Method for detecting grass carp reovirus by using test strip, crisper-cas13a system and application thereof

Abstract

The application discloses a method for detecting grass carp reovirus by using a test strip, a CRISPR-Cas13a system and application thereof, and relates to the technical field of biology. The method comprises the following steps: (1) a primer pair is used to perform RPA amplification on a grass carp reovirus gene target sequence, and an RPA amplification product is obtained; (2) a primer pair for amplifying crRNA is designed according to a target of a grass carp reovirus gene, and crRNA is obtained according to the primer pair; (3) Cas13a protein, crRNA, RNA-reporter, the RPA amplification product, an RNase inhibitor, dNTP and T7 RNA polymerase are mixed and incubated at an isothermal temperature, and CRISPR-Cas13a-LFD detection is performed; and (4) the CRISPR-Cas13a-LFD detection product is transferred into a detection buffer, inserted into a colloidal gold test strip, and after 5-10 min, specific bands are observed. The detection method has high sensitivity and high specificity, can be completed by using a lateral flow strip method, is convenient to carry, results can be read by visual observation, and can be easily quantified, so that the operation procedure during detection is greatly simplified, and the pollution risk is reduced.

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