Sequenced The ORF3 Gene And Established A SYBR Green ? Quantitative Real-time PCR Method For Porcine Torque Teno Sus Virus K2

Torque teno virus is small,non-enveloped virus with a single-stranded,circular DNA genome of negative polarity,belonging to the genus Anellovirus,family Circovirdae.The viruses have been detected not only in humans but in non-human vertebrates,pigs,cattles,sheep,cats,dogs,sea lions and so on.The porcine Torque teno virus which is widespread in most of swine herds in the world has attracted considerable interest.Epidemiogical study has shown that the porcine Torque teno virus partially contributed to the experimental induction of porcine dermatitis and nephropathy syndrome(PDNS)combined with porcine reproductive and respiratory syndrome virus(PRRSV)infection,and also to the experimental induction of postweaning multisystemic wasting syndrome(PMWS)combined with PCV2 infection in a gnotobiotic pig model.And porcine Torque teno viruses maybe take part in infectious hepatitis of pigs with PCV2 or hepatitis E virus(HEV).Because the porcine porcine Torque teno virus is a potential risk to swine industry,it is necessary to study it.Two primers were used to amplify the ORF2 gene of porcine torque teno sus virus k2 from the DNA isolated in the feces with diarrhea at Fujian.The target PCR fragments were cloned to the T-vector and sequence.The sequenced results were identified by BLAST analysis,and splicing the sequences with SeqMan software.The results showed that the cloned ORF2 gene of porcine torque teno sus virus k2 was 600 bp in length,coding an open reading frame(ORF)with 199 amino acids.The nucleotide sequence and the amino acid sequence deduced from the gene were analyzed by the bioinformatics software.Compared with the porcine torque teno sus virus k2 ORF2 genes from GenBank,the sequenced gene shared the highest homogeneity with TTV2XYF7 strain at 99.9%(GenBank accession number JX535334),shared 98.8%homogeneity with German porcine torque teno sus virus 2p strain(GenBank accession number AY823991).However,the isolates shared only 38.8%homogeneity with Japan porcine torque teno sus virus Sd_TTV31 strain(GenBank accession number AB076001),and also shared only 43.1%homogeneity with German porcine torque teno sus virus 1p strain(GenBank accession number AY823990).In order to establish a rapid and quantization detection method for Porcine Torque teno sus virus k2,a SYBR Green I-based quantitative real time PCR method was established using specific primer-pair,targeting to the ORF2 gene of Porcine Torque teno sus virus k2.The detection assay was linear in the range of 1.92×102?1.92×107 copies per microliter.Series of experiments were carried out to assess the sensitivity,specificity and reproducibility of the method,following by the intra-assay and inter-assay CVs for CT values obtained with the standard plasmids.The melting curve analysis using SYBR Green I dye showed one specific peaked with a melting temperature is(83.83±0.08)?,with no primer-dimers peak represent.The intra-assay CVs were equal or less than 1.69%and the inter-assay CVs were equal or less than 2.19%.There was no cross-reaction occurred with nucleic acids extracted from porcine circovirus(type 1 and type 2),porcine parvovirus,porcine bovavirus type 5 and porcine Torque teno virus 1a and 1b.The results demonstrated that the assay was specific and reproducible,which means the quantitative PCR can be used for rapid laboratory diagnosis and epidemiology investigation with Torque teno sus virus k2.