Primer group and kit for detecting characteristic drug-resistant genes of escherichia coli and application of primer group and kit

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a primer group and a kit for detecting Escherichia coli characteristic drug resistance genes and application of the primer group and the kit. The primer group provided by the invention comprises a primer pair for detecting genes floR, tet (A) and sul2. The gene has higher correlation with the drug resistance phenotype, and can reflect the drug resistance condition of a sample to be detected; each primer pair has good specificity and sensitivity, and the detection efficiency of the drug-resistant gene can be improved. The invention also provides a method for rapidly screening the characteristic drug-resistant gene of the animal-derived escherichia coli in the Ningxia region by using the primer group and the kit. According to the method, the screening efficiency of the Ningxia region animal-derived drug-resistant phenotype can be improved, and by detecting and screening the Escherichia coli characteristic drug-resistant gene, guidance can be provided for reasonable application of veterinary antibacterial drugs in farms in the Ningxia region more quickly, more accurately and more pertinently.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a primer set and a kit for detecting characteristic drug-resistant genes of Escherichia coli and applications thereof. Background Art

[0002] Due to the prevalence of drug-resistant genes, medical care, food safety, public health and agriculture have all been greatly affected. In livestock and poultry farming, veterinary personnel need to develop targeted diagnosis and treatment strategies based on the drug-resistant phenotypes of different strains. However, traditional drug-resistant phenotype detection methods, such as bacteriological testing and drug sensitivity testing, have problems such as large workload, long time consumption and poor detection timeliness.

[0003] Rapid detection technology for drug-resistant genes is an important means to solve this problem. Rapid detection of drug-resistant genes can detect the epigenetic characteristics of pathogens as early as possible, which helps to identify the chemical composition of pathogens, especially whether they are drug-resistant. In addition, rapid detection of drug-resistant genes can also help researchers study the mechanism of drug-resistant gene evolution, develop new antibacterial strategies, and promote the development of anti-infective drug research. However, the presence of drug-resistant genes does not necessarily mean that the strain is resistant. The quality and representativeness of drug-resistant genome and drug-resistant phenotypic data are important prerequisites for ensuring the accuracy of drug resistance prediction. At present, the genomic information containing drug-resistant data in public databases mostly comes from North America and Europe. The bacterial genome and drug-resistant data in developing countries are relatively small and insufficiently representative. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a primer set, a kit and its application for detecting characteristic drug resistance genes of Escherichia coli. The primer set provided by the present invention is used to detect drug resistance genes floR, tet (A) and sul2 that are highly correlated with drug resistance phenotypes, has good specificity and sensitivity, can be used to quickly screen characteristic drug resistance genes of Escherichia coli, and thus quickly indicate its drug resistance phenotype. The detection results can be used as one of the bases for clinical drug use in farms, thereby improving the timeliness of clinical drug use.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme:

[0006] The first aspect of the present invention provides a primer set for detecting characteristic drug-resistant genes of Escherichia coli, wherein the drug-resistant genes include floR, tet(A) and sul2, and the primer set includes a PCR primer pair for detecting the sequence of the gene floR as shown in SEQ ID NO.1 and SEQ ID NO.2, a PCR primer pair for detecting the sequence of the gene tet(A) as shown in SEQ ID NO.3 and SEQ ID NO.4, and a PCR primer pair for detecting the sequence of the gene sul2 as shown in SEQ ID NO.5 and SEQ ID NO.6; or includes an RT-PCR primer pair for detecting the sequence of the gene floR as shown in SEQ ID NO.7 and SEQ ID NO.8, an RT-PCR primer pair for detecting the sequence of the gene tet(A) as shown in SEQ ID NO.9 and SEQ ID NO.10, and an RT-PCR primer pair for detecting the sequence of the gene sul2 as shown in SEQ ID NO.11 and SEQ ID NO.12.

[0007] Genes floR, tet(A) and sul2 are florfenicol resistance genes, tetracycline resistance genes and sulfonamide resistance genes, respectively. Among the various resistance genes contained in multidrug-resistant Escherichia coli, they are more highly correlated with the resistance phenotype and can better reflect the resistance phenotype of the strain to florfenicol, tetracycline and sulfamethoxazole.

