Vibrio parahemolyticus dual-real-time fluorescence PCR (Polymerase Chain Reaction) detecting primer, probe, detecting kit and detecting method

Abstract

The invention discloses a vibrio parahemolyticus dual-real-time fluorescence PCR (Polymerase Chain Reaction) detecting primer, a probe, a detecting kit and a detecting method. The primer, the probe and the reaction condition are optimized and designed by using a dual-real-time fluorescence PCR method and adopting a toxR gene and a tdh gene of vibrio parahemolyticus as target genes, so that the toxic gene is detected when specific detection is carried out on the vibrio parahemolyticus in an amplification reaction, the toxR gene is used for specifically detecting the vibrio parahemolyticus, andthe tdh gene is used for detecting whether a toxic gene is carried or not. The invention has the advantages of rapid detection, reliable result, high sensitivity and high specificity, can finish the process from sample preparation to detecting result issuing in 8 to 10 hours, is free from the inference of false positive gender, cross contamination, and the like, provides a favorable tool for carrying out epidemiological survey of vibrio parahemolyticus, is suitable for inspection and quarantine of foods and marine products and can enable an inspection and quarantine bureau, a disease prevention and control center and a quality supervision department to carry out simple, rapid and accurate detection on the sample.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, in particular to dual real-time fluorescent PCR detection primers, probes, detection kits and detection methods for important food-borne pathogenic bacteria Vibrio parahemolyticus, suitable for import and export food inspection, It is used by the Center for Disease Control and Quality Supervision Department. Background technique: [0002] Vibrio parahaemolyticus (VibrioParahaemolyticus) is a Gram-negative halophilic bacillus and one of the important food poisoning pathogens in coastal countries, which can cause typical gastroenteritis reactions such as diarrhea, intestinal cramps, nausea, vomiting, and fever in patients . Vibrio parahaemolyticus mainly exists in seawater and seafood, and food poisoning caused by this bacteria accounts for 40% to 60% of bacterial food poisoning in Japan, ranking first. In addition, the United States, Australia, the United Kingdom, Belgium, France, South ...

AI Technical Summary

Problems solved by technology

These conventional methods have played an important role in the research of Vibrio parahaemolyticus, but with the development of modern science and technology, the traditional method of identifying Vibrio parahaemolyticus based on phenotypic characteristics is far from being able to meet the requirements of the bacteria. The need for rapid diagnosis and epidemiological research has exposed many shortcomings: cumbersome operation, long identification period, which is not conducive to the rapid diagnosis of acute poisoning and the requirements for rapid customs clearance at ports; the pathogenic mechanism of Vibrio parahaemolyticus is complex and diverse Bacterial strains without virulence factors are not necessarily pathogenic, and the traditional culture identification method cannot determine the virulence of the bacteria in a short period of time, and other special biochemical identification tests need to be added, which undoubtedly makes the identification cycle longer

The multiplex real-time fluorescent PCR method for the detection of pathogenic bacteria has just started both at home and abroad. At present, there is no multiplex real-time fluorescent PCR detection kit for Vibrio parahaemolyticus.

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