Detection method of beta-lactamase drug-resistant gene

Abstract

The invention discloses a method for detecting a beta-lactamase drug-resistant gene. The method comprises the following steps: extracting bacterial plasmids; the method comprises the following steps of: downloading sequences of TEM-1 and ampC from GenBank, comparing nucleotide sequences by utilizing Megaalign, finding out a gene conserved region, and designing a primer by utilizing Primer expansion V5; and adding reactants of a PCR reaction system by taking the extracted whole-genome DNA of the bacteria and the extracted plasmid DNA of the bacteria as templates, and carrying out PCR amplification according to the reaction temperature required by the corresponding amplified substances. According to the invention, plasmid-mediated beta-lactam drug resistance detection and analysis are carried out on bovine-derived escherichia coli, so that drug resistance detection rates of TEM-1 type and ampC type genes of a whole genome are 76% and 95% (n is equal to 38); the drug resistance detection rates of the plasmid DNA are 98% and 89% (n = 44), and the drug resistance detection rates of the plasmid DNA are both 100%. The invention provides a scientific basis for further prevention and control of bovine-derived bacterial infectious diseases and reasonable use of antibacterial drugs, and also provides valuable data support and theoretical reference for guiding livestock farm medication and veterinary clinical scientific medication.

Description

Technical Field

[0001] The present invention relates to the field of detection of beta-lactamase resistance genes, and in particular to a method for detecting beta-lactamase resistance genes. Background Art

[0002] In modern animal husbandry, cattle, as an important economic animal, provide humans with abundant dairy and meat products. However, cattle are often infected by various pathogens during breeding, among which Escherichia coli is one of the common pathogens that cause cattle diseases. β-lactam antibiotics are an important class of antibacterial drugs widely used in veterinary clinics and human medicine, with the advantages of broad antibacterial spectrum, strong activity and low toxicity. In the process of cattle breeding, β-lactam antibiotics are used in large quantities to prevent and treat diseases caused by pathogens such as Escherichia coli. With the widespread use of antibiotics, the problem of bacterial resistance is becoming increasingly serious. As a common pathogen, Escherichia coli is also increasingly resistant to β-lactam antibiotics. Plasmid-mediated β-lactamase is one of the important mechanisms for Escherichia coli to develop resistance to β-lactam antibiotics. In recent years, with the continuous development of molecular biology technology, various new detection technologies have emerged, providing more accurate and rapid methods for the detection of plasmid-mediated β-lactamase resistance genes in bovine Escherichia coli. Despite efforts to reduce antibiotic use, global antibiotic use in the livestock sector is expected to increase by 8% between 2020 and 2030, according to an analysis published in PLOS Global Public Health. Global livestock use is expected to reach 107,500 tonnes of antibiotics per year by 2030, up from less than 100,000 tonnes in 2020, the researchers estimate. Antibiotic use is expected to grow fastest in Africa, where it will increase by 25% between 2020 and 2030, due to rising demand for meat products.

[0003] Escherichia coli is one of the important pathogens that cause infectious diseases in cattle, and the resistance of β-lactam drugs, which are widely used clinically, is becoming increasingly serious. Understanding the resistance of bovine Escherichia coli to β-lactam drugs is of great significance for effectively preventing and controlling cattle diseases and ensuring the healthy development of animal husbandry.

[0004] In order to deal with the problem of bacterial resistance, many studies have been conducted abroad on the prevention and control of bovine E. coli resistance. On the one hand, the rational use of antibiotics is strengthened, strict antibiotic use regulations are formulated, and unnecessary antibiotic use is reduced. On the other hand, new antimicrobial drugs and alternative therapies, such as phage therapy and probiotic therapy, are explored and developed.

[0005] Bovine Escherichia coli is an important pathogen that causes cattle diseases, and its drug resistance problem has become increasingly serious in recent years. In animal husbandry, antibiotics are often used as growth promoters and disease prevention agents. This long-term low-dose use increases the risk of the emergence and spread of drug-resistant bacteria. These drug-resistant bacteria not only threaten animal health, but may also be transmitted to humans through the food chain, becoming a major hidden danger to public health. This study focuses on the fields of microbiology and molecular biology, especially the detection and analysis of antibiotic resistance genes. The use of antibiotics in my country varies from region to region, and the drug resistance of isolated Escherichia coli is regional and diverse.

