Fluorescence quantitative PCR method for fast detecting Campylobacter jejuni macrolide drug resistant mutational site

Abstract

The invention belongs to the biotechnical field, relating to a method for detecting drug-resistant related gene mutation of campylobacter jejuni macrolide drugs. The method makes use of a realtime fluorescence quantitative PCR TaqMan probe technology to design a specific primer and a probe according to a nucleic acid sequence of a V zone inside a campylobacter jejuni macrolide drug-resistant mutation zone 23S rDNA; moreover, through optimizing a TaqMan realtime fluorescence quantitative PCR reaction system and conditions, related drug-resistant mutational sites of target genes are accurately detected. The method can realize quick, accurate and simultaneous detection of two main drug-resistant mutational sites of campylobacter jejuni macrolide type, and has better specificity and higher sensitivity than a related molecular biology detection method reported at present; moreover, the method has simple operation, high accuracy and quick speed compared with the prior sequencing and drug sensitive experimental method. The method provides an effective technical means for molecular epidemiological monitoring of macrolide campylobacter jejuni resistance.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue detection in animals, and specifically relates to a method for detecting gene variation related to resistance to macrolides in Campylobacter jejuni by using fluorescent quantitative polymerase chain method, specifically detecting the 2074 and 23S rDNA in Campylobacter jejuni Whether the gene at position 2075 is mutated. Background technique [0002] Campylobacteriosis is a general term for a series of diseases caused by bacterial infection of the genus Campylobacter. There are many species of Campylobacter, among which Campylobacter jejuni and Campylobacter coli are Gram-negative, highly motile, microaerophilic and thermophilic bacteria, which are now recognized as the The main pathogens of human acute bacterial enteritis in various places. Studies have shown that in some developed countries, the diarrhea caused by Campylobacter jejuni infection is even 2-7 times higher than that...

AI Technical Summary

Problems solved by technology

However, this method involves processes such as PCR amplification, preparation of linear probe gel strips, and reverse hybridization, which requires high technical requirements for operation, and there are too many influencing factors in the reaction process, poor stability, and inconvenient. fast

[0010] 2005, Vacher (Vacher S, Menard A, Bernard E, Santos A, Megraud F. Detection of mutations associated with macrolide resistance in thermophilic Campylobacter spp. by real-timePCR. Microb Drug Resist. 2005 Spring; 11(1): 40- 7) et al established a real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) method to detect the macrolide resistance mutation of Campylobacter thermophiles, they were by comparing wild-type and mutant gene amplification products The difference between the melting curve and the Tm value is used to determine whether there is a mutation in the bacterial gene in the sample, so it is difficult to get rid of the common problem of DNA-binding dyes, that is, the interpretation of the test results is not intuitive enough, and it must be combined with the melting curve. The specificity and accuracy of the test are still to be determined. improve

At the same time, they overcome the shortcomings of traditional sequencing techniques such as time-consuming, cumbersome experiments, and professional comparison of sequences.

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