Establishment and Preliminary Application of RAA Assay for the Detection of Porcine Transmissible Gastroenteritis Virus based on S Gene
The aim of this study was to establish a convenient,efficient and specific recombinase-aid amplification(RAA)assay based on the porcine TGEV S gene.Comparing the genome se-quence,selecting specific segment of TGEV S gene to construct the recombinant plasmid pUC57-S as templete.RAA primers and probes were designed and screened.RAA assays were estab-lished.The sensitivity,specificity and repeatability of the method were evaluated.Verifying the established test method by clinical samples of diarrhea,and compare the consistency with the tes-ting results of the fluorescence quantitative PCR assay.Our results showed that the best primer pair F2/R1 and probe were screened out.The assay can be completed in 30 min at 40 ℃.The lowest detection limit of fluorescent RAA assay was 1.34×101 copies·μL-1.There was no cross reaction with other virus nucleic acid,as porcine epidemic diarrhea virus,porcine delta coronavir-us,porcine respiratory coronavirus and other common swine viruses.A total of 107 clinical sam-ples were tested using this method,and positive rate was 5.61％(6/107),which was consistent with the test results of fluorescence quantitative PCR.This study successfully established a sen-sitive,specific and visual RAA test method,which can get rid of the dependence of expensive equipment,the professional and detection technology,and provided a reliable technical basis for the clinical detect of TGEV.
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