A primer composition for detecting drug-resistant genes and its application

Abstract

The present invention discloses a primer composition for detecting drug-resistant genes and its application, comprising primer A and primer B, wherein primer A is selected from primer pairs 1 to 2, and primer B is selected from primer pairs 3 to 5; the primer pair 1 is a primer whose nucleotide sequence is shown in SEQ ID NO.1 to 2; the primer pair 2 is a primer whose nucleotide sequence is shown in SEQ ID NO.3 to 4; the primer pair 3 is a primer whose nucleotide sequence is shown in SEQ ID NO.5 to 6; the primer pair 4 is a primer whose nucleotide sequence is shown in SEQ ID NO.7 to 8; the primer pair 5 is a primer whose nucleotide sequence is shown in SEQ ID NO.9 to 10. The primers provided by the present invention have strong specificity and high sensitivity, simple operation flow for detection, low cost, and can detect multi-drug resistance genes in large quantities, quickly and accurately from environmental samples with low nucleic acid content, greatly saving detection time and cost, and can be promoted for monitoring antibiotic pollution.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a primer composition for detecting drug-resistant genes and an application thereof. Background Art

[0002] Antibiotics are the most widely used class of drugs in clinical applications. In recent years, the overuse of antibiotics in treating human and animal infections has led to the release of a large number of antibiotics into the environment. Under the stimulation of multiple antibiotics, bacteria in the environment express multidrug resistance genes, resulting in multidrug resistance. The main function of multidrug resistance genes is to generate corresponding proteins to pump out antibiotics or protect and replace antibiotic targets. At present, multidrug resistance genes have also been found in aquatic environments, affecting the environment and human health.

[0003] The msbA gene and evgS gene are two multidrug resistance genes that are resistant to five types of antibiotics, including nitroimidazoles, fluoroquinolones, macrolides, penicillins and tetracyclines, and have a relatively high abundance in the environment. Both genes were found in Escherichia coli, and the corresponding msbA protein and evgS protein expressed by them can pump out antibiotics, thus rendering them ineffective.

[0004] In order to deal with the spread of multidrug-resistant genes and the generation of multidrug-resistant bacteria, it is particularly important to use primer pair kits to amplify multidrug-resistant genes to detect the types and abundance of multidrug-resistant genes in the environment. However, existing technologies such as PCR and drug-resistant bacteria culture methods have problems such as complex operation, low primer specificity and low amplification efficiency in detecting environmental multidrug-resistant genes. Summary of the invention

[0005] In order to solve the problems existing in the existing drug-resistant gene detection technology, the present invention provides a primer composition for detecting drug-resistant genes and its application, which can specifically bind to the multi-drug-resistant genes msbA and evgS in various environmental samples and realize their rapid detection.

[0006] The first object of the present invention is to provide a primer composition for detecting drug-resistant genes.

[0007] The second object of the present invention is to provide the use of the primer combination in detecting multi-drug resistant bacteria.

[0008] The third object of the present invention is to provide a method for detecting multi-drug resistant bacteria.

[0009] The fourth object of the present invention is to provide the use of the primer combination in the preparation of a kit for detecting multi-drug resistant bacteria.

[0010] A fifth object of the present invention is to provide a kit for detecting multi-drug resistant bacteria.

[0011] In order to achieve the above object, the present invention is implemented by the following scheme:

[0012] The technical principle of the present invention is to design primer pairs using the gene sequences of two multidrug resistance genes, msbA and evgS, and use the primer pairs and qPCR reaction solution to perform three-step amplification of the two target genes on a fluorescent quantitative PCR platform, thereby obtaining an amplification curve to determine whether the gene is present and its abundance. The concentration of the primer pairs in the system, the temperature and time required for the three-step method of denaturation, annealing and extension, and the DNA and concentration of the samples to be tested from different sources can be adjusted to detect the multidrug resistance genes msbA and evgS in a variety of environmental samples sensitively, accurately, and conveniently.

