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Kit and detection method for detecting eight drug-resistant genes of Staphylococcus aureus

A drug-resistant gene and Staphylococcus technology, which is applied in the field of high-resolution melting curve HRM analysis and fluorescent PCR, can solve the problems that the research of multiple fluorescent PCR detection has not been reported, so as to avoid cross-contamination, simplify the detection process, and shorten the detection time Effect of Cycle Time and Inspection Cost

Active Publication Date: 2017-05-24
江苏睿玻生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For various drug resistance genes of Staphylococcus aureus, researchers have carried out multiple conventional PCR and multiplex fluorescent PCR methods, but the research of multiplex fluorescent PCR detection for these eight main drug resistance genes at the same time has not been reported yet

Method used

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  • Kit and detection method for detecting eight drug-resistant genes of Staphylococcus aureus
  • Kit and detection method for detecting eight drug-resistant genes of Staphylococcus aureus
  • Kit and detection method for detecting eight drug-resistant genes of Staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Use fluorescent PCR of the present invention in conjunction with HRM analysis technology to detect eight kinds of drug-resistant genes of Staphylococcus aureus

[0067] DNA extraction and PCR reaction

[0068] Take 1.5 mL of the culture and centrifuge at 10,000 rpm for 2 min; add 500 μL of TE buffer to the precipitate, resuspend it by pipetting repeatedly, add 30 μL of 10% SDS and 15 μL of proteinase K, mix well, and incubate at 37°C for 1 h; LNaCl, mix well, then add 80ul CTAB / NaCl solution, mix well and then incubate at 65°C for 10min; add an equal volume of phenol / chloroform / isoamyl alcohol and mix well, centrifuge for 4-5min, transfer the supernatant to a In a new tube, add 0.8 times the volume of isopropanol, and mix gently until the DNA precipitates; after the precipitate is washed with 1 mL of 70% ethanol, add TE solution to dissolve the precipitate, and then take 1 μL of the extract and add it to the following reaction system:

[0069]

[0070] Carry out PCR...

Embodiment 2

[0076] The present invention can simultaneously detect eight kinds of drug-resistant gene kits of Staphylococcus aureus, wherein the reaction system in the non-labeled fluorescent PCR amplification process consists of the following components:

[0077]

[0078] Wherein said upstream primers include:

[0079] The upstream primer of methicillin-resistant gene mecA: GCAATCGCTAAAGAACTA

[0080] erythromycin resistance gene ermc upstream primer: TGCCATTGAAATAGACCA

[0081] TetM upstream primer for tetracycline resistance gene: AAAATCCGCACCCCTCTAC

[0082] Aminoglycoside resistance gene aph3 upstream primer: AAAATACCGCTGCGTAAA

[0083] Disinfectant resistance gene qacA / qacB upstream primer: GGGTCGTGTTATTAGGCA

[0084] Vancomycin resistance gene vanA upstream primer: TGAATCGGCAAGACAATA

[0085] Mupirocin resistance gene mupA upstream primer: TCACGAATACGCACCAAG

[0086] The upstream primer of drug resistance accessory gene femB: CGAATCGTGGTCCAGTAA

[0087] Described downstrea...

Embodiment 3

[0099] A detection method of the present invention comprises the following steps:

[0100] A, according to eight kinds of Staphylococcus aureus drug-resistant gene design primer pairs;

[0101] B. After extracting the sample DNA, use the designed primer pair to perform fluorescent PCR amplification;

[0102] C. Use a fluorescent quantitative PCR instrument with an HRM module to perform HRM analysis on the PCR amplification product, determine the Tm value of the amplification product, and then determine the result;

[0103] Wherein the fluorescent PCR amplification reaction system in step B is made up of the following components:

[0104]

[0105]

[0106] Wherein said upstream primers include:

[0107] The upstream primer of methicillin-resistant gene mecA: GCAATCGCTAAAGAACTA

[0108] erythromycin resistance gene ermc upstream primer: TGCCATTGAAATAGACCA

[0109] TetM upstream primer for tetracycline resistance gene: AAAATCCGCACCCCTCTAC

[0110] Aminoglycoside resist...

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Abstract

The invention discloses a kit for detecting eight staphylococcus aureus drug-resistance genes and a detection method. The kit is characterized in that a reaction system in a non-marked fluorescence PCR amplification process consists of an HRM reaction pre-mixing liquid, a dNTPs mixed liquid, Tag polymerase, an upstream primer, a downstream primer, fluorochrome, a DNA template and water. The detection method is characterized by comprising the following steps: A, designing a primer pair according to the eight staphylococcus aureus drug resistance genes; B, extracting sample DNA, and performing fluorescence PCR amplification according to the designed primer pair; and C, performing HRM analysis on a PCR amplification product by using a fluorogenic quantitative PCR with an HRM module, confirming the Tm value of the amplification product, and judging the result. The invention aims at providing a method which is simple, convenient and rapid to operate and low in cost and is used for detecting eight drug resistance genes by using multi-fluorescence PCR, and the detection result is accurate.

Description

technical field [0001] The invention relates to high-resolution melting curve HRM analysis and fluorescent PCR technology. The method is a multiple fluorescent PCR detection kit that can simultaneously detect eight drug-resistant genes of Staphylococcus aureus based on the high-resolution melting curve HRM analysis technology. The eight resistance genes include methicillin resistance gene mecA, erythromycin resistance gene ermc, tetracycline resistance gene tetM, aminoglycoside resistance gene aph3, disinfectant resistance gene qacA / qacB, vancomycin resistance Drug gene vanA and mupirocin resistance gene mupA and drug resistance auxiliary gene femB. Background technique [0002] Due to the use of antibiotics, bacteria are constantly mutating for their own development, forming bacterial resistance to antibacterial drugs, making originally effective antibacterial drugs less effective or even completely ineffective when encountering infections caused by drug-resistant bacteria....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2537/143C12Q2527/107
Inventor 蔡先全鱼海琼岳巧云柏建山李蓉邱德义赵美转张磊萧绮倩
Owner 江苏睿玻生物科技有限公司
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