Method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei

Abstract

The invention discloses a method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei. The method includes the following steps that propidium monoazide mother liquid is added into to-be-detected penaeus-vanmamei-liquid homogenate, and propidium monoazide sample mixed liquid is obtained; the sample mixed liquid is subjected to dark treatment to be exposed and centrifuged at a high speed, and the bacteria are collected; DNA of the bacteria is extracted; a multi-fluorescence quantitative PCR amplified reaction is carried out with the extracted DNA as a template and t1h genes of vibrio parahaemolyticus and orgC genes of salmonella bacteria as target genes,, and corresponding circulation threshold values are output; the circulation threshold values are compared with a standard curve of the vibrio parahaemolyticus and a standard curve of the salmonella bacteria respectively, and the viable-bacterium concentration of the vibrio parahaemolyticus and the viable-bacterium concentration of the salmonella bacteria are obtained. According to the method, the multi-fluorescence quantitative PCR technology and PMA dye are creatively combined, and the aim that the two important viable pathogenic bacteria in the penaeus vanmamei are rapidly quantified is achieved.

Description

technical field [0001] The invention relates to a detection method for food-borne pathogenic bacteria in aquatic products, in particular to a method for simultaneously quantifying two viable pathogenic bacteria in Penaeus vannamei. Background technique [0002] Vibrio parahaemolyticus and Salmonella are two important food-borne pathogens in aquatic products, which have caused great safety hazards to public health. In China, Vibrio parahaemolyticus is the number one foodborne pathogen in aquatic products, because consumption of raw or undercooked aquatic products is highly susceptible to infection with Vibrio parahaemolyticus, which can cause severe acute gastroenteritis, accompanied by Nausea, headache, low-grade fever and other symptoms. Salmonella is the second largest food-borne pathogen causing seafood food poisoning incidents, and about 20.8% of seafood food poisoning incidents are caused by this bacteria every year. [0003] Vannamei is the most important aquatic pro...

AI Technical Summary

Problems solved by technology

However, studies have shown that this new method for live bacteria detection still has certain technical difficulties, and its ability to distinguish dead and live bacteria is often affected by many factors, such as bacterial strains, PMA concentration, dark treatment time, PCR amplification system, and sample matrix, etc.

Therefore, the research on the combination of PMA and multiple real-time fluorescent PCR is still rarely reported, and it has become a hot and difficult issue in the quantitative detection of high-throughput live bacteria.

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