[0006] The purpose of this study is to reveal the distribution and transmission mechanism of these genes and their potential impact on public health by studying plasmid-mediated β-lactamase resistance genes in bovine Escherichia coli from Youjiang and Yulin areas of Guangxi. Summary of the invention

[0007] In view of this, the present invention provides a method for detecting a β-lactamase resistance gene.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The present invention discloses a method for detecting a β-lactamase resistance gene, comprising the following steps:

[0010] Step 1: Extraction of bacterial plasmids

[0011] Step 2: Primer design and synthesis

[0012] The sequences of TEM-1 and ampC were downloaded from GenBank, and the nucleotide sequences were compared using Megalign to find the conserved regions of the genes, and primers were designed using Primer explore V5;

[0013] Step 3: PCR amplification

[0014] The extracted bacterial full gene DNA and bacterial plasmid DNA are used as templates, and each reactant of the PCR reaction system is added, and PCR amplification is performed according to the reaction temperature required by the corresponding amplification product.

[0015] Preferably, in step 1, the specific method is as follows:

[0016] Take 1.0-5.0mL of overnight cultured bacterial solution, centrifuge at 10,000xg for 1min at room temperature to collect bacteria, discard the supernatant medium, add 250μL SolutionI / RNaseA mixed solution, vortex to completely disperse the bacteria, add 250μL SolutionI to the resuspended solution, gently invert several times to mix, add 350μL SolutionIII, gently invert several times to form a white flocculent precipitate, centrifuge at 13,000xg for 10min at room temperature; Place the DNA Mini Column into a 2 ml centrifuge tube and transfer 700 μL of supernatant to DNAMini Column, centrifuge at maximum speed for 1 min at room temperature, discard the filtrate; Put the DNA Mini Column back into the collection tube, add 500 μL HBC Buffer 7 diluted with isopropanol, centrifuge at room temperature at maximum speed for 1 min, and discard the filtrate. Place the DNA Mini Column back into the collection tube, add 700 μL of DNA washing buffer, centrifuge at maximum speed for 1 minute at room temperature, discard the filtrate, and discard the filtrate; Put the DNAMiniColumn back into the collection tube and centrifuge the empty column at maximum speed for 2 minutes to dry the binding column matrix; Place the DNA MiniColumn in a clean 1.5mL centrifuge tube, add 30-100μL Elution Buffer to the binding column matrix, let stand for 1min, and centrifuge at 13,000xg for 1min to elute the DNA; after successful extraction, store in a -20℃ refrigerator.

[0017] Preferably, in step 2, the primer information is as shown in Table 1:

[0018] Table 1 Primer sequence information of drug resistance genes

[0019]

[0020]

[0021] Preferably, in step 3, PCR amplification of TEM-1 and ampC genes is performed according to the following reaction system, as shown in Table 2:

[0022] Table 2 PCR reaction system (25 μL)

[0023]

[0024] Preferably, in step 3, the PCR amplification product is electrophoresed in a 1% agarose gel containing 3 μL of goldview per 100 mL for 30 minutes at a voltage of 120V.

[0025] Compared with the prior art, the present invention has achieved the following technical effects:

[0026] This experiment detected and analyzed the resistance of bovine Escherichia coli to plasmid-mediated β-lactams. The resistance detection rates of TEM-1 and ampC genes in the whole genome were 76% and 95% in Youjiang (n=38); 98% and 89% in Yulin (n=44), while the resistance detection rates of plasmid DNA were 100%. The research results provide a scientific basis for further prevention and control of bovine bacterial infectious diseases and the rational use of antimicrobial drugs. They also provide valuable data support and theoretical reference for guiding the scientific use of drugs in livestock farms and veterinary clinics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A diagram showing the detection rate of TEM-1 and ampC gene resistance of Escherichia coli from cattle in two places according to a method for detecting β-lactamase resistance genes of the present invention;

[0028] Among them, (a) whole genome, (b) flow chart of plasmid DNA;

