基于CRISPR/Cas12a-RT-RAA的猪繁殖与呼吸综合征病毒快速检测方法的建立OA北大核心CSTPCD

[Objective]This study was aimed to establish a rapid detection method for Porcine reproductive and respiratory syndrome virus(PRRSV)based on the combination of CRISPR/Cas12a system and reverse transcription recombinase aided amplification(RT-RAA)technology.[Method]The whole genome sequences of different genotypes of PRRSV were analyzed,and recombinase aided amplification(RAA)primers,crRNA and DNA reporter substrate molecules were designed in the conserved region of the 3'-UTR terminal,and the best primer pairs of RT-RAA were screened.AsCas12a protein was expressed and purified,and the plasmid was used as the standard product.After amplification by RT-RAA,the product was added to CRISPR/Cas12a system to establish the CRISPR/Cas12A-RT-RAA detection method for PRRSV.Nucleic acids of PRRSV,Porcine circovirus type 2(PCV2),Porcine epidemic diarrhea virus(PEDV),Classical swine fever virus(CSFV)and Porcine pseudorabies virus(PRV)were used as templates to verify the specificity of the established method,and different concentrations of pMD18T-PRRSV were used as templates to verify the sensitivity of the established method.The plasmids of 5.62×106,5.62×103 and 5.62 copies/μL were used as templates for repeat tests,and finally the clinical effect was evaluated,and the established method was compared with reverse transcription Real-time quantitative PCR.[Result]This study successfully expressed and purified AsCas12a protein with good shear activity,with a molecular weight of 196 ku.The detection lower limit of the CRISPR/Cas12a RT-RAA method established could reach 5.62 copies/μL.This method had good specificity and could specifically detect PRRSV,but could not detect other porcine disease viruses such as PCV2,PEDV,CSFV and PRV.And it had good repeatability.This method was applied to detect 121 pig tissue samples,the positive conformity rate with reverse transcription Real-time quantitative PCR was 93.75％,and the negative conformity rate was 99.05％,with a total conformity rate of 99.17％.[Conclusion]This study successfully established a rapid detection method for PRRSV based on CRISPR/Cas12a-RT-RAA.The method had strong specificity,high sensitivity,good repeatability,and did not rely on complex equipment.It could be used in both on-site and grassroots laboratories.