RT-RPA primer, probe, kit and detection method for detecting type II grass carp reovirus (GCRV)

Abstract

The invention discloses a real-time fluorescence RT-RPA primer, probe and kit for detecting type II grass carp reovirus (GCRV) and application of the real-time fluorescence RT-RPA primer, probe and kit. The optimal combination of the primer and the probe is obtained by designing and screening a specific conserved region of the gene type II (GCRV), and the primer and the probe are high in detection sensitivity, good in specificity and stable in detection effect; and moreover, the kit and the probe have no cross reaction with 9 fish common viruses including type I GCRV and type III GCRV and bacteria, namely aeromonas veronii and aeromonas hydrophila, which are easily infected by grass carp, and show good specificity. Based on the real-time fluorescence RT-RPA primer, probe and detection kit established by the invention, the detection sensitivity of the gene type II GCRV is 10<1> copy / [mu]l and is 10 times higher than that of a nested PCR method, large-scale instruments and equipment are not needed, the detection only lasts for 4.5-30 min, the detection time is greatly shortened, and the real-time fluorescence RT-RPA primer, probe and detection kit can be used for on-site rapid detection.

Description

Technical field[0001]The present invention belongs to the field of gene detection, and more particularly to RT-RPA primers, probes, kits, and detection methods for detecting II strawder.Background technique[0002]Grass Carp Reovirus (GCRV) is affiliated with aquacine. It is a new member of the Cantorma Virus (REOVIRIDAE), aquareovirus, is the disease One of the strongest strains of power is the first fish virus isolated from mainland China. The virus mainly causes the main varieties of freshwater breeding in Asian countries such as China, North Korea, and Vietnam, and the death rates in the fish species. The mortality is generally 30% ~ 50%, up to 60% ~ 80%, can also infect green fish, wheat Spike and rare 鮈 鮈. The virus is wide, the harm is large, high mortality, and long in the episode, and seriously threatening the production of fisheries. According to incomplete statistics, the economic losses caused by the blood-proof blood of the grassfish at least 1.0 billion yuan per year, se...

AI Technical Summary

Problems solved by technology

[0005] There are many diagnostic methods for grass carp reovirus, each with its own advantages and disadvantages. Nucleic acid detection methods such as virus isolation, electron microscope observation, PCR, and qPCR are still commonly used methods for detecting grass carp reovirus, but these methods are time-consuming and laborious. , The operation is relatively complicated, and the virus cannot be quickly diagnosed, and because the GCRV genome is RNA, the nucleic acid detection methods for GCRV need to extract RNA and continue to reverse transcription, and the obtained cDNA is used as a template for detection, so the operation is relatively cumbersome , is not conducive to the epidemiological investigation of grass carp haemorrhagic disease

There are certain requirements for equipment and operators, and the detection takes about 2 to 5 hours. It is difficult to achieve rapid, batch and simple detection requirements on site.

Images