Establishment Of Multiple Detection System For Porcine Circovirus Type Ⅲ And Epidemic Diarrhea Virus Based On CRISPR/Cas System

Porcine circovirus disease(Porcine circovirus disease,PCVAD)is a widespread and severe disease caused by the porcine circovirus(PCVs)family,its open reading frame 2(Open reading frame 2,ORF2)is a subtype of different strains.The region with the largest difference has a strong correlation with viral infection and immunity.Porcine epidemic diarrhea virus(PEDV)is an enteric coronavirus,and its open reading frame 3(ORF3)is a key factor that determines the yield and virulence of the virus.Existing nucleic acid detection methods have high sensitivity and specificity,but the detection process takes a long time,cumbersome steps and expensive large-scale equipment hinder the detection of viruses.In this study,the combination of recombinase polymerase amplification(RPA)and CRISPR/Cas12a,CRISPR/Cas13a systems,using DNA and RNA fluorescent reporters as indicators of the final reaction,established a dual detection system for PCV3&PEDV.This method targets PCV3open reading frame 2(Openreadingframe2,ORF2)and PEDV open reading frame 3(Openreadingframe3,ORF3),uses cr RNA and RPA amplification primers,and then uses a two-step method at 38°C,first RPA amplification for 30 minutes,and then add the Cas protein detection component to react for 30 minutes to detect the two viruses simultaneously,thus producing results visible to the naked eye under LED blue light.The detection method has high sensitivity and strong specificity,the detection limit is as low as below 10~2copies,and there is no cross-reaction with other porcine pathogens.Overall,this combined RPA preamplification and CRISPR/Cas system lysis assay is a practical tool for rapid detection of PCV3 and PEDV for differential diagnosis.