Establishment And Application Of A Real-time RT-PCR Method Based On SYBR Green Ⅱ For Detection Of Porcine Sapelovirus

Porcine sapelovirus infection is caused by porcine sapelovirus, is a infectious diseases of swine and is associated with diarrhea, pneumonia, severe neurological disorders, reproductive failure or no obvious symptoms in pigs. A TaqMan real-time RT-PCR assay was established for the detection of porcine sapelovirus in this study, The tissue specificity of PSV infection was studied by this method. and Investigations were carried out to identify the PSV in Sichuan province, China, and analyzed the genetic variation of PSV. The availability of this study for PSV will facilitate future research of PSV.A TaqMan real-time RT-PCR assay was established for the detection of porcine sapelovirus. A pair of special primers was designed according to the highly conserved fragmen of the 3D gene in GenBank and a real-time reverse transcription polymerase chain reaction (QRT-PCR) based on SYBR Green II was developed for quantization of porcine sapelovirus. The results indicated that no amplification was detected from other DNA samples by this method, such as CSFV, PRRSV, PKoV, PEDV, TGEV and RV. The detection limit of the method was 1.44×102 copies/μL of initial templates, which was 100-fold more sensitive than that of the conventional PCR. And The melting curve analysis using SYBR Green Ⅱ dye showed one specific peaked with no primer-dimer peak. Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay and inter-assay of less than 1.5%. Samples of tissues were collected from 3 pigs which infected PSV were detected by the established real-time PCR for virus content, the results demonstrated that the PSV were distributed widely, and there is a large difference in different tissue, the virus loading level of intestine and feces is the highest. a total of 428 fecal samples were collected from large-scale pig farm across different districts of Sichuan Province from 2014 to 2015. We detect all samples by real-time PCR, The results show that the infection of PSV among regions,114 (26.6%,114/428) were positive for PSV. And the positive rate in different age group show that the infection rate of 5-9 week old Digs was the highest (37.4%,55/147). The results proved that the ORT-PCR was specific and sensitive to detect porcine sapelovirus, which can support the clinic detection, quantitative analysis and molecular epidemiological investigation.In order to understand the infection situation of PSV and the variation of the strains in Sichuan area. A pair of special primers was designed according to the 1B gene in GenBank. The 14 novel PSV 1B nucleotide sequences were obtained by RT-PCR which was confirmed by sequencing (GenBank accession Numbers: KU323644-KU323657), Nucleotide and the deduced amino acid sequence homologies were between 90.6-100% and 97.1-100% respectively. Phylogenetic analysis indicate that the novel Sichuan strains is belong to a group with the strains from china, there is no major gene mutation, which showed that the gene of PSV are highly conserved in china. the novel Sichuan strains、orea strains、Germany strains and Britain strains are in different branches, which can indicate that the epidemic strains of PSV in our country may be the stable strains rather than the introduction of the new epidemic.