[0013] A primer composition for detecting drug-resistant genes, comprising primer A and primer B, wherein primer A is selected from primer pairs 1 to 2, and primer B is selected from primer pairs 3 to 5;

[0014] The primer pair 1 is a primer having a nucleotide sequence as shown in SEQ ID NO. 1-2;

[0015] The primer pair 2 is a primer having a nucleotide sequence as shown in SEQ ID NO.3-4;

[0016] The primer pair 3 is a primer having a nucleotide sequence as shown in SEQ ID NO.5-6;

[0017] The primer pair 4 is a primer having a nucleotide sequence as shown in SEQ ID NO.7-8;

[0018] The primer pair 5 is a primer having a nucleotide sequence as shown in SEQ ID NOs. 9 to 10.

[0019] The target gene amplified by primer pairs 1 to 2 is the msbA gene (Genbank ID: U00096.3), and the target gene amplified by primer pairs 3 to 5 is the evgS gene (Genbank ID: U00096.3).

[0020] The primer whose nucleotide sequence is shown as SEQ ID NO.1 is the upstream amplification primer of primer pair 1, and the primer whose nucleotide sequence is shown as SEQ ID NO.2 is the downstream amplification primer of primer pair 1;

[0021] The primer whose nucleotide sequence is shown in SEQ ID NO.3 is the upstream amplification primer of primer pair 2, and the primer whose nucleotide sequence is shown in SEQ ID NO.4 is the downstream amplification primer of primer pair 2;

[0022] The primer whose nucleotide sequence is shown in SEQ ID NO.5 is the upstream amplification primer of primer pair 3, and the primer whose nucleotide sequence is shown in SEQ ID NO.6 is the downstream amplification primer of primer pair 3;

[0023] The primer whose nucleotide sequence is shown in SEQ ID NO.7 is the upstream amplification primer of primer pair 4, and the primer whose nucleotide sequence is shown in SEQ ID NO.8 is the downstream amplification primer of primer pair 4;

[0024] The primer whose nucleotide sequence is shown as SEQ ID NO.9 is the upstream amplification primer of primer pair 5, and the primer whose nucleotide sequence is shown as SEQ ID NO.10 is the downstream amplification primer of primer pair 5.

[0025] The above primer sequences and nucleotide sequence variants having more than 85% (eg, 88%, 90% or 98%) homology and the same function can achieve the detection purpose of the present invention.

[0026] Preferably, the primer A is primer pair 1, and the primer B is primer pair 3.

[0027] The use of the above primer combination in detecting multi-drug resistant bacteria in the environment should also be within the protection scope of the present invention.

[0028] Preferably, the multidrug-resistant bacteria are multidrug-resistant bacteria carrying the msbA gene and the evgS gene.

[0029] Preferably, the environmental sample to be detected is a water sample.

[0030] Further preferably, the water sample includes any one or more of natural water bodies such as river water, lake water, sea water, industrial wastewater, aquaculture wastewater, domestic sewage and medical wastewater.

[0031] Further preferably, the salinity range of the water sample should be 0.1-40 g / L, and the pH range should be 6-10.

[0032] Preferably, the multidrug-resistant bacteria is Escherichia coli.

[0033] A method for detecting multi-drug resistant bacteria in an environment, using the primer combination to perform real-time fluorescence quantitative PCR amplification.

[0034] Preferably, a primer combination is used for real-time fluorescence quantitative PCR amplification; in the primer combination, primer A is primer pair 1, and primer B is primer pair 3.

[0035] Preferably, in the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentration of the primers is 6 μM to 10 μM.

[0036] More preferably, in the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentration of the primer is 10 μM.

[0037] Further preferably, the reaction system for real-time fluorescence quantitative PCR amplification contains a primer with a nucleotide sequence as shown in SEQ ID NO.1 at a final concentration of 10 μM and a primer with a nucleotide sequence as shown in SEQ ID NO.2 at a final concentration of 10 μM.

[0038] Further preferably, the reaction system for real-time fluorescence quantitative PCR amplification contains a primer having a nucleotide sequence as shown in SEQ ID NO.5 with a final concentration of 10 μM and a primer having a nucleotide sequence as shown in SEQ ID NO.6 with a final concentration of 10 μM.

[0039] Preferably, the reaction procedure of the real-time fluorescence quantitative PCR amplification is: 80-98°C, 25-35s; 80-98°C, 5-15s, 50-54°C, 5-15s, 70-74°C, 25-35s, 35-45 cycles.