[0008] The above primer pairs are designed for the drug-resistant genes floR, tet(A), and sul2, have good specificity and sensitivity, and will not form dimers in the PCR reaction, and can meet the requirements of the PCR reaction. The detection using the above primer pairs can improve the detection accuracy and efficiency of drug-resistant genes and drug-resistant phenotypes.

[0009] Preferably, the primer set is a primer set for detecting characteristic drug-resistant genes of Escherichia coli from animals in Ningxia. The above-mentioned drug-resistant genes are obtained by screening after analyzing the correlation between drug-resistant genes and phenotypes in multi-drug-resistant strains based on the isolation and identification of Escherichia coli from the main livestock and poultry sources in Ningxia, the detection of drug-resistant phenotypes of drug-resistant bacteria, and whole genome sequencing of drug-resistant strains. They are more suitable for detecting the drug-resistant phenotype of Escherichia coli from animals in Ningxia. By detecting the above-mentioned characteristic drug-resistant genes of Escherichia coli, the test results can be used to guide the scientific and rational use of veterinary antibiotics in livestock and poultry farms in Ningxia, improve treatment efficiency, and reduce the risk of animal-derived drug resistance caused by the abuse of veterinary antibiotics.

[0010] The second aspect of the present invention provides a kit for detecting characteristic drug-resistant genes of Escherichia coli, comprising the above-mentioned primer set.

[0011] Preferably, the kit is a kit for detecting characteristic drug-resistant genes of Escherichia coli of animal origin in Ningxia.

[0012] Preferably, the kit is a PCR detection kit, and the kit further comprises a PCR detection reagent. Optionally, the PCR detection reagent can be selected from 2×Taq PCR Master Mix.

[0013] Further preferably, the kit also includes a nucleic acid extraction reagent.

[0014] Preferably, the kit is an RT-PCR detection kit, and the kit further comprises an RT-PCR detection reagent. Optionally, the RT-PCR detection reagent can be selected from 2×Pro Taq HS Probe Premix and a fluorescent dye, such as ROX (maleimide).

[0015] The third aspect of the present invention provides the use of the above primer set or kit in rapid screening of drug-resistant phenotypes of Escherichia coli.

[0016] Preferably, the Escherichia coli is an animal-derived Escherichia coli from Ningxia that is resistant to florfenicol, tetracycline, and sulfamethoxazole.

[0017] The fourth aspect of the present invention provides a method for rapid screening of drug-resistant phenotypes of Escherichia coli, using the above primer set or kit to screen the drug-resistant phenotype of Escherichia coli; the drug-resistant phenotype is resistance to florfenicol, tetracycline and sulfamethoxazole.

[0018] Optionally, the specific operation of the method is: separating Escherichia coli from the test object to prepare a bacterial solution, using the above primer set or kit to perform drug-resistant gene detection by PCR or RT-PCR, and judging whether the Escherichia coli has a drug-resistant phenotype based on the test results.

[0019] E. coli does not have a lipopolysaccharide wall and can be directly lysed without the need for DNA extraction.

[0020] Preferably, the method is a PCR detection method, and the PCR reaction system is:

[0021] Reagents volume 2×Taq PCR Master Mix 25μL Upstream primer, 0.2~0.5μM 2.5μL Downstream primer, 0.2~0.5μM 2.5μL Template DNA 5μL Double distilled water Add to 50 μL

[0022] The PCR reaction conditions were:

[0023]

[0024] Preferably, the method is an RT-PCR detection method, and the RT-PCR reaction system is:

[0025] Reagents volume 2×Pro Taq HS Probe Premix 10μL Upstream primer, 0.2~0.5μM 0.8μL Downstream primer, 0.2~0.5μM 0.8μL ROX 0.4μL Template DNA 2μL Double distilled water Add to 20 μL

[0026] RT-PCR reaction conditions are:

[0027]

[0028] The beneficial effects of the present invention are as follows: the present invention separates and identifies Escherichia coli from livestock and poultry in Ningxia, and carries out minimum inhibitory concentration detection of 16 antimicrobial drugs on it, and screens out multi-drug resistant strains according to the results of drug sensitivity detection, and screens out drug-resistant genes floR, tet (A), and sul2 with higher correlation with drug-resistant phenotypes by whole genome sequencing and analysis of the correlation between drug-resistant genes and phenotypes in multi-drug resistant strains. These genes can be used to detect the drug-resistant phenotype of Escherichia coli against florfenicol, tetracycline and sulfamethoxazole, and are particularly suitable as characteristic drug-resistant genes of animal sources in Ningxia, and are used to screen the drug-resistant phenotype of Escherichia coli in Ningxia. The primers provided by the present invention, such as the sequences shown in SEQ ID NO.1 to SEQ ID NO.12, have good specificity and sensitivity, and can be used in PCR and RT-PCR detection methods. The method for rapid screening of drug-resistant phenotypes of Escherichia coli provided by the present invention can improve the screening efficiency of animal-derived drug-resistant Escherichia coli resistant to florfenicol, tetracycline, and sulfamethoxazole in Ningxia, and shorten the drug sensitivity detection time from more than 2 days to about 0.5 days. By detecting and screening the characteristic drug-resistant genes and drug-resistant phenotypes of the above-mentioned Escherichia coli, technical guidance can be provided for the rational use of antibiotics in farms more quickly, more accurately, and more targeted, thereby effectively improving the cure rate of sick animals and reducing the mortality rate and clinical treatment costs of sick animals. It is not only of great significance for the treatment of animal diseases, but also helps to indirectly monitor the use of veterinary antibiotics in the breeding industry and reduce the risk of drug resistance caused by the abuse of veterinary antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The drug sensitivity results of Escherichia coli in Example 1 of the present invention are shown in Figure A. Figure A is a bar graph of the overall drug resistance results of Escherichia coli; Figure B is a bar graph of multi-drug resistant strains; Figure C is a bar graph of drug resistance of Escherichia coli of different species;

[0030] Figure 2 A network diagram showing the coexistence of plasmids and drug-resistant genes in Example 1 of the present invention;

[0031] Figure 3 The PCR result of the floR drug resistance gene in Example 1 of the present invention;

[0032] Figure 4 The PCR result of the tet(A) resistance gene in Example 1 of the present invention;

[0033] Figure 5 The PCR result of the sul2 drug resistance gene in Example 1 of the present invention;

[0034] Figure 6 is the PCR result of the primer sensitivity test in Example 1 of the present invention;

[0035] Figure 7 This is the primer dimer verification result in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0037] Traditional methods for detecting drug-resistant phenotypes are labor-intensive and time-consuming. Rapid detection of drug-resistant genes helps determine the drug-resistant phenotype of strains, based on which diagnostic and treatment strategies can be formulated or new antibacterial strategies can be developed, but bacterial genomes and drug-resistant data in public databases are insufficiently representative.

[0038] The invention is based on the separation and identification of Escherichia coli mainly from livestock and poultry in Ningxia, and carries out drug resistance detection of 16 kinds of antibiotics such as ampicillin, amoxicillin / clavulanic acid, gentamicin, spectinomycin, tetracycline, florfenicol, sulfisoxazole, trimethoprim / sulfamethoxazole, ceftiofur, ceftazidime, enrofloxacin, ofloxacin, meropenem, apramycin, colistin, mequindox, etc., and screens out multidrug-resistant strains according to the drug sensitivity test results, obtains drug resistance gene information of the multidrug-resistant strains by whole genome sequencing, analyzes the correlation between drug resistance genes and phenotypes in the multidrug-resistant strains, screens out drug resistance genes with higher correlation with drug resistance phenotypes of florfenicol, tetracycline and sulfisoxazole, designs PCR and RT-PCR primers, and further establishes a drug resistance phenotype screening method.

[0039] Based on the above research, an embodiment of the present invention provides a primer set for detecting characteristic drug-resistant genes of Escherichia coli, wherein the drug-resistant genes include floR, tet(A) and sul2, and the primer set includes a PCR primer pair for detecting the sequence of gene floR as shown in SEQ ID NO.1 and SEQ ID NO.2, a PCR primer pair for detecting the sequence of gene tet(A) as shown in SEQ ID NO.3 and SEQ ID NO.4, and a PCR primer pair for detecting the sequence of gene sul2 as shown in SEQ ID NO.5 and SEQ IDNO.6; or includes an RT-PCR primer pair for detecting the sequence of gene floR as shown in SEQ ID NO.7 and SEQ ID NO.8, an RT-PCR primer pair for detecting the sequence of gene tet(A) as shown in SEQ ID NO.9 and SEQ ID NO.10, and an RT-PCR primer pair for detecting the sequence of gene sul2 as shown in SEQ ID NO.11 and SEQ ID NO.12.

[0040] The embodiment of the present invention also provides a kit for detecting characteristic drug-resistant genes of Escherichia coli.

[0041] The embodiments of the present invention also provide the use of the above primer set or kit in rapid screening of drug-resistant phenotypes of Escherichia coli.

[0042] The embodiment of the present invention also provides a method for rapidly screening the drug-resistant phenotype of Escherichia coli.

[0043] The scheme of the present invention is described below through specific embodiments.

[0044] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0045] Example 1

[0046] This example provides a screening process for characteristic drug-resistant genes of Escherichia coli and a primer pair for detecting the drug-resistant genes.

[0047] 1. Antimicrobial susceptibility results of Escherichia coli isolates

[0048] The results of drug sensitivity test showed that the resistance rate of Escherichia coli isolates to tetracycline reached 89.4%, followed by sulfamethoxazole (85.1%), ampicillin (84%), florfenicol (73.4%), enrofloxacin (53.2%), spectinomycin (52.1%), ofloxacin (44.7%), etc. (as shown in Table 1). The isolates were highly sensitive to polymyxin and meropenem, with sensitivity rates of 93.6% and 91.5%, respectively. Among different species, the resistance rate of chicken-derived isolates to sulfonamides, tetracyclines and ampicillin of β-lactams was high, all at 96.9%; the resistance rate of cattle-derived isolates to tetracycline and ampicillin was high, at 89.2% and 75.7%, respectively; there were 79 multidrug-resistant strains, accounting for 84.0% of the total. There were fewer samples of strains isolated from sheep; the resistance rate of strains isolated from pigs to ampicillin, tetracycline, florfenicol and sulfamethoxazole reached 100% (as shown in Table 2). Among different animals, pig Escherichia coli had the most serious resistance, followed by chicken, cattle and sheep from high to low. Figure 1 shown.

[0049] Table 1 Escherichia coli drug sensitivity results

[0050]

[0051] Note: R, resistant; I, intermediately sensitive; S, sensitive.

[0052] Table 2 Drug sensitivity results of Escherichia coli from various animal sources

[0053]

[0054] Note: R, resistant; I, intermediately sensitive; S, sensitive.

[0055] 2. Screening of characteristic drug resistance genes

[0056] Combining the results of whole genome sequencing and drug sensitivity testing, the correlation between resistance genes and phenotypes in multidrug-resistant strains was analyzed, and resistance genes with a higher correlation with resistance phenotypes were screened out as characteristic resistance genes in Ningxia. PCR and RT-PCR primers were designed for screening characteristic resistance genes and multidrug-resistant strains in Ningxia.

[0057] 2.1 SNP evolutionary tree and characteristics of drug-resistant genes and plasmids carried by indicator bacteria

[0058] (1) SNP evolutionary tree and characteristics of Escherichia coli carrying drug-resistant genes and plasmids

