Development Of A Real-Time Taqman PCR Assay For The Detetion Of Porcine Torque Teno Virus And Investigation Of Torque Teno Virus Prevalence In Swine

The Torque teno virus (TTV) is a small, nonenveloped, single-stranded virus with a circular DNA genome of negative sense.The virus was first isolated in1997from a Japanese patient with post-transfusion hepatitis. Because of its circular genome, TTV was initially classified in the family Circoviridae, which includes the genera Circovirus and Gyrovirus,but was recently reclassified in the’floating’ genus Anellovirus at2008. Nowadays TTV infection is widespread throughout the world, and it is exist in several domestic animals, including pigs,cattle, sheep, cats and dogs and the significantly high prevalence have been discovered. The research about the Porcine TTV reported the most, and it showed highest prevalenc.Porcine TTV occurs in two genogroups,TTV1and TTV2.Two Taqman realtime fluorescence quantitative PCRs were established using two sets of primers and probes based on the conserved untranslated region(UTR) of porcine Torque Teno virus genotype1and2(TTV1and TTV2) The UTR sequences of TTV1and TTV2were amplify by nested PCR and cloned into the pMD18-T vector. The recombinant plasmids is used as template to optimize Taqman real-time PCR conditions. A series of diluted recombinant plasmids were used to generate standard curves.The low detection limit of the TTV1rt-PCR assay we developed was9.2copies per microliter.When the range of external standard was9.2×107copies-9.2×102copies per microliter.,the correlation coefficient of standard between the input copies and fluorescence intensity was1.000,the slope of standard curve was-3.398. The efficiency is96.5%It was found that the specificity was high without any cross-reactions with PRV、PPV、PCV2and TTV2. The intra-assay reproducibility CV were below1%,the inter-assay reproducibility CV were below3%It shows good repeatability.The low detection limit of the TTV2rt-PCR assay we developed was56copies per microliter.When the range of external standard was5.6×106copies-5.6X101 copies per microliter.,the correlation coefficient of standard between the input copies and fluorescence intensity was0.998,the slope of standard curve was-3.440. The efficiency is95.3%It was found that the specificity was high without any cross-reactions with PRV、PPV、PCV2and TTV1. The intra-assay reproducibility CV were less than1%,the inter-assay reproducibility CV were less than3%. It shows highly reproducible.To demonstrate usefulness of the two rt-PCR assay for large-scale testing,220serum samples from domestic pigs were screened for TTV infection. There was no significant difference on the prevalence of genogroups1and2, being83.6%(184/220) and84.5%(187/220) respectively. From all the studied swine71.8%(158/220) were coinfected with both TTV genogroups.To allow high-throughput testing, we describes a development of a duplex real-time PCR assay for efficient simultaneous detection of TTV1and TTV2.The low detection limit of the duplex rt-PCR assay we developed was9.2copies per microliter.for TTV1and56copies per microliter for TTV2. Standard curves of TTV1obtained showed an efficiency of97.8%, a slope of-3.377and regression coefficients (R2) were1.OOO.Standard curves of TTV2obtained showed an efficiency of96.2%, a slope of-3.416and regression coefficients (R2) were0.998.It was found that the specificity was high without any cross-reaetions with PRV、PPV、PCV2. The intra-assay reproducibility CV were below1%,the inter-assay reproducibility CV were below3%.It shows highly reproducible.When using the duplex rt-PCR assay we developed for large-scale testing, The prevalence of TTV1and TTV2being82.72%(182/220) and83.18%(183/220) respectively. From all the studied swine,68%(150/220) were coinfected with both TTV genogroups.In order to compare the Taqman real-time PCR assay developed in this study,we also choose the other two molecular methods to detect the TTV prevalence of the220serum samples. The real-time PCR assay we developed provided better detection results for the two TTV genogroups in swine compared to the other two molecular methods. These new Taqman real time PCR assays has the advantages of sensitivity and specificity and efficiency for large-scale screening,and to evaluate the viral load in swine herds.