猪δ冠状病毒Basic RT-RPA和LF-RT-RPA方法的建立与应用OACSTPCD

This study was purposed to establish a recombinase polymerase amplification(RPA)method for rapid detection of porcine deltacoronavirus(PDCoV).In total,seven pairs of primers and one probe were designed according to the PDCoV N gene.First of all,the Basic RT-RPA method was established to identify the best primers.Then,the optimal system,reaction temperature and time were determined for the LF-RT-RPA reaction,while the specificity and sensitivity of both methods were verified simultaneously.The established RPA method combined with the RT-PCR method was adopted to detect 162 clinical porcine diarrhea materials collected from some regions of Jilin province.According to the test results,the optimal reaction conditions of the Basic RT-RPA assay were 41.6℃for 25 min,and the detection limit was 3.34×10 copies/µL of viral nucleic acid.The optimal reaction conditions of the LF-RT-RPA assay were 39℃for 12 min,and the detection limit was 3.34×102 copies/µL of viral nucleic acid.Both methods were demonstrated with high specificity and sensitivity,and the nucleic acid positive rate was 12.34%of PDCoV in diarrhea piglets from some regions of Jilin province.The LF-RT-RPA assay proposed in this study was applicable for on-site clinical detection for infection with PDCoV to prevent and control of the disease of pigs in Jilin province.