[0029] Figure 2 This is a graph showing the PCR amplification results of a TEM-1 gene-positive strain in a method for detecting a β-lactamase resistance gene of the present invention;

[0030] Among them, (a) the whole genome of Youjiang area, 1-9: TEM-1 type positive partial strains; (b) the whole genome of Yulin area, 1-11: TEM-1 type partial positive strains; M: DNA Maker; N: negative control;

[0031] Figure 3 This is a diagram showing the PCR amplification results of ampC gene-positive strains in a method for detecting β-lactamase resistance genes of the present invention;

[0032] Among them, (a) the whole genome of Youjiang area, 2-6: some positive strains of ampC type; (b) the whole genome of Yulin area, 1-6: some positive strains of ampC type; M: DNA Maker; N: negative control;

[0033] Figure 4 This is a graph showing the PCR amplification results of a TEM-1 gene-positive strain in a method for detecting a β-lactamase resistance gene of the present invention;

[0034] Among them, (a) plasmid DNA from Youjiang area, 2-6: partial positive strains of TEM-1 type; (b) plasmid DNA from Yulin area, 1-6: partial positive strains of TEM-1 type; M: DNA Maker; N: negative control;

[0035] Figure 5 This is a diagram showing the PCR amplification results of ampC gene-positive strains in a method for detecting β-lactamase resistance genes of the present invention;

[0036] Among them, (a) plasmid DNA from Youjiang area, 1-11: partial positive strains of ampC type; (b) plasmid DNA from Yulin area, 1-7: partial positive strains of ampC type; M: DNA Maker; N: negative control. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] A method for detecting a β-lactamase resistance gene comprises the following steps:

[0039] Step 1: Extraction of bacterial plasmids

[0040] The specific method is as follows:

[0041] Take 1.0-5.0mL of overnight cultured bacterial solution, centrifuge at 10,000xg for 1min at room temperature to collect bacteria, discard the supernatant medium, add 250μL SolutionI / RNaseA mixed solution, vortex to completely disperse the bacteria, add 250μL SolutionI to the resuspended solution, gently invert several times to mix, add 350μL SolutionIII, gently invert several times to form a white flocculent precipitate, centrifuge at 13,000xg for 10min at room temperature; Place the DNA Mini Column into a 2 ml centrifuge tube and transfer 700 μL of supernatant to DNAMini Column, centrifuge at maximum speed for 1 min at room temperature, discard the filtrate; Put the DNA Mini Column back into the collection tube, add 500 μL HBC Buffer 7 diluted with isopropanol, centrifuge at room temperature at maximum speed for 1 min, and discard the filtrate. Place the DNA Mini Column back into the collection tube, add 700 μL of DNA washing buffer, centrifuge at maximum speed for 1 minute at room temperature, discard the filtrate, and discard the filtrate; Put the DNAMiniColumn back into the collection tube and centrifuge the empty column at maximum speed for 2 minutes to dry the binding column matrix; Place the DNA MiniColumn in a clean 1.5mL centrifuge tube, add 30-100μL Elution Buffer to the binding column matrix, let stand for 1min, centrifuge at 13,000xg for 1min to elute the DNA; after successful extraction, store in a -20℃ refrigerator;

[0042] Step 2: Primer design and synthesis

[0043] The sequences of TEM-1 and ampC were downloaded from GenBank, and the nucleotide sequences were compared using Megalign to find the conserved regions of the genes, and primers were designed using Primer explore V5;

[0044] Primer information is shown in Table 1:

[0045] Table 1 Primer sequence information of drug resistance genes

[0046]

[0047] Step 3: PCR amplification

[0048] Using the extracted bacterial full gene DNA and bacterial plasmid DNA as templates, adding each reactant of the PCR reaction system, and performing PCR amplification at the reaction temperature required by the corresponding amplification product;

[0049] The PCR of TEM-1 and ampC genes were amplified according to the following reaction system, as shown in Table 2:

[0050] Table 2 PCR reaction system (25 μL)

[0051]

[0052]

[0053] The PCR amplification products were electrophoresed in 1% agarose gel containing 3 μL goldview per 100 mL at a voltage of 120V for 30 min.

