background Art

[0002] In recent decades, antimicrobial drugs have been used in livestock and poultry farming to promote growth, prevent and treat diseases. However, the irrational or excessive use of antimicrobial drugs has led to the emergence and widespread spread of resistant bacteria and resistant genes, exacerbating the problem of bacterial resistance and posing a huge threat to livestock and poultry farming and human public health. It is estimated that 700,000 deaths worldwide are related to pathogen resistance each year. By 2050, the number of deaths due to antibiotic resistance will exceed 10 million each year, more than the number of deaths caused by cancer. The emergence of super-resistant bacteria has brought great risks to public health and caused huge economic losses. Understanding the prevalence and transmission mechanism of bacterial resistance genes is of great significance for formulating bacterial resistance prevention and control policies and guiding clinical drug use.

[0003] The oxazolidinone drugs linezolid and tedizolid were approved for clinical use in humans in 2000 and 2014, respectively. They are the only antibacterial drugs with completely new structures in the past 50 years. Linezolid has strong antibacterial activity against Gram-positive bacteria, while tedizolid has strong antibacterial activity against methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococci, and linezolid-resistant bacteria. Therefore, this class of drugs is considered to be the "last line of defense" for antibacterial drugs after vancomycin. Due to the continuous use of oxazolidinone drugs in clinical practice, bacterial resistance has also emerged. Researchers have successively discovered oxazolidinone resistance genes in human and animal bacteria. In 2000, Professor Stefan Schwarz of Germany discovered the cfr gene from Staphylococcus aureus, which can mediate bacterial resistance to amine alcohols, oxazolidinones, lincosamides, pleuromutilins and streptogramin A drugs. Subsequently, multiple cfr gene variants, cfr(B), cfr(C), cfr(D) and cfr(E), were reported. The optrA gene and poxtA gene were discovered in 2015 and 2018, respectively, helping bacteria to achieve resistance to amide alcohols, tetracyclines and oxazolidinone antibiotics. Currently, the oxazolidinone resistance genes with higher detection rates in human and animal bacteria include cfr, cfr(D), optrA and poxtA.

[0004] Traditional methods for detecting drug-resistant genes are mostly based on single-gene polymerase chain reaction (PCR) or whole genome sequencing technology. The advantages of the PCR method are simplicity, ease of operation and low cost, but the disadvantage is that only a single gene can be detected in one reaction, which is inefficient. Whole genome sequencing technology can provide a comprehensive spectrum of bacterial resistance genes, but it has high requirements for instruments, a long detection cycle and is expensive. There are many types of drug-resistant genes, and sometimes a strain of bacteria can carry multiple drug-resistant genes. Therefore, it is necessary to develop a method that can quickly detect multiple drug-resistant genes. So far, there has been no report on a multiplex PCR method that can simultaneously detect multiple oxazolidinone resistance genes. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a multiplex PCR primer set and a detection method for rapid detection of oxazolidinone resistance genes. The present invention designs 4 pairs of specific primers, and the above primers are added to the multiplex PCR reaction system at the same time for amplification, so as to achieve the purpose of rapid detection of 4 oxazolidinone resistance genes at the same time.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention provides a multiplex PCR primer set for rapid detection of oxazolidinone resistance genes. The multiplex PCR primer set consists of four pairs of primers, which are respectively used for detecting oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA. The primer sequences for detecting the oxazolidinone resistance gene cfr are shown in SEQ ID NO.1 and SEQ ID NO.2, the primer sequences for detecting the oxazolidinone resistance gene cfr(D) are shown in SEQ ID NO.3 and SEQ ID NO.4, the primer sequences for detecting the oxazolidinone resistance gene optrA are shown in SEQ ID NO.5 and SEQ ID NO.6, and the primer sequences for detecting the oxazolidinone resistance gene poxtA are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0008] The present invention also provides a multiplex PCR method for detecting oxazolidinone resistance genes, wherein the multiplex PCR method is used to detect oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA, and comprises the following steps:

[0009] (1) Extraction of bacterial DNA template: Use a bacterial DNA extraction kit to extract the total DNA of the strain;

[0010] (2) using the above-mentioned multiplex PCR primer set, amplifying the oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA by multiplex PCR reaction;

[0011] (3) Identification of drug resistance gene detection results: The PCR product obtained in step (2) is separated by agarose gel electrophoresis and compared with the agarose gel electrophoresis patterns of cfr, cfr(D), optrA and poxtA genes to identify the drug resistance gene type of the bacteria to be detected.

[0012] Based on the above technical solution, further, the total system of the multiplex PCR reaction described in step (2) is 20 μL, and the reaction system is as follows:

[0013]

[0014] Based on the above technical solution, further, in step (2), the concentration of the primers shown in SEQ ID NO.1-8 is 5 to 15 μmol / L.

