Non-diagnostic method for detecting Vibrio vulnificus through TaqMan probe fluorescence RT-PCR

Abstract

The invention discloses a non-diagnostic method for detecting Vibrio vulnificus through TaqMan probe fluorescence RT-PCR. The method comprises the following steps: comparing through adopting the DNA sequence of a Vibrio vulnificus hemolysin A (vvhA) gene as a target sequence, selecting a conserved region, determining the conserved sequence of the vvhA gene, and designing the real time PCR primers and the probes of the Vibrio vulnificus. The Vibrio vulnificus RT-PCR detection method which utilizes the specific fluorescence probes to implement a completely-closed tube type operation enables the amplified product pollution opportunity to be substantially reduced, the experiment steps, the experiment time and the reagent consumption to be reduced, the PCR in the invention to be different from routine PCR, electrophoretic identification to be not needed, and the appearance of a false positive result to be difficult because of difficult environmental protection. The method which can rapidly detect pathogenic bacteria has the advantages of high sensitivity, high specific degree and high lower limit of detection, can be applied to the detection of the Vibrio vulnificus in frontier port environment water, aquatic products and night soil of diarrhoea patients, and has a very high application value.

Description

technical field [0001] The invention relates to a non-diagnostic method for detecting Vibrio vulnificus by fluorescent RT-PCR with a TaqMan probe. Background technique [0002] Vibrio vulnificus (Vibrio vulnificus) is a Gram-negative halophilic Vibrio that widely exists in warm water environments, and is a common pathogen in estuaries and marine environments. Infecting healthy humans and animals mainly by contaminating seafood and infecting wounds, Vibrio vulnificus can cause sepsis and wound infections. In recent years, there have been reports of gastroenteritis caused by consumption of seafood contaminated by Vibrio vulnificus. The main symptoms are vomiting, diarrhea and abdominal pain. The case fatality rate of wound infection caused by Vibrio vulnificus is as high as 50%, and the case fatality rate of sepsis caused by Vibrio vulnificus infection is as high as 75%, especially in immunosuppressed patients and people with excessive iron loading in the body. In the monito...

AI Technical Summary

Problems solved by technology

For example, Mark S. Campbell established a real-time PCR detection method based on SYBR Green based on the Vibrio vulnificus hemolysin gene (vvhA), but this method needs the help of melting curves to distinguish specific amplification and non-specific amplification, and the specificity is not high

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