Detection method of flounder rhabdovirus

Abstract

The invention relates to a detection method of a bastard halibut rhabdovirus in the aquatic livestock pathogen detection and diagnosis technical field. A real-time quantitative reverse transcription-polymerase chain reaction technology is adopted to detect the bastard halibut rhabdovirus of fish. A specific primer sequence and a fluorescence probe sequence are designed firstly and then nucleic acid of a sample to be detected is extracted; secondly, the real-time quantum reverse transcription-polymerase chain reaction is carried out, and ribonucleic acid containing target amplified fragments by in vitro transcription is used as a standard substance to draft a standard curve; finally, whether the sample to be detected contains the bastard halibut rhabdovirus is judged according to circulating valve value of the sample to be detected calculated by an apparatus software. The method of the invention can be widely applied to epidemic disease monitoring over aquatic plants of bastard halibut, weever, turbot, snapper sea bream, etc. and detection of the bastard halibut bullet-shaped virus in import and export trade. Compared with the prior art, the method of the invention is characterized by rapid detection, high efficiency, accurate quantum, strong specificity, wide detection scope, high sensitivity, etc.

Description

Technical field: [0001] The invention belongs to the technical field of detection and diagnosis of aquatic animal pathogens, and relates to a detection method of flounder rhabdovirus, in particular to a method for detecting fish by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR for short) technology. Method for rhabdoviruses in flounder. Background technique: [0002] The flounder rhabdovirus (Hirame rhabdovirus, referred to as HRV) virus particle is bullet-shaped, with a size of 80nm×160-180nm, which was first reported in Japan in 1986. This disease has been found in flounder bred in the Yellow Sea, Bohai Sea and other places in my country. The onset season is winter and early spring. When the fish body is onset, the water temperature is generally below 15°C. The disease mainly harms flounder, and artificial infection is highly pathogenic to red sea bream and black sea bream. The virus has also been isolated from sweetfish and unprepa...

AI Technical Summary

Problems solved by technology

Currently, fish virus detection methods mainly include three categories: cytological diagnostic technology, immunological diagnostic technology, and molecular biological diagnostic technology. , the detection cycle is long, and the sensitivity is low; immunological diagnostic techniques mainly include immunofluorescence detection and immune dot hybridization, which have the advantages of strong specificity and high sensitivity, but the method is quite cumbersome and is not suitable for detection of a large number of samples; Biological diagnostic techniques mainly include polymerase chain reaction (PCR), which is relatively fast and sensitive, but requires agarose gel electrophoresis and ethidium bromide staining to observe the results. Ethidium bromide is a carcinogen and is harmful to the human body and the environment , and the problem of cross-contamination is relatively serious; in addition, PCR can only be used for qualitative detection, and cannot be used for quantitative detection of viruses

[0004] Existing methods for the detection of rhabdoviruses in flounder generally use cytological diagnostic methods, and real-time quantitative PCR technology has been widely used to detect pathogens in aquatic animals, such as bacteria (Vibrio prawns, Vibrio parahaemolyticus), shrimp viruses ( Shrimp white spot syndrome virus, Tao La syndrome virus, yellow head virus), fish viruses (infectious hematopoietic organ necrosis virus, koi herpes virus, infectious pancreatic necrosis virus, red sea bream iridescent virus), etc., according to retrieval, There is no report on detection of rhabdovirus in flounder by real-time quantitative RT-PCR