猫传染性腹膜炎病毒CRISPR-Cas13a检测方法的建立及应用OA

Feline infectious peritonitis(FIP)caused by feline infectious peritonitis virus(FIPV)is highly lethal,posing a serious threat to the health of cat populations,especially kittens and young cats.To develop a FIPV diagnostic method with high sensitivity,low cost and easy operation,this study combined RT-RPA with the CRISPR detection system.Based on laboratory isolation,sequencing and NCBI database alignment,the N gene sequence of FIPV strains was selected and cloned into the pUC57 vector to construct a positive control plasmid;specific RPA primers and reaction temperature were screened,CRISPR reaction parameters were optimized,and the specificity and sensitivity of the method were evaluated;LwCas13a protein was induced,expressed and purified by the Escherichia coli(E.coli)prokaryotic expression system.The active LwCas13a protein was successfully obtained;the F1R1 primer pair showed the optimal amplification efficiency,and the established FIPV-Cas13a detection method could specifically recognize FIPV nucleic acid without cross-reaction;the optimal reaction conditions were 37℃,crRNA dosage of 7.5 ng,LwCas13a protein dosage of 250 ng,the fluorescence was stable after 40 min of reaction,the limit of detection reached 101 copies·μL-1,and visual detection could be achieved by combining with test strips.The detection of 38 clinical samples of anal swabs showed that the positive detection rates of both the fluorescence method and the test strip method were 50%(19/38),which was highly consistent with the industry standard RT-PCR at 42%(16/38).In conclusion,the FIPV-Cas13a detection system provides a robust and visual alternative detection method,which is suitable for on-site FIPV monitoring.