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PCR-ELISA detection method of vibrio parahaemolyticus in aquatic product

A hemolytic vibrio and detection method technology, applied in the field of PCR-ELISA detection and quantification of vibrio parahaemolyticus in aquatic products, can solve the problems of heavy workload, long detection cycle, high cost of detection samples, etc., and achieve low cost and high specificity High sensitivity and low cost of consumables

Inactive Publication Date: 2009-05-20
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

This method has a long detection cycle and a large workload; in 1999, British scientist Asim et al. established a method for the detection of Vibrio parahaemolyticus by common PCR, which can detect tlh, tdh, and trh genes in Vibrio parahaemolyticus simultaneously, but this The first method can only detect Vibrio parahaemolyticus qualitatively but cannot perform quantitative analysis; in 2003, American scientist Geroge et al. established a method for detecting the tdh gene of Vibrio parahaemolyticus by fluorescent quantitative PCR. This method has high detection sensitivity and fast detection speed , can carry out quantitative analysis on Vibrio parahaemolyticus, but this method needs to purchase an expensive fluorescent quantitative PCR instrument, and the cost of testing samples is high, which cannot meet the requirements of large-scale detection in practice

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  • PCR-ELISA detection method of vibrio parahaemolyticus in aquatic product
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  • PCR-ELISA detection method of vibrio parahaemolyticus in aquatic product

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Embodiment 1

[0041] Artificial contamination of Vibrio parahaemolyticus standard bacteria (BJ1997, ATCC1615, ATCC1616) and control bacteria (Vibrio alginolyticus, Escherichia coli, Salmonella, Shigella) oyster samples detection.

[0042] 1. Preparation of DNA template:

[0043] a. Aseptic operation of the specimen to be tested. For the oyster sample, take all shellfish meat containing internal organs, weigh 25g of the specimen to be tested, add 225mL of 3.5% sterile NaCl solution, after homogenization, take 10mL and add 90mL of modified alkaline peptone water (MAPW, adjust the NaCl concentration in APW to 3.5 %)middle. Placed in a constant temperature incubator at 37°C, enriched at 120r / min for 7h.

[0044] b. Take 1.0 mL of the enriched bacterial culture, put it in a 1.5 mL centrifuge tube, centrifuge at 12,000 r / min for 5 min, discard the supernatant; add 100 μL of sterile water, vortex and mix well, bathe in 100°C water for 10 min, and immediately freeze Bath for 2 minutes; centrifug...

Embodiment 2

[0066] Quantitative detection of Vibrio parahaemolyticus in aquatic samples:

[0067] 1. Preparation of DNA template:

[0068] a. Aseptic operation of the specimen to be tested. Shellfish refers to all the shellfish including viscera, fish refers to different parts of the fish body, and crustaceans refers to the part or whole including gills and intestines. Weigh 25 g of the specimen to be tested, add 225 mL of 3.5% sterile NaCl solution, and after homogenization, take 10 mL and add 90 mL of modified alkaline peptone water (MAPW, adjust the NaCl concentration in APW to 3.5%). Placed in a constant temperature incubator at 37°C, enriched at 120r / min for 7h.

[0069] b. Take 1.0 mL of the enriched bacterial culture, put it in a 1.5 mL centrifuge tube, centrifuge at 12,000 r / min for 5 min, discard the supernatant; add 100 μL of sterile water, vortex and mix well, bathe in 100°C water for 10 min, and immediately freeze Bath for 2 minutes; centrifuge at 12000r / min for 5 minutes, ...

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Abstract

A PCR-ELISA detection method for the vibrio parahaemolyticus in aquatic products is achieved by four steps: the extraction of sample DNA, the PCR reaction, the ELISA reaction and the result detection. By providing two groups of specific digoxigenin-labeled primers of vibrio parahaemolyticus tlh, tdh, a biotin-labeled probe for distinguishing two genes, and plasmid used in a quantitative specific sequence containing the tlh gene, the invention can further detect the vibrio parahaemolyticus in the aquatic products. The invention can be carried out in a 96-hole ELISA plate; and the provided method has low cost, good specificity and high sensitivity, and is suitable for conducting large-scale, high-throughput detection on the vibrio parahaemolyticus.

Description

Technical field: [0001] The invention relates to a safety detection technology for microorganisms in aquatic products, and is a PCR-ELISA method for detecting and quantifying Vibrio parahaemolyticus in aquatic products. Background technique: [0002] Vibrio parahaemolyticus is an important food-borne pathogen, which mainly exists in coastal seawater, seawater sediments, fish, shellfish and other seafood, and can cause acute gastroenteritis and primary sepsis. According to data from the Foodborne Disease Surveillance Network (including 13 provinces) from 1992 to 2001, the disease outbreak rate caused by Vibrio parahaemolyticus ranks first among microbial foodborne diseases. In addition, Vibrio parahaemolyticus is also one of the main pathogens threatening the mariculture industry. [0003] Vibrio parahaemolyticus can produce a variety of hemolytic toxins, including thermostable direct hemolysin (TDH), thermolabile hemolysin (TLH) and relatively thermostable direct hemolysin ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06
CPCY02A50/30
Inventor 周德庆赵峰刘琦柳淑芳苏来金曹慧慧马丽萍
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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