[0059] By establishing a SNP phylogenetic tree of Escherichia coli and conducting corresponding research, it was found that strains from different animal sources are closely related and there may be horizontal transmission of drug-resistant genes. The present invention analyzed multiple antibiotic resistance genes (ARGs) of 94 sequenced samples, from which 72 ARG types were detected, including aminoglycoside resistance genes [aadA5 (n=8), aac(3)-IId (n=16), aadA2 (n=9), aac(3)-IV (n=24), aadA1 (n=11), aac(6')-Ib-cr (n=4), aac(3)-IIa (n=2), aadA22 (n=6), aac(6')-Ib3 (n=2), aph(3")-Ib (n=41), aph(3')-Ia (n=35), aph( 6)-Id (n=45), aph(4)-Ia (n=24), aph(3')-IIa (n=2), rmtB (n=3)], β-lactam resistance genes [blaTEM-1B (n=18), blaOXA-10 (n=26), blaCTX-M-65 (n=14), blaOXA-1 (n=3), blaCTX-M-55 (n=13), blaTEM-176 (n=1), blaCTX-M-14 (n=8), blaLAP-2 (n=8), blaCMY-2 (n=3), blaTEM-1A (n=5)] , sulfonamide resistance genes [sul1(n=9), sul3(n=34), sul2(n=41), dfrA27(n=1), dfrA1(n=5), dfrA14(n=41), dfrA12(n=6), dfrA17(n=10), dfrA5(n=1)], tetracycline resistance genes [tet(A)(n=35), tet(M)(n=5), tet(B)(n=4), tet(C)(n=2), tet(X4)(n=1), mef(B)(n=1)], enrofloxacin resistance genes [oqxA(n=5), oqxB(n=5) , qnrS1(n=42), qnrS2(n=2), qnrS13(n=1)], chloramphenicol resistance genes [catB3(n=3), ARR-2(n=30), ARR-3(n=4)], florfenicol resistance genes [floR(n=50), cmlA1(n=16), cml(n=2)], streptomycin resistance genes [erm(B)(n=2), erm(42)(n=1)], colistin resistance gene [mcr-1.1(n=2)], lincomycin resistance gene [lnu(F)(n=30)], macrolide resistance gene [mph(A)(n=18)].

[0060] A total of 53 different serotypes were found in E. coli in this study, among which serotypes O8, O9, O63, O166, O101 and O13 accounted for a high proportion. O8 was the main serotype in the E. coli isolates, which is the most common enterotoxigenic E. coli (ETEC) serogroup that can cause diseases in other animals.

[0061] At the same time, this study found that strains carrying ESBLs genes carried more drug-resistant genes than other strains. This study also found two mcr-1.1-positive strains and tet(X4)-positive strains.

[0062] (2) SNP evolutionary tree and characteristics of drug-resistant genes and plasmids carried by E. coli isolates

[0063] In this study, multiple antibiotic resistance genes (ARGs) of 89 sequenced samples were analyzed, and 27 ARG types were detected. The top 16 ARGs included aminoglycoside resistance genes [aph(3')-III (n=34), aac(6')-Il (n=28), ant(6)-Ia (n=20)], macrolide resistance genes [erm(A) (n=16), erm(B) (n=26), msr(C) (n=18)], tetracycline resistance genes [t et(L)(n=47,tet(M)(n=55)],chloramphenicol resistance gene[cat(n=22,fexA(n=15)],lincomycin resistance gene[lsa(A)(n=59,lsa(E)(n=22)],linezolid resistance gene[optrA(n=17)],streptomycin resistance gene[str(n=11)],methoxazole resistance gene[dfrG(n=35)]and lincomycin resistance gene[lun(B)(n=22)]. This study also found 17 isolates harboring the oprtA gene, and it was found that the strains carrying the optrA resistance gene contained more resistance genes than the strains without the optrA resistance gene.

[0064] (3) Coexistence and correlation analysis of Escherichia coli plasmids and drug resistance genes

[0065] In this study, 24 plasmid types were detected, and the top 10 were rep10 (36), rep9c (21), rep9b (19), rep9a (15), repUS15 (11), repUS11 (7), rep1 (7), rep2 (3), rep29 (3), and rep22 (3).

[0066] Plasmid and drug resistance gene coexistence network diagram (such as Figure 2The results show that AMR co-occurs with plasmids. Twelve drug-resistant genes, including blaOXA-1, floR, dfrA14, and sul1, are present in all plasmids in the figure. Among them, tet(X4) co-occurs with plasmids IncX1, IncFIC, CoiRNAI, and IncFB, suggesting that the transmission of drug-resistant genes by plasmids plays a very important role.

