Multiple qPCR (quantitative Polymerase Chain Reaction) method for rapidly detecting five kinds of diarrheogenic Escherichia coli, kit and application thereof

Abstract

The invention discloses a kit for rapidly detecting five kinds of diarrheogenic Escherichia coli through multiple qPCR (quantitative Polymerase Chain Reaction). The kit comprises primer and probe groups capable of respectively performing specific amplification on eae genes of EPEC or EHEC, escV genes of EPEC or EHEC, bfpB genes of EPEC, stx1 or stx2 genes of EHEC lt, sth or stp genes of ETEC, aggR, astA or pic genes of EAEC and internal quality control IAC genes. Moreover, the invention further discloses a method and application for rapidly detecting the five kinds of diarrheogenic Escherichiacoli by using the kit. A method for simultaneously detecting the five kinds of diarrheogenic Escherichia coli in foods in the same reaction tube by virtue of fourteen real-time fluorescence quantitative PCR is realized, all characteristic genes in the national standard of the five DEC (diarrheogenic Escherichia coli) can be totally detected, and phenomena such as missing detection and false negative are avoided. The kit has the advantages of being short in detection time, simple and convenient to operate, easily decipherable in results, high in sensitivity, excellent in specificity and the like.

Description

technical field [0001] The invention relates to the technical field of biological detection. More specifically, the present invention relates to a multiplex qPCR rapid detection method, kit and application of five kinds of diarrhea-causing Escherichia coli. Background technique [0002] Diarrheogenic Escherichia coli (DEC) is a general term for a type of Escherichia coli that can cause diarrhea in the human body. Common diarrhea-causing Escherichia coli mainly include enteropathogenic Escherichia coli ( Enteropathogenic Escherichia coli (EPEC), Shigatoxin-producing Escherichia coli (STEC) including enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (Enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), enteroinvasive Escherichia coli (EIEC), which can cause human disease by contaminating food. Diarrheogenic Escherichia coli infection is more common in infants and young children. In recent years, the detection rate in a...

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Problems solved by technology

The patent "a kit for multiplex PCR detection of six types of Escherichia coli and its preparation method" (CN 105779605A) reports a method that can detect six types of Escherichia coli at the same time, but it uses ordinary multiplex PCR and then runs the gel, so Not only will there be a phenomenon that similar bands of amplified fragments cannot be distinguished, but also there will be pollution caused by opening the cover during the gel running process after PCR, and the operation steps are complicated, which has defects in practical application; the patent "diarrheic large intestine Escherichia detection kit and its detection method" (CN 102605068A) has reported the method that can detect four kinds of diarrhea-causing Escherichia coli simultaneously, and what it used is common PCR and then (DHPLC) denatured high performance liquid chromatography method, which also has the problem of pollution caused by opening the cover after PCR, and the chromatography method has high requirements for operators and instruments, and the cost is relatively high; the patent "diarrheogenic E. coli detection kit and its detection and typing method" (CN 103215358A) is a method using fluorescent quantitative PCR, but it needs to be divided into two tubes for detection, and the detection of pathogenic genes is incomplete, and it cannot better achieve the purpose of fast and easy detection

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