Multiplex PCR detection method and kit for Shiga toxin-producing Escherichia coli and application thereof

Abstract

The invention discloses a multiplex PCR detection method and kit for Shiga toxin-producing Escherichia coli and application thereof. The method and the kit thereof can simultaneously detect the genes consisting of stx1, stx2 and wzyO157 through a triplex PCR reaction, electrophoresis staining and ultraviolet visualization, subtypes consisting of stx1 and stx1c can be amplified by using a detection primer for the stx1 gene, subtypes consisting of stx2, stx2c and stx2d can be amplified by using a detection primer for the stx2 gene, and an O157 serotype strain can be identified by using a detection primer for the wzyO157 gene. According to the invention, the method and the kit can effectively detect stx and main variants thereof and can be used for distinguishing O157 from other non-O157 Escherichia coli, for directly detecting Shiga toxin-producing Escherichia coli in a food sample and for identifying a clinical strain and a food isolate.

Description

technical field [0001] The invention relates to the technical field of molecular biology applications, in particular to a Shiga toxin-producing Escherichia coli multiplex PCR detection method, kit and application. Background technique [0002] Shiga toxin-producing Escherichia coli is an important food-borne pathogen, mainly represented by Escherichia coli O157:H7. Shiga toxin-producing Escherichia coli can produce Shiga toxin, which can be transmitted by beef, chicken, fruits and vegetables, etc., causing severe food poisoning complications such as hemorrhagic enteritis and hemolytic uremic syndrome, and even acute Kidney failure and death. The main virulence factors of Shiga toxin-producing Escherichia coli are the stx1 and stx2 genes located on the phage, which encode Shiga toxin Stx1 and Stx2 respectively. [0003] Since the first report of Escherichia coli O157:H7 infection in my country in 1986, there have been two outbreaks of infectious diarrhea caused by Escherich...

AI Technical Summary

Problems solved by technology

However, the existing E. coli O157 detection method only detects the O157 marker gene, such as rfb O157 Gene (PCR method for rapid detection of multiple pathogenic bacteria in food, SN / T 1869-2007), cannot further determine whether E. coli O157 is virulent

In addition, because the nucleotide sequences of different subtypes of the stx1 and stx2 genes are quite different, the variants of the stx1 gene are divided into stx according to the difference in gene sequence. 1 , stx 1c , stx 1d , and variants of the stx2 gene can be classified into stx 2 , stx 2c , stx 2d , stx 2e and stx 2f , it is difficult to simultaneously amplify all genotypes or major genotypes with one universal primer

Furthermore, the more primers used in a reaction, the easier it is for the primers to interfere with each other, forming primer hairpin loops or primer dimers, which makes PCR amplification difficult and the detection sensitivity is greatly reduced.

Therefore, for the PCR detection of Shiga toxin-producing Escherichia coli, it is necessary to design a method that can complete the detection of Shiga toxin coding gene and O157 characteristic gene in one PCR reaction, and can identify the pathogenic bacteria and detection of O157 serotype Escherichia coli. It is difficult to identify stx1 and stx2 genes and their common subtypes, specific and broad-spectrum multiplex PCR primers

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