[0067] 3. Primer Design

[0068] Based on the correlation analysis between drug-resistant genes and drug-resistant phenotypes, three drug-resistant genes with the highest correlation with drug-resistant phenotypes were screened out, namely floR (53.2%), tet (A) (37.2%), and sul2 (43.6%), and their phenotype and genotype consistency reached more than 85%. The above three drug-resistant genes were used as characteristic drug-resistant genes for the phenotypic drug resistance of animal-derived Escherichia coli in Ningxia.

[0069] Primers were designed based on the genomic sequences of the above three drug-resistant genes, as shown in Table 3.

[0070] Table 3 Primers for characteristic drug resistance genes of Escherichia coli

[0071]

[0072] 4. Primer Verification

[0073] Five strains carrying each target gene were randomly selected for PCR and RT-PCR experiments to verify the effect and specificity of the primers. The PCR reaction system was 50 μL, including 2×Taq PCR Master Mix 25 μL, 2.5 μL of upstream primer (0.2 μL) and downstream primer (0.2 μL), 5 μL of template DNA (10-100 ng), and double distilled water was added to 50 μL. PCR reaction conditions: 95℃5min; 95℃1min, annealing (temperature see Table 3) 40s, 72℃40s, 30 cycles; 72℃5min. The RT-PCR reaction system was 20 μL, including 2×Pro Taq HS Probe Premix 10 10 μL, 0.8 μL of upstream primer (0.2 μL) and downstream primer (0.2 μL), 0.4 μL of ROX, 2 μL of template (10-100 ng), and double distilled water was added to 6 μL. RT-PCR reaction conditions: 95°C for 3 min, 95°C for 10 s, annealing at 60°C for 15 s, and 72°C for 30 s, for 40 cycles.

[0074] Specific test results such as Figure 3 to Figure 5 As shown, the above primers have good specificity.

[0075] Primer sensitivity experiments were performed on Enterobacter NXGC30, which contained specific resistance genes of Enterobacter in Ningxia, and all of them were single-copy resistance genes in the genome annotation. 8 After the concentration was 100 / mL, the dilution was repeated seven times. Figure 6 As shown, from left to right, 1 to 7 correspond to the bacterial solution concentrations of 1.5×10 8 , 1.5×10 7 , 1.5×10 6 , 1.5×10 5 , 1.5×10 4 , 1.5×10 3 , 1.5×10 2 The results showed that the primers had good sensitivity. 4 The primers for the floR resistance gene had the best sensitivity, reaching 1.5×10 2 Pieces / mL.

[0076] The primers were verified by primer dimer PCR. The results were as follows Figure 7 As shown, from left to right 1 to 4 are Enterobacterial resistance genes floR, tet(A), sul2 and negative control, all of which show no primer dimer formation.

[0077] Example 2

[0078] The present embodiment provides a kit for detecting characteristic drug-resistant genes of Escherichia coli, which is a PCR detection kit, including a primer pair for detecting the sequence of gene floR as shown in SEQ ID NO.1 and SEQ ID NO.2, a primer pair for detecting the sequence of gene tet(A) as shown in SEQ ID NO.3 and SEQ ID NO.4, and a primer pair for detecting the sequence of gene sul2 as shown in SEQ ID NO.5 and SEQ ID NO.6, and also includes a PCR detection reagent 2×TaqPCR Master Mix.

[0079] Example 3

[0080] The present embodiment provides a kit for detecting characteristic drug-resistant genes of Escherichia coli, which is an RT-PCR detection kit, including a primer pair for detecting the sequence of gene floR as shown in SEQ ID NO.7 and SEQ ID NO.8, a primer pair for detecting the sequence of gene tet(A) as shown in SEQ ID NO.9 and SEQ ID NO.10, and a primer pair for detecting the sequence of gene sul2 as shown in SEQ ID NO.11 and SEQ ID NO.12, and also includes RT-PCR detection reagent 2×Pro Taq HS Probe Premix and fluorescent dye ROX.