[0054] Embodiment 1:

[0055] Analysis of drug-resistant genes of Escherichia coli from cattle

[0056] like Figure 1As shown in the figure, the drug resistance gene detection of bovine Escherichia coli in Youjiang and Yulin areas was carried out using TEM-1 and ampC resistance genes. The experimental results showed that the drug resistance detection rate of the whole genome TEM-1 gene in Youjiang area was 76%, and the drug resistance detection rate of the ampC gene was 95% (n=38); the drug resistance detection rate of the whole genome TEM-1 gene in Yulin area was 98%, and the drug resistance detection rate of the ampC gene was 89% (n=44). The drug resistance detection rate of the TEM-1 gene and ampC gene of the plasmid DNA in the two areas was 100%. Among them, a total of 38 plasmid DNAs were extracted from Youjiang area, and a total of 43 plasmid DNAs were extracted from Yulin area.

[0057] Embodiment 2:

[0058] PCR amplification results

[0059] Both TEM-1 and ampC screened out positive strains and amplified target fragments of corresponding sizes. The PCR amplification products were sequenced and compared with known sequences in the NCBI database, and the similarities reached 100% and 99%, respectively, as shown in Table 3.

[0060] The electrophoresis diagrams of TEM-1 gene and ampC gene of the whole genome DNA of the two places are shown in Figure 2 and Figure 3 shown.

[0061] The electrophoresis diagrams of TEM-1 gene and ampC gene of plasmid DNA from two species are shown in Figure 4 and Figure 5 shown.

[0062] Table 3 Comparison of PCR products of drug resistance genes with Genbank sequences

[0063]

[0064] The present invention performs plasmid-mediated β-lactam resistance detection and analysis on bovine Escherichia coli from beef cattle farms in Youjiang and Yulin areas. The experiment found that compared with the resistant strains of bovine Escherichia coli from Yulin, the resistant strains of bovine Escherichia coli from Youjiang area are less. This reflects from a macroscopic perspective that different breeding environments in different regions have a certain influence on the drug resistance and drug resistance genes of bovine Escherichia coli, reflecting the regional differences.

[0065] In Shihezi City, Xinjiang, a study of local cattle-derived E. coli isolates found that their resistance to cefazolin and amoxicillin exceeded 50%, but the resistance rates of ampicillin, cephalexin and cefepime were lower, all below 5%, and all isolates were sensitive to cefepime. It is worth noting that some strains were detected with β-lactam resistance genes, which means that a small number of normal cattle-derived E. coli in the area already carry resistance genes and have developed resistance to some β-lactam drugs. The test results of 782 strains of E. coli from 22 dairy farms in Qingdao showed that the resistance rate of E. coli producing extended-spectrum β-lactamases to ceftriaxone, ceftiofur, cefuroxime and ampicillin reached 100%, and they also had varying degrees of resistance to some other antibiotics, but they were more sensitive to drugs such as gentamicin and enrofloxacin. In northern Xinjiang, samples were collected from cattle farms in Shihezi, Urumqi, Yili and other places. The resistance rate of isolated extended-spectrum β-lactamase-producing Escherichia coli to cefotaxime, ampicillin, piperacillin and ciprofloxacin was as high as 90%, but it was sensitive to imipenem and polymyxin B. Studies in Ningxia have shown that the resistance rate of bovine Escherichia coli to chloramphenicol, streptomycin, tetracycline, doxycycline, ceftriaxone and sulfamethoxazole is high, and the positive rate of extended-spectrum β-lactamase-producing strains is 13.64%. The enzyme-producing strains have obvious multidrug resistance and the resistance rate to various antimicrobial drugs is higher than that of non-enzyme-producing strains. The Escherichia coli isolated from the liver of a calf that died of diarrhea in a dairy farm in Shanxi Province was resistant to multiple antibiotics such as penicillin, amoxicillin, and ampicillin, and was sensitive to some antibiotics. The isolate was a positive strain that produced both extended-spectrum β-lactamase and cephalosporinase, and carried a plasmid-mediated β-lactamase gene. In Heilongjiang Province, the resistance rate of bovine Escherichia coli to streptomycin, amoxicillin, tetracycline, cotrimoxazole, and doxycycline was high, reaching 100%. Among the plasmid-mediated resistance genes, the positive rate of blaTEM gene was 84.62%, while the positive rate of blaCTX-M gene was 19.23%. In a study in Sichuan Province, among 467 strains of Escherichia coli from food animals, the proportions of ESBLs and AmpC were 36.83% and 13.70%, respectively. Among them, the positive rate of blaTEM gene was 84.09%, and the positive rate of blaCTX-M gene was 60.80%.

