Triple real-time fluorescence PCR (Polymerase Chain Reaction) detection kit of vibrio parahaemolyticus

Abstract

The invention provides a triple real-time fluorescence PCR (Polymerase Chain Reaction) detection kit of vibrio parahaemolyticus. The triple real-time fluorescence PCR detection kit comprises three pairs of specific primers, EvaGreenPCR premixed liquid, negative control and positive control, wherein the specific primers comprise thermostable direct hemolysin gene (tdh), thermostable related hemolysin gene (trh) and toxin expression and regulation protein gene (toxR) of the vibrio parahaemolyticus. The triple real-time fluorescence PCR detection kit not only has the advantages of fast real-time fluorescence PCR, sensitivity and high degree of automation, but also carries out triple real-time fluorescence detection on the basis of a melting curve technology, does not need to synthesize an expensive detection probe, is low in detection cost, can finish detection of three target genes only by the most conventional FAM / SYBR channel, is low in requirement on a real-time fluorescence PCR instrument, is almost applicable to all the real-time fluorescence PCR instruments sold on market, and is short in detection period, low in detection cost and reliable in detection result.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a triple real-time fluorescent PCR detection kit and a detection method for vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a Gram-negative halophilic Vibrio isolated from a food poisoning patient in Japan by Fujino et al. in 1953. It is one of the more important pathogenic bacteria in the Vibrio genus. Eating food contaminated by this bacterium can cause food poisoning. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea and watery stool. Vibrio parahaemolyticus is a marine bacterium mainly derived from seafood such as fish, shrimp, crab, shellfish and seaweed. The bacterium has strong survival ability and is the most common pathogenic bacterium in bacterial food poisoning in coastal areas. The traditional detection methods of Vibrio parahaemolyticus include pre-enrichment, selective enrichment...

AI Technical Summary

Problems solved by technology

Real-time fluorescent PCR is mainly divided into two types: probes and dyes. Probe-based real-time fluorescent PCR needs to synthesize fluorescently labeled probes. Due to the high synthesis cost of probes, the detection cost of probe-based real-time fluorescent PCR is high; Although the detection cost of dye-based real-time fluorescent PCR is low, due to the early use of unsaturated fluorescent dye SYBR Green I, there are problems such as poor stability and dye redistribution, low detection sensitivity and not suitable for multiple real-time fluorescent PCR, which limits the use of dyes. The application of real-time fluorescent PCR in the field of inspection and detection, with the emergence of saturated fluorescent dyes such as Eva Green and LC Green, the detection sensitivity of dye-based real-time fluorescent PCR has been significantly improved, and since the problem of dye redistribution is solved, it can be used in combination with melting curve technology Multiple real-time fluorescent PCR detection, so it has shown great advantages in the field of inspection and detection

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