Method for detecting vibrio parahaemolyticus

Abstract

The present invention relates to a method to detect bibrio parahemolyticus belonging to the technical field of biology, which is characterized in that: DNA template is prepared by centrifugating pure bacterial culture, discarding supernatant fluid, adding 100MuL sterile water for water bathing, ice bathing, centrifugation, removal of supernatant fluid as standby for expansion of the template. In addition, the method adopts primer 5.0 software to design four primer sequences of LAMP primer for bibrio parahemolyticus tlh gene M36437. Besides, loop-mediated isothermal amplification of bibrio parahemolyticus is applied. In detail, it is necessary to prepare a 25uL reaction system and carry out incubation and inactivation. Outcome detection refers to that turbidity of the reaction system is observed with naked eyes. Compared with prior arts, the detecting method of the present invention has the advantages of convenience, reliability, high sensitivity, short time and lower cost as a simple conventional detecting method, particularly adapts to grass-roots inspection and quarantine authorities and breed aquatics and brings great significance to improve food sanitation and boost development of international food trade.

Description

[technical field] [0001] The invention relates to the field of biotechnology, in particular to a method for detecting Vibrio parahaemolyticus. [Background technique] [0002] Vibrio parahaemolyticus belongs to Vibrio family Vibrio genus, is a Gram-negative bacteria with a single flagella at one end, active movement, halophilic bacteria, it is an important pathogenic bacteria, widely distributed In coastline areas, seawater sediments, fish and shellfish and other aquatic animals and seafood, it can infect humans and various aquatic animals such as fish, prawns, and crustaceans. People who eat food contaminated by this bacteria will cause diarrhea, Intestinal cramps, nausea, vomiting, fever and other typical gastroenteritis reactions, severe patients may also suffer from dehydration, shock, coma, and even death. [0003] The incidence of Vibrio parahaemolyticus is distributed worldwide, especially in coastal areas where the incidence is high. It is an important item for the i...

AI Technical Summary

Problems solved by technology

[0004] The detection methods for Vibrio parahaemolyticus are mainly traditional culture and biochemical identification methods, which are cumbersome, time-consuming and labor-intensive, and the detection cycle takes 5 to 7 days, and it is impossible to detect Vibrio parahaemolyticus in an unculturable state. It affects the accuracy of detection, and the polymerase chain reaction method, that is, PCR method, has the characteristics of specificity and sensitivity, and it also has a good application prospect for a large number of "not to be detected" in the current food hygiene standards. However, this Such methods usually require expensive and sophisticated instruments and equipment, and have relatively high requirements for the experimental environment and operator skills; at the same time, because PCR-related reactions need to undergo thermal denaturation of DNA, long-term temperature cycles, etc., it takes a long time, from It ranges from 3 hours to 20 hours, which is not conducive to the on-site rapid diagnosis and treatment of food poisoning. Therefore, as far as the current situation is concerned, it is widely used in laboratory research. In the actual inspection of food hygiene, due to relatively high requirements, it is popularized and applied. there is some difficulty

[0005] Loop-Mediated Isothermal Amplification Technology Loop-Mediated Isothermal Amplification, LAMP is a novel nucleic acid amplification technology that relies on 4 primers that can recognize 6 specific regions on the target DNA and a DNA polymer with strand displacement activity Enzyme, which efficiently amplifies nucleic acids under constant temperature conditions, has the characteristics of high specificity, fast sensitivity, high efficiency, and easy operation. At present, there is no rapid detection of parahaemolysis using the heat-labile hemolytic toxin gene tlh through loop-mediated isothermal amplification technology Vibrio reports

Images