[0066] In general, the research in these regions fully demonstrates that there are significant differences in the resistance of bovine Escherichia coli to β-lactamase drugs in cattle farms in different regions of the country. In general, there is a certain degree of drug resistance, and the problem of drug resistance in some areas is even more serious. This difference and severity are closely related to the breeding environment and antibiotic use in various places. For example, in areas with high breeding density, complex environment, frequent and unreasonable use of antibiotics, drug resistance may be more prominent. Understanding these drug resistance situations plays a vital role in the rational use of antibiotics, prevention and control of bovine Escherichia coli infections, and ensuring the healthy development of the cattle breeding industry. At the same time, it also provides a basis for further in-depth research on the mechanism of drug resistance and the formulation of targeted response strategies.

[0067] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for detecting a β-lactamase resistance gene, characterized in that: The steps include: Step 1: Extraction of bacterial plasmids Step 2: Primer design and synthesis The sequences of TEM-1 and ampC were downloaded from GenBank, and the nucleotide sequences were compared using Megalign to find the conserved regions of the genes, and primers were designed using Primer explore V5; Step 3: PCR amplification The extracted bacterial full gene DNA and bacterial plasmid DNA are used as templates, and each reactant of the PCR reaction system is added, and PCR amplification is performed according to the reaction temperature required by the corresponding amplification product.

2. The method for detecting a β-lactamase resistance gene according to claim 1, characterized in that: In the step 1, the specific method is as follows: Take 1.0-5.0mL of overnight cultured bacterial solution, centrifuge at 10,000xg for 1min at room temperature to collect bacteria, discard the supernatant medium, add 250μL Solution I / RNaseA mixed solution, vortex to completely disperse the bacteria, add 250μL Solution I to the resuspended solution, gently invert several times to mix, add 350μL Solution III, gently invert several times to form a white flocculent precipitate, centrifuge at 13,000xg for 10min at room temperature; Place the DNA Mini Column into a 2 ml centrifuge tube and transfer 700 μL of supernatant to DNAMini Column, centrifuge at maximum speed at room temperature for 1 min, discard the filtrate; Put the DNAMini Column back into the collection tube, add 500 μL HBC Buffer 7 diluted with isopropanol, centrifuge at room temperature at maximum speed for 1 min, and discard the filtrate. The DNA Mini Column was placed back into the collection tube, 700 μL of DNA wash buffer was added, and the column was centrifuged at top speed for 1 min at room temperature, and the filtrate was discarded; Bundle Put the DNAMiniColumn back into the collection tube and centrifuge the empty column at maximum speed for 2 minutes to dry the binding column matrix; The DNA MiniColumn was placed in a clean 1.5 mL centrifuge tube, 30-100 μL of Elution Buffer was added to the binding column matrix, allowed to stand for 1 min, and the DNA was eluted by centrifugation at 13,000 x g for 1 min; After successful extraction, store in a -20℃ refrigerator.

3. The method for detecting a β-lactamase resistance gene according to claim 1, characterized in that: In step 2, the primer information is shown in Table 1: Table 1 Primer sequence information of drug resistance genes 4. The method for detecting a β-lactamase resistance gene according to claim 1, characterized in that: In step 3, PCR of TEM-1 and ampC genes are amplified according to the following reaction system, as shown in Table 2: Table 2 PCR reaction system (25 μL) 5. The method for detecting a β-lactamase resistance gene according to claim 1, characterized in that: In the step 3, the PCR amplification product was electrophoresed in a 1% agarose gel containing 3 μL of goldview per 100 mL for 30 minutes at a voltage of 120V.

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