NASBA-microwell plate detection kit and detection method for five vibrios in mariculture

Abstract

The invention relates to an NASBA-microwell plate detection kit and a detection method for five vibrios in mariculture. The kit comprises an enzyme mixed solution, a reaction liquid containing a forward primer and a reverse primer, and five probe solutions. The five probe solutions are a vibrio parahaemolyticus probe solution, a vibrio alginolyticus probe solution, a vibrio harveyi probe solution, a vibrio vulnificus probe solution and a vibrio anguillarum probe solution. The detection method comprises the steps of extracting total RNA by using a cracking liquid, amplifying the total RNA to obtain an NASBA amplification product by using the reaction solution; and at the same time, coating the probes in the microwell plate; subjecting the NASBA amplification product and the probes to hybrid chromogenic reaction; and then detecting absorption value of A405 nm. The detection kit is sensitive and specific, is convenient for operation in detection and can detect five vibrios in the mariculture rapidly.

Description

technical field [0001] The invention relates to a detection technology of pathogenic vibrio, in particular to a NASBA-micropore plate detection kit and a detection method of five kinds of vibrio in mariculture. Background technique [0002] With the rapid development of the mariculture industry, the problem of disease has become increasingly apparent. The annual incidence rate is over 50%, and the loss rate is about 20%. The direct economic loss caused by this is as high as tens of billions of yuan every year, and it is on the rise. At present, the main characteristics of mariculture diseases in my country are: there are many mariculture species that are affected, such as fish, crustaceans, shellfish and other cultured species; there are many types of diseases and fast mutations, such as skin ulcer disease, enteritis disease, rotten disease of fish, etc. Tail disease, gill rot, columnar flexibacillosis and sarcoidosis, etc., crustacean red leg disease, bacterial larval sepsis...

AI Technical Summary

Problems solved by technology

At present, the detection of pathogenic Vibrio has PCR, ELISA (enzyme-linked immunosorbent assay), LAMP and gene chip etc., as publication number is the invention patent application of CN1012025557, just detects Vibrio alginolyticus by triple PCR primer; Publication number is CN101020926 Invention patent application for the detection of Vibrio cholerae by LAMP technology; the invention patent with the announcement number CN101178363 uses primers and probes for constant temperature amplification and enzyme-linked detection of Vibrio parahaemolyticus; but ELISA is sensitive, specific, simple, fast and stable It has the characteristics of easy automatic operation and other characteristics, and its shortcomings are also obvious. Its experimental cycle is longer, generally takes 1-2 days, and it is difficult to prepare specific monoclonal antibodies. At the same time, it detects proteins, so it is suitable for bacterial detection. Said that it is impossible to judge whether the target bacteria are dead or alive, and it is easy to make mistakes

RT-PCR is fast, sensitive, and specific. It detects the PCR amplification process in real time and online without post-processing the PCR amplification products. It overcomes the shortcomings of conventional PCR technology that is easy to contaminate. However, RT-PCR requires expensive instruments and equipment. , high operating requirements for technicians, not suitable for grassroots use

LAMP constant temperature amplification is extremely fast, but it also has disadvantages, such as troublesome primer design, unstable reaction system, and inability to perform multiple amplification

Gene chips can achieve accurate identification results of multiple pathogenic bacteria through one experimental operation, and meet the requirements for parallel detection of multiple coexisting background bacteria in complex samples, but require expensive professional equipment, and the current detection cost is extremely expensive. It is not yet possible to deploy in the laboratory, let alone promote the application at the grassroots level