| Porcine Rotavirus(PRo V),Porcine Sapovirus(Po Sa V)and Porcine Astrovirus(PAst V)are not only common intestinal pathogens that the pig industry,but also related to human infant diarrhea.The clinical symptoms and pathological changes are similar and difficult to distinguish.So it is necessary to relay on laboratory testing techniques for differential diagnosis.Therefore,it is not only of great significance to establish a rapid and accurate method for the identification of PRo V,Po Sa V and PAst V,but also can provid technical support for veterinary public health.At the same time,the small intestine is the main invading site for most enteroviruses.Isolation and immortalization of the epithelial cells of the small intestine will contribute to the development of enteroviruses.1.Establishment of triplex PCR detection methods for porcine rotavirus,sapvirus and astrovirusSpecific primers were designed with reference to the conserved sequences of VP6 gene of PRo V,the Rd Rp gene of Po Sa V and ORF1 gene of PAst V respectively.The PCR reaction conditions were explored and optimized to establish a triplex PCR that could simultaneously identify and diagnose PRo V,Po Sa V and PAst V.The method has good specificity and the test of common porcine diseases,such as TGEV,PEDV,CSFV,PRRSV,PRV,PCV2 were all negative and highly senstive.The detection limits of PRo V,Po Sa V and PAst V were 1.38×10~2,8.33×10~2 and 4.4×10~3copies/μL,respectively,which were consistent with the result of single-plex-PCR,253 samples from intensive swine farms were detected by using accurate and reliable method.The positive rates of PRo V,Po Sa V and PAst V were 9.09%,2.37%and 5.14%,indicating that,in the presence of mixed infection phenomenon,in which 2 samples were positive for both PRo V and Po Sa V.In addition,we also found that PRo V was often mixed with swine epidemic diarrhea virus and Po Sa V could be mixed infection with common pathogens.2.Primary isolation of intestinal epithelial cells from Bama miniature pigBama miniature pigs without colostrum within 12 hours of birth were selected.The small intestine was treated by the method of tissue block,combining with cell scraped,trypsin digestion and 96-well dilution to separate and purify the small intestine cells,whereas tightly packed cells displayed a cobblestones morphological,which were identified and confirmed by cytokeratin 18.At the same time,two restriction sites Xba I and Sal I were added at both ends of the sequence by designing primers.The identified SV40LT and h TERT gene fragments were connected to the plenti CMV GFP Hygro vector to construct recombinant immortalized plasmids containing plenti-SV40LT and plenti-h TERT.After the identification,The recombinant plasmid was transfected into 293T cells,and the recombinant lentivirus was successfully obtained,which provided materials for further screening of small intestinal epithelial cell lines.In this study,a triplex PCR detection method of PRo V,Po Sa V and PAst V were successfully established to provide guidance for the surveillance and prevention of these three zoonotic diseases and have public health significance.At the same time,the small intestinal epithelial cells of Bama miniature pig from Guangxi were successfully isolated,and the recombinant immortalized plasmids containing plenti-SV40LT and plenti-h TERT were constructed to package the recombinant lentivirus,which laid the foundation for the construction of the small intestinal epithelial cell line of local pig breeds in Guangxi. |