[0040] More preferably, the reaction program of the real-time fluorescence quantitative PCR amplification is: 95°C, 30s; 95°C, 10s, 52°C, 10s, 72°C, 30s, 30 cycles.

[0041] The use of the above primer combination in the preparation of a kit for detecting multi-drug resistant bacteria in an environment should also be within the protection scope of the present invention.

[0042] A kit for detecting multi-drug resistant bacteria in an environment comprises the above primer combination and a real-time fluorescence quantitative PCR reaction reagent.

[0043] Preferably, the method for using the kit comprises the following steps:

[0044] S1. Obtain DNA of the sample to be tested;

[0045] S2. Using the DNA of the sample to be tested as a template, the above primer combination and real-time fluorescence quantitative PCR reaction reagent are used to prepare a reaction system, and real-time fluorescence quantitative PCR is performed to determine whether the sample to be tested contains multidrug-resistant bacteria;

[0046] The method for determining whether the sample to be tested contains multidrug-resistant bacteria is as follows: if the result shows that the Ct value is ≤35, then there are multidrug-resistant Escherichia coli bacteria in the sample to be tested; if the result shows that the Ct value is >35, then there are no multidrug-resistant Escherichia coli bacteria in the sample to be tested.

[0047] Preferably, in the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentration of the primers in the primer composition is 6 μM to 10 μM.

[0048] More preferably, in the reaction system of real-time fluorescence quantitative PCR amplification, the final concentration of the primers in the primer composition is 10 μM.

[0049] Further preferably, the reaction system for real-time fluorescence quantitative PCR amplification contains a primer with a nucleotide sequence as shown in SEQ ID NO.1 at a final concentration of 10 μM and a primer with a nucleotide sequence as shown in SEQ ID NO.2 at a final concentration of 10 μM.

[0050] Further preferably, the reaction system for real-time fluorescence quantitative PCR amplification contains a primer having a nucleotide sequence as shown in SEQ ID NO.5 with a final concentration of 10 μM and a primer having a nucleotide sequence as shown in SEQ ID NO.6 with a final concentration of 10 μM.

[0051] Preferably, the reaction procedure of the real-time fluorescence quantitative PCR amplification is: 80-98°C, 25-35s; 80-98°C, 5-15s, 50-54°C, 5-15s, 70-74°C, 25-35s, 35-45 cycles.

[0052] More preferably, the reaction program of the real-time fluorescence quantitative PCR amplification is: 95°C, 30s; 95°C, 10s, 52°C, 10s, 72°C, 30s, 30 cycles.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The primers provided by the invention have good amplification and specificity for samples with low nucleic acid content, and the amplification cycle thresholds for the multidrug resistance genes msbA and evgS reach 15 to 30.

[0055] The primers provided by the present invention have strong specificity and high sensitivity, and the operation process for detection is simple and low-cost. At the same time, they can detect multidrug-resistant genes in large quantities, quickly and accurately from environmental samples with low nucleic acid content, greatly saving detection time and cost, and can be promoted for monitoring antibiotic pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the fluorescence curve obtained by amplifying the drug-resistant genes msbA and evgS in positive samples using primer pairs SEQ ID NO. 1 to 10 through real-time fluorescence quantitative PCR.

[0057] Figure 2 This is the fluorescence curve obtained by using primer pairs SEQ ID NO.1-10 to amplify the drug-resistant genes msbA gene and evgS gene in environmental water samples using real-time fluorescence quantitative PCR.

[0058] Figure 3 This is the fluorescence curve obtained by amplifying the drug-resistant genes msbA and evgS in Staphylococcus aureus samples using primer pairs SEQ ID NO. 1 to 10 through real-time fluorescence quantitative PCR. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0060] Example 1 Determination of primers for amplification of multidrug resistance genes

[0061] 1. Experimental methods

[0062] (1) Primer design

[0063] According to the msbA gene sequence of Escherichia coli (Genbank ID: U00096.3), two pairs of amplification primers were designed. The specific sequences are as follows:

[0064] Upstream primer msbA-1-F: 5'-tctbtacgtbgcagabattcc-3' (SEQ ID NO. 1);

[0065] Downstream primer msbA-1-R: 5'-tggbttaagbagcgabaaca-3' (SEQ ID NO. 2);

[0066] Upstream primer msbA-2-F: 5′-gctdcccaadtgtcadggat-3′ (SEQ ID NO. 3);

[0067] Downstream primer msbA-2-R: 5′-tgahttacchgaacchgagc-3′ (SEQ ID NO. 4).

