NoV G Ⅱ.2亚型RT-RPA-CRISPR/Cas12a检测方法的建立OA

In recent years,a novel G Ⅱ.2 subtype of Norovirus(NoV G Ⅱ.2)has appeared,which causes human acute gastroenteritis through raw seafood such as oysters,is widely distributed in the world.In order to achieve on-site inspection of NoV infection and rapidly response to an outbreak,it was urgent to establish a fast and convenient method for detection of nucleic acids of NoV G Ⅱ.2.In the study,a RT-RPA-CRISPR/Cas12a assay for NoV G Ⅱ.2 was established through designing primers and crRNA and optimizing reaction conditions.The results showed that RT-RPA amplification was conducted for NoV G Ⅱ.2 standard RNA using primers of NoV-G Ⅱ.2-F1B and NoV-G Ⅱ.2-R1,and the detection could be completed within 40 min in combination with crRNA1-mediated CRISPR/Cas12a reporting system;the method,with a detection limit of 10 copies/μL,failed to interact with rotavirus,adenovirus,astrovirus,human parvovirus,bocavirus and zarovirus;30 clinical simulated samples were tested separately by the established method and qRT-PCR,it was found that the overall compliance rate was 96.67%.In conclusion,the established method was characterized by strong sensitivity,specificity and accessibility without any expensive equipment,contributing to the on-site detection,prevention and control of NoV G Ⅱ.2 infection.