[0081] Example 4

[0082] This embodiment provides a method for rapid screening of drug-resistant phenotypes of Escherichia coli in Ningxia.

[0083] Escherichia coli was separated from the test object and prepared into bacterial solution, and PCR and RT-PCR reactions were performed using the primers in Table 3 of Example 1. The PCR reaction system is shown in Table 4, and the reaction conditions are shown in Table 5. The RT-PCR reaction system is shown in Table 6, and the reaction conditions are shown in Table 7.

[0084] Table 4 PCR reaction system

[0085] Reagents volume 2×Taq PCR Master Mix 25μL Upstream primer 2.5μL Downstream primer 2.5μL Template DNA 5μL Double distilled water Add to 50 μL

[0086] Table 5 PCR reaction conditions

[0087]

[0088] Table 6 RT-PCR reaction system

[0089] Reagents volume 2×Pro Taq HS Probe Premix 10μL Upstream primer 0.8μL Downstream primer 0.8μL ROX 0.4μL Template DNA 2μL Double distilled water Add to 20 μL

[0090] Table 7 RT-PCR reaction conditions

[0091]

[0092]

[0093] After the reaction is completed, the PCR or RT-PCR results are used to determine whether the strain to be tested contains the characteristic drug resistance genes floR, tet (A) and sul2 in Ningxia, and then to determine whether the E. coli in the sample to be tested is resistant to florfenicol, tetracycline, and sulfamethoxazole. If the test result is positive, the E. coli is very likely to be resistant, and the antimicrobial drug corresponding to the positive result needs to be replaced with another type of antimicrobial drug.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer set for detecting characteristic drug resistance genes of Escherichia coli, characterized in that: The drug-resistant genes include floR, tet(A) and sul2, and the primer set includes a PCR primer pair for detecting the sequence of the gene floR as shown in SEQ ID NO.1 and SEQ ID NO.2, a PCR primer pair for detecting the sequence of the gene tet(A) as shown in SEQ ID NO.3 and SEQ ID NO.4, and a PCR primer pair for detecting the sequence of the gene sul2 as shown in SEQ ID NO.5 and SEQ ID NO.6; or includes a RT-PCR primer pair for detecting the sequence of the gene floR as shown in SEQ ID NO.7 and SEQ ID NO.8, a RT-PCR primer pair for detecting the sequence of the gene tet(A) as shown in SEQ ID NO.9 and SEQ ID NO.10, and a RT-PCR primer pair for detecting the sequence of the gene sul2 as shown in SEQ ID NO.11 and SEQ ID NO.

12.

2. The primer set according to claim 1, characterized in that The primer set is a primer set used to detect characteristic drug-resistant genes of Escherichia coli from animal sources in Ningxia.

3. A kit for detecting characteristic drug resistance genes of Escherichia coli, characterized in that: Comprising the primer set according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The kit is a kit for detecting characteristic drug-resistant genes of Escherichia coli from animal sources in Ningxia.

5. The kit according to claim 3 or 4, characterized in that The kit is a PCR detection kit, which also includes a PCR detection reagent; or the kit is a RT-PCR detection kit, which also includes a RT-PCR detection reagent.

6. Use of the primer set according to claim 1 or 2 or the kit according to any one of claims 3 to 5 in rapid screening of drug-resistant phenotypes of Escherichia coli.

7. The use according to claim 6, characterized in that: The Escherichia coli is an animal-derived Escherichia coli from Ningxia that is resistant to florfenicol, tetracycline, and sulfamethoxazole.

8. A method for rapid screening of drug-resistant phenotypes of Escherichia coli, characterized in that: The primer set according to claim 1 or 2 or the kit according to any one of claims 3 to 5 is used to screen the drug resistance phenotype of Escherichia coli; the drug resistance phenotype is resistance to florfenicol, tetracycline and sulfamethoxazole.

9. The method according to claim 8, characterized in that The method is a PCR detection method, and the PCR reaction system is: The PCR reaction conditions were:

10. The method according to claim 8, characterized in that The method is an RT-PCR detection method, and the RT-PCR reaction system is: RT-PCR reaction conditions are:

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