[0068] According to the evgS gene sequence of Escherichia coli (Genbank ID: U00096.3), three pairs of amplification primers were designed. The specific sequences are as follows:

[0069] Upstream primer evgS-1-F: 5'-cgavaagcavtctcgvattc-3' (SEQ ID NO. 5);

[0070] Downstream primer evgS-1-R: 5'-tcabtaatabcttgcbaaccaca-3' (SEQ ID NO. 6);

[0071] Upstream primer evgS-2-F: 5'-ggthttagchaggaghaacg-3' (SEQ ID NO. 7);

[0072] Downstream primer evgS-2-R: 5'-aacvgcgtgvttgtcvtcaa-3' (SEQ ID NO. 8);

[0073] Upstream primer evgS-3-F: 5'-aatdtgcagdgcgatdtgtc-3' (SEQ ID NO. 9);

[0074] Downstream primer evgS-3-R: 5′-attdtgctcdcggagdttgc-3′ (SEQ ID NO. 10).

[0075] (2) Primer amplification

[0076] Real-time fluorescence quantitative PCR amplification was performed using genomic DNA of Escherichia coli (strain number: ATCC25404) as a template and nuclease-free water as a negative sample using the designed 5 pairs of primers.

[0077] The reaction system is: template (concentration 30 ng / μL) or nucleic acid-free water negative sample, 2 μL; 10 μM upstream primer and 10 μM downstream primer (SEQ ID NO.1-2 or SEQ ID NO.3-4 or SEQ ID NO.5-6 or SEQ ID NO.7-8 or SEQID NO.9-10), 1 μL each; qPCR premix, 10 μL; nucleic acid-free water is supplemented to 20 μL.

[0078] Real-time fluorescence quantitative PCR was used for analysis, and the reaction procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 52°C for 10 s, extension at 72°C for 30 s, and 40 cycles.

[0079] 2. Experimental results

[0080] like Figure 1As shown, the upstream primer msbA-1-F (SEQ ID NO.1) and the downstream primer msbA-1-R (SEQ ID NO.2) as well as the upstream primer msbA-2-F (SEQ ID NO.3) and the downstream primer msbA-2-R (SEQ ID NO.4) have very high specificity and amplification efficiency for positive samples containing the drug-resistant gene msbA, while maintaining a low Ct value for negative samples; similarly, the upstream primer evgS-1-F (SEQ ID NO.5) and the downstream primer evgS-1-R (SEQ ID NO.6), the upstream primer evgS-2-F (SEQ ID NO.7) and the downstream primer evgS-2-R (SEQ ID NO.8) as well as the upstream primer evgS-3-F (SEQ ID NO.9) and the downstream primer evgS-3-R (SEQ ID NO.10) have very high specificity and amplification efficiency for positive samples containing the drug-resistant gene msbA, while maintaining a low Ct value for negative samples; similarly, the upstream primer evgS-1-F (SEQ ID NO.5) and the downstream primer evgS-1-R (SEQ ID NO.6), the upstream primer evgS-2-F (SEQ ID NO.7) and the downstream primer evgS-2-R (SEQ ID NO.8) NO.10), it also has very high specificity and amplification efficiency for positive samples containing the drug-resistant gene evgS, while maintaining a low Ct value for negative samples.

[0081] The above results indicate that primers msbA-1-F (SEQ ID NO.1) and msbA-1-R (SEQ ID NO.2) as well as msbA-2-F (SEQ ID NO.3) and msbA-2-R (SEQ ID NO.4) can be used to amplify the msbA gene, and primers evgS-1-F (SEQ ID NO.5) and evgS-1-R (SEQ ID NO.6), evgS-2-F (SEQ ID NO.7) and evgS-2-R (SEQ ID NO.8) as well as evgS-3-F (SEQ ID NO.9) and evgS-1-R (SEQ ID NO.10) can be used to amplify the evgS gene.

