PCR detection method of enterohemorrhagic escherichia coli in vegetables

Abstract

The invention discloses a PCR detection method of enterohemorrhagic escherichia coli in vegetables, in particular to an optimized micro-droplet digital PCR detection reaction system. The micro-dropletdigital PCR detection method comprises the following steps: a, preparing a sample, and performing the enriched culture; b, extracting a bacterial template DNA; and c, adopting the extracted DNA as atemplate, performing the real-time fluorescent PCR by adding the primer and a probe with fluorescent dye as well as enzyme reaction liquid, after the reaction is ended, detecting a fluorescent signalvalue of all micro droplets, setting a threshold value, determining whether the micro droplets cover the template DNA or not according to the threshold value of the fluorescent signal, wherein the micro droplet covering the template DNA greater than the threshold value is a positive micro droplet, and the micro droplet not covering the template DNA lower than the threshold value is a negative micro droplet. The detection method provided by the invention is simple in process, also has the advantage of the real-time fluorescent quantitative PCR method, can realize the absolute quantitative analysis of nucleic acid, also has high-flux sample treatment capability and is high in practicability.

Description

technical field [0001] The invention belongs to the technical field of food hygiene detection, in particular to a vegetable enterohemorrhagic Escherichia coli O 157: H 7 Droplet digital PCR detection method. Background technique [0002] Enterohemorrhagic E. coli (EHEC) is a subtype of E. coli. The main pathogenic mechanism of EHEC is that the toxins produced can cause lesions in vero cells, so it is called vero toxins. It is also called Shigella-like toxin (SLT) or Shigella toxin Stx because of its similarity to the toxin of Shigella in terms of biological properties, physical properties and antigenicity. Enterohemorrhagic Escherichia coli O 157 :H 7 is the main pathogenic strain. Enterohemorrhagic Escherichia coli O has been isolated from feces of patients with hemorrhagic enteritis since 1982. 157 :H 7 Enterohemorrhagic Escherichia coli O 157 :H 7 Infection outbreaks and sporadic outbreaks have spread to more than 30 countries on 6 continents. Enterohemorrhagi...

AI Technical Summary

Problems solved by technology

[0003] At present, the identification of enterohaemorrhagic Escherichia coli O at home and abroad 157 :H 7 The methods can be divided into traditional methods, immunological methods and molecular detection methods. Traditional separation and culture methods and biochemical identification take a long time and have low sensitivity; in comparison, immunological methods shorten the detection time, but are prone to enterohemorrhagic large intestine Cross-reaction of multiple serotypes of Bacillus; with the development of polymerase chain reaction (PCR) method, real-time fluorescence quantitative PCR technology has been successfully used in enterohaemorrhagic Escherichia coli O 157 :H 7 It has the advantages of strong specificity, high sensitivity and rapidity in the detection of

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