Establishment And Application Of Double PCR With Porcine Torque Teno Virus ?and ?

ObjectiveWe designed 2 pairs of specific primers according to complete conservative genome sequences and established duplex PCR method to investigate porcine infections in Liaoning province.At the same time,we made genetic variation analysis on the complete genomic sequence,and the results provided certain theory basis on the studies of PTTV molecular epidemiology in Liaoning province.MethodsWe used Oligo7.0 to design two pairs of specific primers in reference to the complete conservative genome sequences of PTTVI(GU570202)and PTTVII(AY823991)in Gen Bank.On the basis of single PCR,we established duplex PCR detection method.We differentiated two genotypes of infection of PTTVI and PTTVII,and optimized the duplex PCR reaction conditions to verify the accuracy of the method through the specificity,sensitivity and repeatability test.We made it clear about PPTV infections in Liaoning area,using the established duplex PCR detection method to test collected blood samples of 14 cities in Liaoning province.We used PCR segmented amplification to amplify complete genomic sequence of PTTVI and PTTVII.We made cloning sequencing respectively of PCR products and got complete genomic sequence by DNAMAN6.0 software for Mosaic.Then we used DNASTAR 7.1(By Jotun Hein Method)software to make homology comparison analysis with the isolate gene sequence at domestic and abroad.We used MEGA software(Neighbor-joining method)to build system evolutionary tree,making analysis of the genetic variation features of the strains in Liaoning.Results1.We used Oligo7.0 software to design two pairs of specific primers according to PTTVI and PTTVII conservative gene sequences and established duplex PCR detection method.This method can got specifically amplified PTTV two different strains of purpose gene,and the differences of fragment size were above100 bp which was convenient to observe electrophoresis.Recombinant plasmid standard detection was under the limit of 1.0 x 105 copies.2.The results showed that healthy porcine PTTVI and PTTVII infection rates were 36.44% and 60.16% respectively;PTTVI and PTTVII mixed infection rate was 21.46%;unhealthy porcine PTTVI and PTTVII infection rates were 39.76% and 63.94% respectively;PTTVI and PTTVII mixed infection rate was 29.25%,after testing 501 samples of healthy and unhealthy porcine blood with the established duplex PCR method.3.The full-length gene of Liaoning strain TTV1 LNJZ and TTV2 LNSY were 2894 bp and 2804 bp respectively,and the accession number of Gen Bank were KT968712 and KU156681 respectively.Two genotypes both had four open reading frames(ORFs).Compared with 20 PTTVI type strains at domestic and abroad,the homology were 58.5% ~ 96.4%.Compared with 20 PTTVII type strains at domestic and abroad,the homology were 70.5% ~ 96.7%.Conclusions1.This study successfully constructed PTTV duplex PCR detection method which had the advantages of rapidity,accuracy and sensitivity.2.There were both infections of PTTVI and PTTVII in Liaoning province and the infection rate was high.Healthy and unhealthy porcine infection rates of PTTVI were 36.44% and 39.76% respectively.Healthy and unhealthy porcine infection rates of PTTVII were 60.16% and 63.94% respectively.3.The homology of the complete genomic sequence of Liaoning strain TTV1-LNJZ and TTV2-LNSY with strains at domestic and abroad were 58.5% ~96.4% and 96.4% ~ 70.5% respectively.The genetic evolutionary relationship of TTV1-LNJZ strain and 1p strain(AY823990.1)was closest.The genetic evolutionary relationship of TTV2-LNSY strain and TTV2XYF7 strain(JX535334.1)was closest.