[0015] Based on the above technical solution, further, the cycle parameters of the multiple PCR reaction described in step (2) are as follows: denaturation: 97-99°C, 9-11s; annealing: 51-53°C, 4-6s; extension: 71-73°C, 4-6s, for 22-30 consecutive cycles.

[0016] Based on the above technical solution, further, in step (3), the PCR product obtained in step (2) is separated by 2% agarose gel electrophoresis, and the electrophoresis condition is 150V voltage electrophoresis for 30 minutes, and the DNA molecular weight is compared using DL1000 Marker. The appearance of a 725bp band indicates the presence of cfr, the appearance of a 287bp band indicates the presence of cfr(D), the appearance of a 389bp band indicates the presence of optrA, and the appearance of a 586bp band indicates the presence of poxtA.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention can conveniently and quickly detect four common oxazolidinone resistance genes (cfr, cfr(D), optrA and poxtA) from bacterial samples. All resistance gene results can be obtained through four pairs of specific primers and one PCR reaction. Compared with the single gene PCR results, the accuracy, specificity and sensitivity can reach 100%, effectively improving the detection efficiency and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The figures are the electrophoresis diagrams of PCR products of oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA, wherein A is the electrophoresis diagram of single primer PCR products, M: 1000bp DNA molecular weight standard, lane 1 is the electrophoresis result of cfr(D) gene; lane 2 is the negative control of cfr(D) gene; lane 3 is the electrophoresis result of optrA gene; lane 4 is the negative control of optrA gene; lane 5 is the electrophoresis result of poxtA gene; lane 6 is the negative control of poxtA gene; lane 7 is the electrophoresis result of cfr gene; lane 8 is the negative control of cfr gene; B is the electrophoresis diagram of multiple PCR products, M: 2000bp DNA molecular weight standard, lanes 1 to 3 are the electrophoresis diagrams of multiple PCR products; lane 4 is the negative control. DETAILED DESCRIPTION

[0020] The following embodiments can make the purpose, technical solutions and advantages of the present invention clearer and facilitate a better understanding of the present invention. However, the embodiments are only exemplary and do not limit the scope of the present invention. Based on the embodiments of the present invention, many other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0021] The Enterococcus faecalis strain used in the examples is an oxazolidinone-resistant strain.

[0022] Example 1 Establishment of a multiplex PCR method for rapid detection of oxazolidinone resistance genes

[0023] Bacterial DNA template extraction: A bacterial DNA extraction kit was used to extract and purify the total DNA of clinically isolated Enterococcus faecalis strains. For specific methods, please refer to the manufacturer's instructions. The extracted total bacterial DNA was stored in a -20°C refrigerator for future use.

[0024] The present invention designs 4 pairs of specific primers according to the gene sequences of oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA disclosed by NCBI (National Center for Biotechnology Information), as shown in Table 1.

[0025] Table 1 Specific primer information

[0026]

[0027] The multiplex PCR reaction system was 20 μL, and 10 μL PrimeSTAR Max Premix (2×) was added to the PCR tube in sequence, followed by 0.6 μL of 10 μmol / L primers shown in SEQ ID NO.1-2, 0.3 μL of 10 μmol / L primers shown in SEQ ID NO.3-4, 0.4 μL of 10 μmol / L primers shown in SEQ ID NO.5-6, 0.5 μL of 10 μmol / L primers shown in SEQ ID NO.7-8, and 1 μg of bacterial DNA. Finally, ddH 2 Add 4% HO to 20 μL, mix the reaction system with a pipette, centrifuge for 10 seconds, and then put it into the PCR instrument.

[0028] The multiplex PCR reaction cycle parameters were as follows: denaturation: 98°C, 10 s; annealing: 52°C, 5 s; extension: 72°C, 5 s, for 25 consecutive cycles and stored at 4°C.

[0029] Identification of drug resistance gene detection results: After PCR amplification, 5 μL of PCR product was subjected to 2% agarose gel electrophoresis for identification. The electrophoresis condition was 150 V for 30 min. After electrophoresis, the gel was observed under ultraviolet light and DNA molecular weight was compared using DL1000 Marker. The bands of 725 bp, 287 bp, 389 bp, and 586 bp indicated cfr, cfr(D), optrA, and poxtA genes, respectively. The results are shown in Figure 1 .

[0030] Figure 1 A is the electrophoresis diagram of single primer PCR product, M: 1000bp DNA molecular weight standard, lane 1 is the electrophoresis result of cfr(D) gene; lane 2 is the negative control of cfr(D) gene; lane 3 is the electrophoresis result of optrA gene; lane 4 is the negative control of optrA gene; lane 5 is the electrophoresis result of poxtA gene; lane 6 is the negative control of poxtA gene; lane 7 is the electrophoresis result of cfr gene; lane 8 is the negative control of cfr gene; Figure 1 B is the electrophoresis of multiple PCR products, M: 2000bp DNA molecular weight standard, lanes 1 to 3 are the electrophoresis of multiple PCR products; lane 4 is the negative control. Figure 1 It can be seen that the bands of 725 bp, 287 bp, 389 bp and 586 bp indicate cfr, cfr(D), optrA and poxtA genes, respectively, indicating that the multiplex PCR method of the present invention has successfully amplified the oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA.

