Composition used for detection of methicillin-resistant and/or tetracycline-resistant staphylococcus aureus

A methicillin and staphylococcus-resistant technology, applied in the field of mPCR detection of methicillin- and/or tetracycline-resistant Staphylococcus aureus resistance genes, can solve the problems of increased risk, superinfection, unreasonable, etc., and achieve savings The effect of labor and financial resources, simple operation, and short time-consuming inspection

Inactive Publication Date: 2014-12-17
郑秋月 +4
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Tetracycline is a broad-spectrum bacteriostatic antibiotic, long-term, irrational, and abuse can not only cause double infection, but also accelerate the emergence of drug-resistant bacteria. It is reported in China that the resistance rate of food-borne Staphylococcus aureus to tetracycline is close to 50%. In animal breeding, tetracycline is often mixed into feed, resulting in a higher resistance rate of animal-derived bacteria to tetracycline
The emergence of such a large number of drug-resistant strains is often ignored by people, but its harm is extremely serious. On the one hand, it can cause the failure of animal disease prevention and control, and on the other hand, there is a risk that a large number of animal-derived drug-resistant bacteria will be transferred to humans through the food chain. greatly increase

Method used

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  • Composition used for detection of methicillin-resistant and/or tetracycline-resistant staphylococcus aureus
  • Composition used for detection of methicillin-resistant and/or tetracycline-resistant staphylococcus aureus
  • Composition used for detection of methicillin-resistant and/or tetracycline-resistant staphylococcus aureus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] (1) Primer design, synthesis and kit assembly:

[0069] Primers were designed for the species-specific gene thermostable nucase nuc gene, MRSA resistance determinant mecA gene and tetracycline resistance determinant tetM of Staphylococcus aureus. The present embodiment determines that the primer sequences and amplified fragment lengths used for detection are shown in Table 2:

[0070] Table 2

[0071]

[0072]

[0073] On this basis, a kit for detection is 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 , dNTP (dATP, dGTP, dCTP and dTTP) 2.5 mM each and the above three pairs of primers for Staphylococcus aureus (concentrations are shown in Table 2).

[0074] (2) mPCR:

[0075] Use the multiple primers designed in the above step (1) and the corresponding kit to carry out PCR amplification, including the following steps:

[0076] ①Prepara...

Embodiment 2

[0104] Embodiment 2. Specificity test

[0105] Genomic DNA was extracted from all the reference strains in Table 1, and then using the genomic DNA as a template, the method established in Example 1 was used for detection and analysis. The results of mPCR-electrophoresis are attached image 3 As shown, among the test strains, only the corresponding bands were detected for Staphylococcus aureus, and the rest of the strains showed negative test results. The detection result of the mPCR-electrophoresis method established in Example 1 has high specificity and no false positive and false negative results.

[0106] The results of mPCR-DHPLC are attached Figure 4 As shown, among the test strains, only Staphylococcus aureus detected the corresponding absorption peak, and the rest of the strains showed negative test results. The PCR-DHPLC detection method established in Example 1 has high specificity and no false positive and false negative results.

Embodiment 3

[0107] Embodiment 3 detection sensitivity test

[0108] Inoculate Staphylococcus aureus ATCC51153 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 6 cfu / ml, and the enrichment solution was carried out 10 times -2 、10 -3 、10 -4 Gradual dilution, the number of bacteria obtained was 5.4×10 5 cfu / ml, 5.4×10 4 cfu / ml, 5.4×10 3 cfu / ml, 5.4×10 2 The cfu / ml bacterial liquid was extracted and analyzed respectively, and the results of mPCR-electrophoresis / DHPLC are attached Figure 5 and 6 As shown, it shows that the nuc gene can be detected as low as 5.4×10 3 cfu / ml.

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Abstract

The invention provides a composition used for detection of methicillin-resistant and / or tetracycline-resistant staphylococcus aureus. The composition used for detection of the methicillin-resistant and / or tetracycline-resistant staphylococcus aureus comprises a primer pair shown in SEQ ID No.1 / 2 and at least one of primer pairs shown in SEQ ID NO.3 / 4 and SEQ ID NO.5 / 6; meanwhile, an mPCR-DHPLC / electrophoresis detection kit and method are further constructed on the basis, and pathogenic staphylococcus aureus, MRSA and tetracycline-resistant staphylococcus aureus drug resistant strains in food are detected; and bacterial strain identification is carried out on staphylococcus aureus by utilizing the composition and the method which are disclosed by the invention, and MRSA and a tetracycline-resistant gene can be detected at the same time, so that staphylococcus aureus, MRSA and erythromycin-resistant staphylococcus aureus in a sample can be identified at the same time during one-step detection. The composition used for detection of methicillin-resistant and / or tetracycline-resistant staphylococcus aureus has the advantages of high speed and high throughput; and meanwhile, detection time is short, operation is simple, labour and cost can be greatly saved, and the rapid detection requirement can be met.

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 Staphylococcus aureus in food and biological products, in particular for mPCR detection of drug-resistant genes of methicillin-resistant and / or tetracycline-resistant Staphylococcus aureus . technical background [0002] Antimicrobial resistance of food-borne bacteria has gradually attracted people's attention in recent years. The World Health Organization has made "fighting antimicrobial resistance" the theme of World Health Day 2011. Since 1997, international organizations such as the Food and Agriculture Organization of the United Nations (FAO), the World Health Organization (WHO) and the World Organization for Animal Health (OIE) have held meetings for many times to discuss the impact of antimicrobial drugs on human medical and public health after they are used in food animals. Global guidelines devel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11
CPCC12Q1/686C12Q1/689C12Q2537/143C12Q2565/137C12Q2565/125
Inventor 郑秋月于珂高鹭马惠蕊战晓微
Owner 郑秋月
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