[0082] Example 2 Determination of the PCR reaction system and reaction procedure for multidrug resistance genes

[0083] 1. Experimental methods

[0084] (1) Determination of primer concentration

[0085] Escherichia coli (strain number: ATCC25404) carries the drug resistance gene msbA and the drug resistance gene evgS. Its genomic DNA was used as a template and nuclease-free water was used as a negative sample. Real-time fluorescence quantitative PCR amplification was performed using 5 pairs of primers (SEQ ID NOs. 1 to 10) obtained in Example 1 with different concentrations.

[0086] The reaction system was prepared according to the method of Example 1, except that the concentrations of the upstream primer and the downstream primer were changed to 10 μM, 8 μM, and 6 μM, respectively.

[0087] Real-time fluorescence quantitative PCR analysis was performed according to the reaction procedure of Example 1.

[0088] (2) Determination of annealing temperature

[0089] Using genomic DNA of Escherichia coli (strain number: ATCC25404) as a template and nuclease-free water as a negative sample, different annealing temperatures were set and the five pairs of primers (SEQ ID NOs. 1-10) obtained in Example 1 were used for real-time fluorescence quantitative PCR amplification.

[0090] The reaction system was prepared according to the method of Example 1.

[0091] According to the reaction procedure of Example 1, only the annealing temperature was changed to 52°C, 55°C or 58°C, and fluorescence quantitative PCR analysis was performed. According to the formula: amplification efficiency = 100% × (sample CT value-optimal positive sample CT value) / optimal positive sample CT value, the amplification efficiency of each primer pair was statistically calculated.

[0092] (3) Determination of annealing time

[0093] Using genomic DNA of Escherichia coli (strain number: ATCC25404) as a template and nuclease-free water as a negative sample, different annealing times were set and the five pairs of primers (SEQ ID NOs. 1-10) obtained in Example 1 were used for real-time fluorescence quantitative PCR amplification.

[0094] The reaction system was prepared according to the method of Example 1.

[0095] According to the reaction procedure of Example 1, only the annealing time was changed to 10s, 20s or 30s, and real-time fluorescence quantitative PCR analysis was performed. The amplification efficiency of each primer pair was statistically calculated according to the formula: amplification efficiency = 100% × (sample CT value-optimal positive sample CT value) / optimal positive sample CT value.

[0096] 2. Experimental results

[0097] Table 1 Amplification results of different primer concentrations

[0098]

[0099]

[0100] As shown in Table 1, when other conditions in the real-time fluorescence quantitative PCR reaction system remain unchanged, the optimal concentration of the primer is 10 μM.

[0101] Table 2 Amplification results at different annealing temperatures

[0102]

[0103] As shown in Table 2, when other conditions of the real-time fluorescence quantitative PCR reaction program remain unchanged, the optimal annealing temperature is 52°C.

[0104] Table 3 Amplification results at different annealing times

[0105]

[0106] As shown in Table 3, when other conditions remain unchanged, the annealing time is changed and the optimal annealing time is 20s.

[0107] The above results show that the optimal primer concentration for analyzing the multidrug resistance genes msbA and evgS by fluorescent quantitative PCR using primer pairs of SEQ ID NO.1 to 10 is 10 μM; the optimal annealing temperature is 52° C.; and the optimal annealing time is 20 s.

[0108] Example 3 Detection of multidrug resistance genes in environmental samples

[0109] 1. Experimental methods

[0110] (1) Sample collection

[0111] Coastal aquaculture seawater (salinity of 37.6 g / L, pH of 8.2), natural river water (salinity of 0.1 g / L, pH of 8.1), lake water, industrial wastewater, aquaculture wastewater, domestic sewage, and medical wastewater were collected respectively, and the bacterial DNA in the samples was extracted using a DNA extraction kit (manufacturer Shanghai Shenggong, product number B618763-0050) according to the kit instructions.

