Vibrio parahaemolyticus detection method based on nanogold discoloration response isothermal nucleic acid amplification
An isothermal nucleic acid amplification, hemolytic vibrio technology, applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc. Application limitations and other problems, to achieve the effect of short detection time, simple equipment and easy operation
Inactive Publication Date: 2017-09-08
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology
Although there are a variety of microbial detection methods, most of them require high laboratory conditions, and the operation is complicated and time-consuming. Only those who have mastered the operation skills can complete the test, which is not suitable for promotion and use at the grassroots level. Applications in developing countries and areas with limited clinical conditions in my country are limited
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Embodiment
[0047] This embodiment relates to a method for detecting Vibrio parahaemolyticus based on nano-gold discoloration response isothermal nucleic acid amplification; it specifically includes the following steps:
[0048] 1. Preparation of gold nanoparticles
[0049] In a flask, 50 mL of 254 μM (0.01% wt) chloroauric acid solution was heated to vigorous boiling and stirred, and 500 μL of 388 mM trisodium citrate solution was added to the boiling and stirring solution, and the color of the solution changed from yellow to yellow within 2 minutes. After boiling and stirring for 15 minutes, stop heating, continue stirring until the reaction solution returns to room temperature, filter the reaction solution through a 0.8 μm gelman filter to obtain wine red gold nanoparticles, and measure its absorption peak at 527nm by ultraviolet-visible spectroscopy . Store the prepared gold nanoparticles in a brown bottle, store them in a refrigerator at 4°C, and usually use them up within one month...
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The invention discloses a vibrio parahaemolyticus detection method based on nanogold discoloration response isothermal nucleic acid amplification. According to the invention, biotin labeled primers of tlh gen are designed for rapid amplification of the tlh gen by loop-mediated isothermal amplification technology, a lot of biotin labeled repeated sequences are generated in the amplification process and achieve fast aggregation of streptavidin modified nanogold, and an intense color change effect is produced to reach the purpose of detection of specific genes. The invention establishes an economical, rapid, accurate, specific, sensitive and simple detection method for vibrio parahaemolyticus in aquatic products, and improves the detection quality and efficiency of medical health inspection, disease control and prevention, aquatic product processing, import and export inspection and other departments.
Description
technical field [0001] The invention belongs to the field of detection of food disease microorganisms, and in particular relates to a detection method for Vibrio parahaemolyticus based on nano-gold discoloration response isothermal nucleic acid amplification. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus, VP) is a Gram-negative halophilic bacterium belonging to the genus Vibrio in the Vibrio family. It is one of the important food-borne pathogenic bacteria in offshore fish, shrimp, crab, shellfish and other seafood. Important pathogens of regional food poisoning and summer diarrhea. In order to ensure food safety and protect people's health, developed countries such as the European Union and the United States have made clear regulations on Vibrio parahaemolyticus in aquatic products. ×10 4 / g. my country has listed the bacteria as the main inspection or monitoring object. At present, this bacterium is one of the pathogenic bacteria that ...
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Login to View More IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/137C12Q2563/155Y02A50/30
Inventor 孔聪沈晓盛王媛史永富刘志东
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI