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Composition for detecting methicillin-resistant and/or erythromycin-resistant staphylococcus aureus

A methicillin- and staphylococcus-resistant technology is applied in the field of mPCR detection of methicillin- and/or erythromycin-resistant Staphylococcus aureus resistance genes, and can solve the problems of complicated operation, false resistance or false sensitivity, Problems such as missed detection, to achieve the effect of simple operation, short detection time, saving labor and financial resources

Inactive Publication Date: 2014-12-10
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
However, the automatic microbial identification and drug sensitivity system 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 sensitivity test cannot directly evaluate bacterial drug resistance The degree of harm to the human body

Method used

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  • Composition for detecting methicillin-resistant and/or erythromycin-resistant staphylococcus aureus
  • Composition for detecting methicillin-resistant and/or erythromycin-resistant staphylococcus aureus
  • Composition for detecting methicillin-resistant and/or erythromycin-resistant staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] ⑴ Primer design, synthesis and kit assembly:

[0070] Primers were designed for the species-specific heat-resistant nuclease nuc gene of Staphylococcus aureus, the mecA gene of MRSA drug resistance determinant, and the two common drug resistance genes ermA and ermC genes that are resistant to erythromycin. The sequence of the primers and the length of the amplified fragments determined in this embodiment are shown in Table 2:

[0071] Table 2

[0072]

[0073]

[0074] On this basis, a test kit is designed. The kit includes Taq DNA polymerase at a concentration of 5U / μL and PCR reaction solution; among them, the PCR reaction solution contains 10mM Tris·HCl, 50mM KCl, 25mM MgCl 2 , DNTPs (dATP, dGTP, dCTP and dTTP) 2.5mM each and the above 4 pairs of Staphylococcus aureus primer pairs (concentrations shown in Table 2).

[0075] ⑵mPCR:

[0076] Using the multiple primers designed in the above step (1) and the corresponding kits to carry out PCR amplification includes the followin...

Embodiment 2

[0105] Example 2. Specificity test

[0106] Extract DNA from erythromycin-resistant MRSA strains carrying ermA, ermC genes, Escherichia coli, Salmonella, Listeria monocytogenes, Vibrio parahaemolyticus, Enterococcus faecalis, Pseudomonas aeruginosa (see Table 1) according to the examples 1 The established method was used for quadruple PCR amplification, and the results of electrophoresis analysis showed that only the erythromycin-resistant MRSA strains carrying ermA and ermC genes detected specific amplified bands, and none of the other strains had amplified bands ( image 3 ). DHPLC analysis results such as Figure 4 , It can be seen that only erythromycin-resistant MRSA strains carrying ermA, ermC genes have detected positive absorption peaks, and the four absorption peaks are consistent with the size of the amplified fragments, indicating that the established DHPLC detection method is effective for pathogenic Staphylococcus aureus, MRSA And erythromycin-resistant Staphylococcu...

Embodiment 3

[0107] Embodiment 3. Detection sensitivity test

[0108] The erythromycin-resistant MRSA strain carrying the ermA, ermC gene was inoculated into TSB medium, cultured at 36±1°C for 24 hours, and the number of bacteria in the enrichment solution was measured by the gradient dilution method or counting method to be 2.3×10 8 cfu / ml, and perform 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Dilution of, the number of bacteria is 2.3×10 7 cfu / ml, 2.3×10 6 cfu / ml, 2.3×10 5 cfu / ml, 2.3×10 4 cfu / ml, 2.3×10 3 cfu / ml, 2.3×10 2 cfu / ml bacterial solution, for 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 DNA extraction and quadruple PCR-electrophoresis and DHPLC analysis were performed on the bacterial liquid.

[0109] mPCR-electrophoresis results such as Figure 5 As shown, the lowest detection limit of electrophoresis detection method is 10 -5 (I.e. 2.3×10 3 cfu / ml).

[0110] The results of mPCR-DHPLC are as follows Image 6 As shown, the lowest detection limit of DHPLC method is 10 -5 (I.e. 2.3×10 3 cf...

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Abstract

The invention provides a composition for detecting methicillin-resistant and / or erythromycin-resistant staphylococcus aureus. The composition comprises a primer pair SEQ ID NO.1 / 2, a primer pair SEQ ID NO.3 / 4, a primer pair SEQ ID NO.5 / 6 and a primer pair SEQ ID NO.7 / 8. When the staphylococcus aureus is subjected to bacterial genus identification by using the composition disclosed by the invention as well as a corresponding detection method, MRSA and erythromycin drug-resistant genes can be simultaneously detected. The composition has the advantages of rapidness, high throughput and the like, and the requirement for rapidly and accurately detecting microbes in food is met. By utilizing an mPCR-DHPLC / electrophoresis method established by the invention, the staphylococcus aureus, MRSA and erythromycin-resistant staphylococcus aureus in samples can be simultaneously identified through one time of detection. Moreover, by using the composition, the detection consumes less time, is easy and rapid to operate and suitable for rapid detection requirement and can save lots of labor force and financial resources.

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 foods and biological products, especially for methicillin-resistant and / or erythromycin-resistant Staphylococcus aureus genes mPCR detection. technical background [0002] In recent years, with the extensive use of antibiotics and the abuse of veterinary drugs in the process of animal feeding, the isolation rate of Methicillin Resistant Staphylococcus aureus (MRSA) and erythromycin-resistant Staphylococcus aureus in food and animals Keep rising. The drug-resistant strains of Staphylococcus aureus in foods are difficult to treat due to their drug resistance when infecting humans and are difficult to cure; their drug-resistant factors will also spread and spread between strains, causing the incidence of drug-resistant strains to continue to rise and serious harm . Drug-resistant strain...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11
CPCC12Q1/689C12Q1/686C12Q2537/143C12Q2565/125C12Q2565/137
Inventor 郑秋月孙瑶李一尘苏明明战晓微
Owner 郑秋月
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