[0112] (2) Real-time fluorescence quantitative PCR

[0113] The obtained bacterial DNA was used as a template and the nuclease-free water was used as a negative sample. Real-time fluorescence quantitative PCR amplification was performed using the five pairs of primers obtained in Example 1 (SEQ ID NOs. 1 to 10).

[0114] The reaction system is: 2 μL of bacterial DNA extracted from coastal aquaculture seawater (concentration of 38 ng / μL), bacterial DNA extracted from coastal aquaculture seawater (concentration of 112 ng / μL), bacterial DNA extracted from natural river water (concentration of 40 ng / μL), or negative sample of nucleic acid-free water; 10 μM upstream primer and 10 μM downstream primer (SEQ ID NO.1-2 or SEQ ID NO.3-4 or SEQID NO.5-6 or SEQ ID NO.7-8 or SEQ ID NO.9-10), 1 μL each; qPCR premix, 10 μL; nucleic acid-free water is supplemented to 20 μL.

[0115] Real-time fluorescence quantitative PCR was used for analysis, and the reaction procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 52°C for 20 s, extension at 72°C for 30 s, and 40 cycles.

[0116] 2. Experimental results

[0117] like Figure 2 As shown, using bacterial DNA extracted from coastal aquaculture seawater at a concentration of 38 ng / μL as a template, the five pairs of primers (SEQ ID NOs. 1-10) obtained in Example 1 were used for real-time fluorescence quantitative PCR amplification, and a single fluorescence curve was amplified. Other water samples also had similar fluorescence curve results.

[0118] Table 4 Test results of different environmental water samples

[0119]

[0120]

[0121] As shown in Table 4, the primer pairs (SEQ ID NOs. 1-10) provided by the present invention can specifically and efficiently amplify the multidrug resistance genes msbA and evgS in different water environment samples, and accurately detect the multidrug resistance genes therein.

[0122] The above results show that the five pairs of primers (SEQ ID NO. 1-10) can detect the presence of multidrug resistance genes in environmental water.

[0123] Example 4 Sensitivity determination of primers for multidrug resistance genes

[0124] 1. Experimental methods

[0125] (1) DNA template serial concentration dilution

[0126] Using genomic DNA of Escherichia coli (strain number: ATCC25404) as a template, gradient dilution was performed to concentrations of 5 ng / μL, 10 ng / μL and 20 ng / μL, and nuclease-free water was used as a negative sample. Real-time fluorescence quantitative PCR amplification was performed using the five pairs of primers obtained in Example 2 (SEQ ID NOs. 1 to 10).

[0127] The reaction system is: template (concentration of 5 ng / μL, 10 ng / μL or 20 ng / μL) or nucleic acid-free water negative sample, 2 μL; 10 μM upstream primer and 10 μM downstream primer (SEQ ID NO.1-2 or SEQ ID NO.3-4 or SEQ ID NO.5-6 or SEQID NO.7-8 or SEQ ID NO.9-10), 1 μL each; qPCR premix, 10 μL; nucleic acid-free water is supplemented to 20 μL.

[0128] Real-time fluorescence quantitative PCR was used for analysis, and the reaction procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 52°C for 20 s, extension at 72°C for 30 s, and 40 cycles.

[0129] 2. Experimental results

[0130] Table 5 Amplification results of different template concentrations

[0131]

[0132]

[0133] As shown in Table 5, even if the DNA template concentration is as low as 5 ng / μL, the Ct values ​​detected by the five pairs of primers (SEQ ID NOs. 1 to 10) are still below 27, indicating that the primers and detection methods provided by the present invention have good sensitivity and can efficiently amplify the multidrug resistance genes msbA and evgS in low-concentration DNA template samples.

[0134] Example 5 Primer specificity determination of multidrug resistance genes

[0135] 1. Experimental methods

[0136] (1) DNA template serial concentration dilution

[0137] Using genomic DNA of Staphylococcus aureus (strain number: ATCC6538) as a template at a concentration of 39 ng / μL and nuclease-free water as a negative sample, real-time fluorescence quantitative PCR amplification was performed using the five pairs of available primers (SEQ ID NOs. 1 to 10) obtained in Example 2.