[0031] Example 2 Verification of single gene PCR and multiplex PCR results of the present invention

[0032] The single gene PCR method and the multiplex PCR method of the present invention were used to simultaneously detect 7 known clinical streptococcal strains. The specific results are shown in Table 2.

[0033] Table 2 Verification of single gene PCR and multiplex PCR results

[0034]

[0035] The results in Table 2 showed that the results of single gene PCR and multiplex PCR were consistent, with an accuracy rate of 100%. The single gene results showed that 6 strains (85.7%) did not contain any of them, and 1 strain carried the optrA gene (14.3%, n = 1). The multiplex PCR results showed that there were no strains carrying multiple oxazolidinone resistance genes at the same time, and only 1 strain carried the optrA gene alone.

[0036] Example 3 Specificity and sensitivity test of the multiplex PCR method of the present invention

[0037] The multiplex PCR method of the present invention was used to detect 7 clinical streptococci and 23 clinical staphylococci with known gene distribution to verify the specificity and sensitivity of the multiplex PCR method of the present invention. The specific results are shown in Table 3;

[0038] Table 3 Specificity and sensitivity experimental results of multiplex PCR method

[0039] test Positive = A False negative = B False Positive = C Negative = D Specificity (%) Sensitivity (%) Multiplex PCR method 2 0 0 28 100 100

[0040] Sensitivity = (A / A+B)×100, specificity = (D / C+D)×100.

[0041] The results in Table 3 show that according to the equation (Parikh R, Mathai A, Parikh S, Chandra Sekhar G, Thomas R. Understanding and using sensitivity, specificity and predictive values. Indian J Ophthalmol. 2008; 56(1): 45-50.), the specificity and sensitivity of the multiplex PCR method of the present invention are both 100%.

Claims

1. A multiplex PCR primer set for rapid detection of oxazolidinone resistance genes, characterized in that: The method consists of four pairs of primers, which are respectively used to detect the oxazolidinone resistance gene cfr, cfr(D), optrA and poxtA. The primer sequences for detecting the oxazolidinone resistance gene cfr are shown in SEQ ID NO.1 and SEQ ID NO.2, the primer sequences for detecting the oxazolidinone resistance gene cfr(D) are shown in SEQ ID NO.3 and SEQ ID NO.4, the primer sequences for detecting the oxazolidinone resistance gene optrA are shown in SEQ ID NO.5 and SEQ ID NO.6, and the primer sequences for detecting the oxazolidinone resistance gene poxtA are shown in SEQ ID NO.7 and SEQ ID NO.

8.

2. A multiplex PCR method for detecting oxazolidinone resistance genes, characterized in that: The multiplex PCR method is used to detect the oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA, and comprises the following steps: (1) Extraction of bacterial DNA template: Use a bacterial DNA extraction kit to extract the total DNA of the strain; (2) using the multiplex PCR primer set of claim 1 to amplify the oxazolidinone resistance genes cfr, cfr(D), optrA and poxtA by multiplex PCR reaction; (3) Identification of drug resistance gene detection results: The PCR product obtained in step (2) is separated by agarose gel electrophoresis and compared with the agarose gel electrophoresis patterns of cfr, cfr(D), optrA and poxtA genes to identify the drug resistance gene type of the bacteria to be detected.

3. The multiplex PCR method according to claim 2, characterized in that: The total system of the multiplex PCR reaction described in step (2) is 20 μL, and the reaction system is as follows:

4. The multiplex PCR method according to claim 3, characterized in that: In step (2), the concentration of the primers shown in SEQ ID NO. 1-8 is 5 to 15 μmol / L.

5. The multiplex PCR method according to claim 2, characterized in that: The cycle parameters of the multiplex PCR reaction described in step (2) are as follows: denaturation: 97-99° C., 9-11 s; annealing: 51-53° C., 4-6 s; extension: 71-73° C., 4-6 s, for 22-30 consecutive cycles.

6. The multiplex PCR method according to claim 2, characterized in that: In step (3), the PCR product obtained in step (2) is separated by 2% agarose gel electrophoresis at 150 V for 30 min. DNA molecular weight comparison is performed using DL1000 Marker. The presence of a 725 bp band indicates the presence of cfr, the presence of a 287 bp band indicates the presence of cfr(D), the presence of a 389 bp band indicates the presence of optrA, and the presence of a 586 bp band indicates the presence of poxtA.

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