Development And Application Of Visible Assay For Rapid Detection Of Seven Porcine Pathogens

With the number of large-scale pig farms increasing,seven swine diseasescaused by Porcine circovirus type 2/3,Porcine epidemic diarrhea virus,Porcine transmissible gastroenteritis virus,Porcine deltacorona virus,Porcine rotavirus and Swine vesicular disease virus,have brought huge economic losses to pig industry,and serious threats the high-quality and healthy development of the pig industry in China.To facilitate the purification of swine disease-associated pathogens,on-site detection methods of these pathogens has become a research hotspot.This study intends to develop LFB RPA assays for swine disease-associated pathogens through recombinase polymerase amplification combined with lateral flow biosensor,therefore a set of feasible assays for rapidly detect to swine disease-associated pathogens in field can be provided.1.Development of the LFB RPA assays for PCV2 and PCV3.Specific RPA primers and nfo probes were designed Cap gene,and then the specificity,sensitivity and applicability of the developed LFB RPA assays were validated using standard samples and clinical samples.The results showed that the two assays were only positive for PCV2 or PCV3,and no cross-reaction with other common pig pathogens;the two assays limit of detection were 10 copies and 11 copies,respectively;the detection results of 30 clinical samples confirmed that the coincidence rates between LFB RPA and qPCR were 86.7%(26/30)and 90.0%(27/30)for PCV2 and PCV3.2.Development of the LFB RT-RPA assays for PDCoV,TGEV,PoRV and PEDV.Specific RPA primers and probes were designed based on PDCoV M gene,TGEV S gene,PoRV VP6 gene and TGEV N gene.The specificity,sensitivity and applicability of the developed LFB RT-RPA assays were evaluated by testing standard samples and clinical samples.The results showed that the four LFB RT-RPA assays were only positive for PDCoV,TGEV,PoRV and PEDV,and had no cross reaction to PRV,PPV and PRRSV;the limit of detection for PDCoV,TGEV,PoRV and PEDV were 12 copies,12 copies,13 copies and 120 copies,respectively;the detection results of 40 clinical samples of pigs with diarrhea symptoms confirmed that the coincidence rate between the four LFB RT-RPA assays and RT-qPCR were 100.0%(40/40)respectively.3.Development of the LFB RT-RPA assays for SVD.Specific RPA primers and probe were designed based on SVD VP1 gene,and the specificity,sensitivity and effectiveness of the developed LFB RT-RPA assay were evaluated by testing standard samples and clinical samples.The results showed that the developed LFB RT-RPA assay was only positive for SVD and no cross-reaction with other common pig pathogens such as PPV,PRV and M hyo;the limit of detection was 12 copies;the detection results of 60 viscera samples confirmed that the coincidence rate between SVD LFB RT-RPA and RT-qPCR was 100.0%(60/60).To sum up,the LFB RPA assays for seven common pig epidemics were established in this study.These assays are user-friendly,highly instrument-ind ependent,and intuitive results could be provided in a short time.The developed LFB RPA assays would be promising on-site detection methods to rapidly detect swine diseaseassociated pathogens,and therefore have great important practical significance for prevention and control of common diseases in grassroots pig farms.