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Composition for detecting four common drug-resistant genes of escherichia coli

A drug-resistant gene and Escherichia coli technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome operation, pseudo-sensitivity, and pseudo-resistance, and save labor and financial resources, good detection effect, and short detection time

Inactive Publication Date: 2014-12-17
郑秋月 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The in vitro bacterial culture susceptibility test is cumbersome, not suitable for the detection of a large number of samples, and can only detect the drug-resistant phenotype of the strain, and the drug-resistant strains that have not yet expressed the drug-resistant gene may easily cause missed detection
Moreover, the fully automatic biochemical identification instrument for drug susceptibility identification is expensive, and the identification results are greatly affected by the operator, and it is easy to cause false drug resistance or false sensitivity; in addition, the results of the in vitro drug susceptibility test cannot evaluate bacterial drug resistance The dangers of sex to the human body

Method used

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  • Composition for detecting four common drug-resistant genes of escherichia coli
  • Composition for detecting four common drug-resistant genes of escherichia coli
  • Composition for detecting four common drug-resistant genes of escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] (1) Primer design, synthesis and kit assembly:

[0071] bla CTX-M-1 、bla CTX-M-9 、bla OXA , aadA designed primers, and the determined primer sequences and amplified fragment lengths for detection are shown in Table 2:

[0072] Table 2

[0073]

[0074]

[0075] On this basis, a kit for mPCR was designed. The kit includes Taq DNA polymerase and PCR reaction solution at a concentration of 5U / μL; the PCR reaction solution contains 10mM Tris HCl, 50mM KCl, 25mM MgCl 2 2.5 mM each of dNTP (dATP, dGTP, dCTP and dTTP) and the above 4 pairs of primers for drug-resistant Escherichia coli (concentrations are shown in Table 2).

[0076] (2) mPCR:

[0077] Use the multiple primers designed in the above steps (1) and corresponding kits to carry out PCR amplification, including the following steps:

[0078] ①Preparation of the sample to be tested: the DNA genome of the sample to be tested is prepared by the kit extraction method:

[0079] a. Food samples:

[0080] Take...

Embodiment 2

[0106] Embodiment 2. Specificity test

[0107] Separately extract Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Enterococcus faecalis, Escherichia coli (ATCC25922 sensitive strain), Escherichia coli producing CTX-M-1 type ESBLs, and producing CTX-M-9 type ESBLs Escherichia coli, produce OXA type ESBLs Escherichia coli, carry the aminoglycoside antibiotic-resistant Escherichia coli (see Table 1) DNA of aadA gene, according to the detection method established in embodiment 1, get and produce CTX-M-1 type ESBLs Escherichia coli , CTX-M-9 ESBLs-producing Escherichia coli, OXA-producing ESBLs Escherichia coli, and aminoglycoside antibiotic-resistant Escherichia coli DNA templates carrying the aadA gene were each 1 μL as Escherichia coli positive DNA templates, and 1 μL of Staphylococcus aureus, The DNA templates of Listeria monocytogenes, Salmonella typhimurium, Enterococcus faecalis, and Escherichia coli (ATCC25922 sensitive strain) were amplified by mPCR,...

Embodiment 3

[0109] Embodiment 3 detection sensitivity test

[0110] Inoculate Escherichia coli positive drug-resistant strains (5-5 in Table 1) into TSB medium, culture at 36±1°C for 24 hours, and measure the number of bacteria in the enrichment solution by gradient dilution method or counting method to be 5.4×10 8 cfu / mL, and carry out 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 The serial dilution of the obtained bacterial count was 5.4×10 7 cfu / ml, 5.4×10 6 cfu / ml, 5.4×10 5 cfu / ml, 5.4×10 4 cfu / ml, 5.4×10 3 cfu / ml, 5.4×10 2 cfu / ml, 5.4 × 10cfu / ml bacterial solution, and according to the method established in Example 1 respectively for 5.4 × 10 6 cfu / ml, 5.4×10 5 cfu / ml, 5.4×10 4 cfu / ml, 5.4×10 3 cfu / ml, 5.4×10 2 cfu / ml, 5.4×10cfu / ml bacteria liquid was extracted and detected by electrophoresis, the results are as attached Figure 5 shown. Figure 5 Display: The lowest detection limit of mPCR-electrophoresis detection is 5.4×10 3 cfu / ml.

[0111] According to the...

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Abstract

The invention provides a composition for detecting four common drug-resistant genes of escherichia coli. The composition comprises primer pairs as shown in SEQ ID NO. 1 / 2, SEQ ID NO.3 / 4, SEQ ID NO.5 / 6 and SEQ ID NO.7 / 8. An mPCR-DHPLC / electrophoresis detection kit and an mPCR-DHPLC / electrophoresis detection method are further established on the basis to detect the escherichia coli capable of producing CTX-M-1 / CTX-M-9 / OXA type ESBLs (Extended Spectrum Beta Lactamases) and aminoglycoside antibiotics resistant escherichia coli which are contained in foods. The established method can simultaneously complete the detection on four drug-resistant genes, namely blaCTX-M-1, blaCTX-M-9, blaOXA and aadA, of the escherichia coli contained in a sample during one-step detection at high specificity and high sensitivity, has the advantages of less time consumption, high flux and the like, is easy and convenient to operate and fast, can conserve a large quantity of labor power and resources, meets the requirement for fast detection and meets the requirement for fast and accurate detection of microbes contained in the foods.

Description

technical field [0001] The invention belongs to the technical field of biological detection and relates to the detection of drug-resistant genes carried by drug-resistant Escherichia coli in food and biological products. technical background [0002] Escherichia coli is a common pathogenic bacteria that can cause zoonotic diseases. Drug-resistant strains derived from food animals may pose a huge potential hazard to humans. Research on the detection of drug resistance of E. coli from food sources and animal sources is particularly important. important. Since the research on bacterial drug resistance is mainly in the field of clinical medicine, the research on drug resistance of animal-derived and food-borne bacteria started relatively late, and because bacteria are often transmitted to humans through the food chain at the same time, people pay more attention to the middle link of the food chain It focuses on the detection of bacterial pathogenicity, but ignores bacterial res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCC12Q1/686C12Q1/689C12Q2537/143C12Q2565/125C12Q2565/137
Inventor 郑秋月刘冉肖珊珊曹际娟战晓微
Owner 郑秋月
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