Detection method and detection kit of gene II type grass carp reovirus

Abstract

The invention discloses a detection method of gene II type grass carp reovirus. The detection method comprises the following steps: (1) extracting the RNA of the grass carp sample, adding RNA into a premix reaction solution, and performing loop-mediated isothermal nucleic acid amplification on the conserved sequence of the gene II type grass carp reovirus by two pairs of primers, wherein the primers are respectively a primer GCRVII-F3 with the sequence of SEQ ID NO:1, a primer GCRVII-B3 with the sequence of SEQ ID NO:2, a primer GCRVII-F1P with the sequence of SEQ ID NO:3 and a primer GCRVII-BIP with the sequence of SEQ ID NO:4; labeling an FITC probe, wherein the sequence of the probe GCRVII-FITC-Probe is SEQ ID NO:5; and (2) dropwisely adding the amplified product on a sample spotting position of the LFD test paper, dropwisely adding a buffer solution in the front end of the sample position, finishing the reaction after the buffer solution is flown to the other end of the test paper, and judging the detection result according to the color development tape on the test paper. By combining the isothermal amplification and the nucleic acid test strip rapid detection technology, the detection method and the kit provided by the invention have the characteristics of simple operation, high detection reliability, good specificity and high sensitivity.

Description

technical field [0001] The invention relates to an epidemic disease diagnosis technology in the field of aquatic animal viruses, in particular to an RT-LAMP-LFD detection method of genotype II grass carp reovirus and a detection kit thereof. Background technique [0002] Hemorrhage of Grass Carp (Hemorrhage of Grass Carp) is a serious viral hemorrhagic disease in the grass carp aquaculture industry, which poses a major threat to the grass carp aquaculture industry. Its epidemic season is between the beginning of May and the end of October every year, and the water temperature begins to be popular at 20°C. The most suitable prevailing water temperature is 27°C-30°C, and the mortality rate can reach 50%-80%. According to incomplete statistics, the economic loss caused by grass carp haemorrhagic disease reaches 2.126 billion yuan every year. List of animal diseases. The pathogen of the disease is Grass carp reovirus (GCRV), which is the first fish virus isolated and identified...

AI Technical Summary

Problems solved by technology

However, in the actual detection work, the existing detection technology has the following problems: first, the sensitivity is not enough to detect latent infection samples; second, it is easy to produce false positives, which requires high template quality; High, not conducive to farmers and front-line production technicians to operate

[0005] Although the above two technologies have been initially applied in recent years, the applicable conditions for specific virus detection still need extensive and in-depth exploration. At present, there is no use of the above technologies in the detection of genotype II grass carp reovirus in this field. precedent

In the early days, the grass carp reovirus RT-LAMP detection kit CN201210186709.6 of genotype III was studied in China. On the one hand, genotype III is rare in clinical practice, and there is no market demand for this detection kit; on the other hand, the program The primers can not be used to amplify other grass carp reoviruses, and the specificity of the above-mentioned disclosed primers to amplify gene type II grass carp reoviruses is too poor to be used for LFD test strip detection

There have also been attempts in the field to study the RT-LAMP-LFD detection method for HIV, but due to the huge sequence differences between HIV and genotype II grass carp reovirus, the probes used have no reference value for other detections

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