Establishment And Preliminary Application Of Multiple RT-PCR Detection Methods For Porcine Group A Rotavirus, Porcine Group C Rotavirus And Porcine Astrovirus

To detect the porcine group A rotavirus group A porcine rotavirus(GARV),porcine group C rotavirus group C porcine rotavirus(GCRV)and porcine Astroviridae(PAstV),Rapidly),and find out the infection of GARV?GCRV and PastV in Sichuan province.we proposed to establish a multiplex Reverse Transcription-PCR for GARV?GCRV and PAstV.Molecular diversity features of VP6 and ORF2 were learned ai the same time,which defined the population of three virus.The study would make the evolution about GARV?GCRV and PastV between procine and others,Contributed to prevent three disease.1 Established a Multiplex Reverse Transcription-PCR for GARV?GCRV and PAstVAccording to the published sequences of GARV,GCRV and PAstV in GenBank,primers were designed based on the conserved regions of VP6(GARV,GCRV)and ORF2(PAstV)by software Primer 5.0,respectively.As a result,the specific PCR products were 229 bp,166bp and 410 bp for GARV,GCRV and PAstV.The Multiplex RT-PCR was established based on the optimized condition of the individual RT-PCR.Meanwhile the Multiplex RT-PCR was applied to the detection of 115 piglets' diarrhea samples about GARY,GCRV and PAstV in Sichuan Province.PCR and cloning were carried out for the positive strains.Sequence analysis was refered the gene relational and impressed on software MEGA5.05.Drawing phylogeny trees analyzs genetic evolution.The result displayed that multiplex RT-PCR could propagate specific DNA segment from GARV,GCRV and AstV positive stencil,instead of PRV,TGEV,PEDV,E.coli;The minimum cDNA concentration of GARV,GCRV and AstV was 5.47×10-4g?g/?L?3.8×10-3 ?g/?L and 4.8×10-4?g/?L;We detected the same positive stencil three time,and the results are in one mind.So the multiplex PCR was charactered with specificity,rapidity and accuracy Detection results of multiplex RT-PCR were 100%,100%and 96.7%which were in accordance with those of individual RT-PCR.The overall coincidencerate achieved to 98.9%.Moreover the specificity of the multiplex RT-PCR was up to 100%.It could be used for the laboratory detection of GARV,GCRV and AstV.2 Applied the Multiplex RT-PCR to detect clinical samplesThe positive rates of GARV,GCRV and PAstV were 20%(23/115),3.38%(4/115)and 12.17%(14/115),respectively.The count of creole infection aboutGARV,GCRV is 8,meanwhile GCRV,PAstV mix infection.is 2;GARV,GCRV and PAstV were detected simultaneously in sole sample.Our investigation about epidemiology of GARV,GCRV and PastV overlaided Sichuan province.the Prevalent season of the virus is winter;the main infected group was two month old;SuiNing and DeYang were the badly infected area.3 Analysised the genetic diversity of GARV,GCRV and PAstVAccording to the analysis of homology and evolutionary,we can be aware of slight difference between the GARV,GCRV virulent strain,respectively.It was 88.5%?95.4%homologous among GARV,88.7%?92%homologous comparedporcine A OSU(F317123),Si Chuan GARY strain had closer relationship with chinese virulent strainZZ-12and GD;It was 88.7%?95.5%homologous among GCRV,89%?93%homologous comparedporcine C CUK-5(EU002785),Si Chuan GCRV strain had closer relationship with south korea virulent strain.Otherwise,The epidemical disease serotypes of PAstV were 2 and 5.It was 97.7%?99.5%homologous among PAstV2,87.9%?89.2%homologous compared PastV5.The genetic homology was 87.9%?89.2%between PAstV2 and PoAstV/Krv7/cro,70%among others PAstV5.They had closer relationship with Croatia virulent strain.The consequent would make contributio to enrich molecular epidemiological data and control and Prevent GARV,GCRV and PastV in Sichuan province.