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Compositions for the detection of drug resistance of staphylococcus aureus

A Staphylococcus, drug-resistant technology, applied in the field of PCR detection of Staphylococcus aureus drug-resistant genes, can solve the problems of pseudo-sensitivity, pseudo-drug resistance, and the high price of automatic biochemical identification instruments, so as to prevent pollution, The effect of good applicability

Inactive Publication Date: 2016-06-15
中华人民共和国辽宁出入境检验检疫局
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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; and the results of the in vitro drug susceptibility test cannot be used to evaluate bacteria The harm of drug resistance to the human body

Method used

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  • Compositions for the detection of drug resistance of staphylococcus aureus
  • Compositions for the detection of drug resistance of staphylococcus aureus
  • Compositions for the detection of drug resistance of staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] (1) Primer design, synthesis and kit assembly:

[0079] This embodiment determines the primer sequences and amplified fragment lengths used for detection as shown in Table 2-1 and Table 2-2:

[0080] table 2-1

[0081]

[0082] Table 2-2

[0083]

[0084]

[0085] Among them: FAM (Carboxyfluorescein, carboxyfluorescein), TAMRA (Carboxytetramethylrhodamine, carboxytetramethylrhodamine);

[0086] On this basis, a real-time fluorescent PCR detection kit is designed. The kit includes TaqDNA polymerase at a concentration of 5 U / μL, 10 μmol / L dNTP and 10×PCR buffer; 20 μmol / L of the above six primer pairs, and 20 μmol / L of the above six probes.

[0087] (2) Establishment of real-time fluorescent PCR detection method:

[0088] This detection method uses the detection kit established in this embodiment, comprising the following steps:

[0089] ①Preparation of the sample to be tested: the DNA genome of the sample to be tested is prepared by the kit extraction metho...

Embodiment 2

[0108] Embodiment 2, specificity test

[0109] The nuc gene was used as the specific identification gene of Staphylococcus aureus, the mecA gene was used as the MRSA identification gene, the ermA and ermC genes were used as the target genes for the detection of erythromycin-resistant strains, and the aacA-aphD, aph(3')-Ⅲa The gene is used as the target gene for the detection of aminoglycoside antibiotic-resistant strains. According to the method established in Example 1, real-time fluorescent PCR detection is performed on Staphylococcus aureus drug-resistant strains, sensitive strains, sensitive strains of other species, and blank controls. Among the detection results of nuc, mecA, ermA, ermC, aacA-aphD, and aph(3')-Ⅲa genes, only the drug-resistant strain of Staphylococcus aureus detected the increase of fluorescence signal, while other sensitive strains and blank control were all See growth with fluorescent signal.

[0110] The specific detection results of real-time fluore...

Embodiment 3

[0112] Embodiment 3, detection sensitivity test

[0113] Take Staphylococcus aureus ATCC51153 (carrying mecA, ermA, aph(3')-Ⅲa, aacA-aphD genes), and Staphylococcus aureus isolates (carrying ermC gene) (see Table 1) DNA template in the ultraviolet spectrophotometer Measure the content of the DNA template in the sample mentioned above, with a 10-fold gradient from 10 -1 to 10 -8 Dilute, take DNA templates of different dilutions for real-time PCR amplification, and finally determine that the detection limit of nuc gene and mecA gene by real-time fluorescent PCR detection method is 2.3×10 2 cfu / ml, ermA gene, ermC gene, aph(3′)-Ⅲa gene, aacA-aphD gene detection limit is 2.3×10 3 cfu / ml.

[0114] The test results are shown in Figure 2.

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Abstract

The invention provides a composition for detecting the drug resistance of staphylococcus aureus. The composition comprises primer-probe groups for PCR amplification, wherein the primer-probe groups comprise SEQ ID NO.1 / 2 and SEQ ID NO.3 as well as at least one of SEQ ID NO.4 / 5 / 6, SEQ ID NO.7 / 8 / 9, SEQ ID NO.10 / 1 / 12, SEQ ID NO.13 / 14 / 15 and SEQ ID NO.16 / 17 / 18. According to the composition and a detection method, drug-resistant genes can be effectively detected and analyzed while the staphylococcus aureus is subjected to strain identification; the detection and drug resistance analysis of the staphylococcus aureus, MRSA (methicillin-resistant staphylococcus aureus), erythromycin-resistant staphylococcus aureus and aminoglycoside antibiotic-resistant staphylococcus aureus can be simultaneously finished in a one-time detection process; the composition has the advantages of quickness, high flux and the like, and meets the requirements of quick and accurate detection of microorganisms in 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 Staphylococcus aureus in food and biological products, especially for methicillin-resistant Staphylococcus aureus and erythromycin-resistant Staphylococcus aureus , and PCR detection of drug-resistant genes of Staphylococcus aureus resistant to aminoglycoside antibiotics. Background technique [0002] In recent years, with the extensive use of antibiotics in animal feeding and the abuse of veterinary drugs, methicillin-resistant Staphylococcus aureus (MRSA), aminoglycoside antibiotic-resistant Staphylococcus aureus, and erythromycin-resistant bacteria in food and animals The isolation rate of Staphylococcus aureus is increasing continuously. Drug-resistant Staphylococcus aureus strains in food, when infecting humans, are difficult to treat and cure due to their drug resistance; their drug-resistant factor...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12R1/445
CPCC12Q1/686C12Q1/689C12Q2561/101C12Q2561/113
Inventor 郑秋月那晗董振霖徐凤敏战晓微
Owner 中华人民共和国辽宁出入境检验检疫局
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