Method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei

A technique for white shrimp and pathogenic bacteria, which is applied in the field of simultaneous quantification of two types of pathogenic bacteria in Penaeus vannamei

Inactive Publication Date: 2016-05-04
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • Method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei
  • Method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei
  • Method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei

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Embodiment

[0031] This embodiment relates to a method for simultaneously quantifying live bacteria of two pathogenic bacteria in Penaeus vannamei; specifically, it includes the following steps:

[0032] 1. Materials and methods

[0033] 1.1 Tested strains and culture conditions

[0034] A total of 18 strains of tested bacteria were used to verify the specificity of the primer and probe sequences used in this experiment, and the specific strain information is shown in Table 1.

[0035] Culture conditions of Vibrio parahaemolyticus: Streak and inoculate the seed solution in the glycerol tube stored at -80°C on the TCBS plate, pick a single colony in a 10ml TSB (3% NaCl, pH8.0) test tube, and rotate at 37°C Cultivate in a shaker at 180r / min for 18h to obtain the bacterial cell culture liquid in the initial stage of stabilization, which is used as a seed liquid for future use.

[0036] Salmonella culture conditions: Streak and inoculate the bacterial seed solution in the glycerol tube stor...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/63C12R1/42
CPCC12Q1/6851C12Q2537/143C12Q2563/107C12Q2531/113C12Q2563/173
Inventor 赵勇肖莉莉张昭寰娄阳杜苏萍潘迎捷
Owner SHANGHAI OCEAN UNIV
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