[0138] The reaction system is: template or nucleic acid-free water negative sample, 2 μL; 10 μM upstream primer and 10 μM downstream primer (SEQID NO.1-2 or SEQ ID NO.3-4 or SEQ ID NO.5-6 or SEQ ID NO.7-8 or SEQ ID NO.9-10), 1 μL each; qPCR premix, 10 μL; nucleic acid-free water is supplemented to 20 μL.

[0139] Real-time fluorescence quantitative PCR was used for analysis, and the reaction procedure was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 52°C for 20 s, extension at 72°C for 30 s, and 40 cycles.

[0140] 2. Experimental results

[0141] Table 6 Amplification results of Staphylococcus aureus samples

[0142]

[0143] As shown in Table 6 and Figure 3 As shown, the CT values ​​of the five pairs of primers (SEQ ID NO.1-10) for amplifying different concentrations of Staphylococcus aureus genomic DNA were all above 35, and the test results were all negative, and were not interfered by the DNA in other strains, indicating that these five pairs of primers only amplified Escherichia coli containing the multidrug resistance genes msbA and evgS, with high specificity.

[0144] Example 6 A kit for detecting multidrug-resistant bacteria in the environment

[0145] 1. Composition

[0146] A kit for detecting multi-drug resistant bacteria comprises the five pairs of primers (SEQ ID NOs. 1 to 10) of Example 1 and a real-time fluorescence quantitative PCR reaction reagent, wherein the real-time fluorescence quantitative PCR reaction reagent may be a qPCR premix.

[0147] 2. Usage

[0148] (1) Extraction of DNA from test samples

[0149] This kit does not specify the extraction method for the DNA of the sample to be tested. Generally, the DNA of the sample to be tested can be extracted using conventional laboratory methods (phenol-chloroform extraction method) or commercially available kits.

[0150] (2) Preparation of real-time fluorescence quantitative PCR reaction system

[0151] 2 μL of sample DNA to be tested; 10 μL of 10 μM upstream primer and 10 μM downstream primer (SEQ ID NO.1-2 or SEQ ID NO.3-4 or SEQ ID NO.5-6 or SEQ ID NO.7-8 or SEQ ID NO.9-10), each; 10 μL of qPCR premix; supplement to 20 μL with nuclease-free water.

[0152] (3) Setting up the real-time fluorescence quantitative PCR reaction program

[0153] Pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 52°C for 20 s, extension at 72°C for 30 s, and cycle 40 times.

[0154] (4) Interpretation of results

[0155] The method for determining whether the sample to be tested contains multidrug-resistant bacteria is: if the result shows that the Ct value is ≤35, then there are multidrug-resistant Escherichia coli in the sample to be tested; if the result shows that the Ct value is >35, then there are no multidrug-resistant Escherichia coli in the sample to be tested.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. 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 claims of the present invention.

Claims

1. A method for detecting multidrug-resistant bacteria in the environment, It is characterized in that Perform real-time fluorescence quantitative PCR amplification using the primer combination; The primer composition comprises primer A and primer B, wherein primer A is primer pair 1, and primer B is primer pair 3; the primer pair 1 is a primer having a nucleotide sequence as shown in SEQ ID NO. 1-2; The primer pair 3 is a primer having a nucleotide sequence as shown in SEQ ID NO.5-6; The detection environment is to detect water samples, and the water samples are coastal aquaculture seawater or natural river water; The multi-drug resistant bacteria are Escherichia coli carrying the msbA gene and the evgS gene.

2. The method according to claim 1, It is characterized in that In the reaction system of real-time fluorescence quantitative PCR amplification, the final concentration of the primers in the primer composition is 6 μM to 10 μM.

3. The method according to claim 2, It is characterized in that The reaction procedure of the real-time fluorescence quantitative PCR amplification is: 80-98° C., 25-35 s; 80-98° C., 5-15 s, 50-54° C., 5-15 s, 70-74° C., 25-35 s, 35-45 cycles.

4. Use of the primer combination described in claim 1 in detecting multi-drug resistant bacteria in the environment, It is characterized in that The detection environment is to detect water samples, and the water samples are coastal aquaculture seawater or natural river water; The multi-drug resistant bacteria are Escherichia coli carrying the msbA gene and the evgS gene.

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