Analysis Of The Full Genome And Establishment Of Florescence Quantitative PCR Detection Of Porcine Kobuvirus

Picornaviruses are small, non-enveloped viruses with asingle-stranded, positive-sense genomic RNA. Porcine kobuvirus (PKoV)is a member of the gennus Kobuvirus of the family Picornaviridae. Thereis no evidence that PKoV can case disease, the long-term existence ofPorcine kobuvirus in the pigs may be pathogenic on host. So, it is necessaryto do research into PKoV. In this paper, we do researchs into PKoV fromepidemiological investigation, the whole genome sequence analysis,quantitative PCR method for detection of and preliminary applications, theresults are as follows:A pair primer was designed based on the3D region of genomeseuence of PKoV that were previously submitted to GenBank. A total of116stool specimens were collected from pigs of different ages in three pigfarms located in different districts around Shanghai (MinhangDistrict,Qingpu District and Fengxian District), from October to December,2010. Of all the specimens tested,45(38.8%) were positive forPKoV using RT-PCR. The prevalence rate of PKoV in the three pig farmswas46.7%(21/45),35.1%(13/37) and32.4%(11/34), respectively. Wedemonstrated that PKoV infections are existent in certain domestic pigs inShanghai. Statistical analysis of PKoV positive rate suggested that pigletsare more susceptible to PKoV than adults and the diarrhea may be relatedto the PKoV by Pearson’s chi-square test using SPSS13.0software. Thephylogenetic analysis revealed the presence of multiple PKoV lineages inShanghai using MEGA4.0software.Ten pair primes were designed based on complete genome seuence ofPKoV on GenBank, the sequence of SH-W-CHN in our study wasamplificated using RT-PCR. The complete RNA genome of SH-W-CHN isconsisted of8210nt, excluding its3’end poly-A tail and a large openreading frame of7467nt that encodes a potential polyprotein of2488aawas detected. More nucleotide mutations were found in structural than innonstructural regions compared with Y-1-CH(GU292559) andS-1-HUN(EU787450) in this study. Certain possible recombination signalswere identified using SimPlot software, and may be contributed to the highlevels of genetic diversity of porcine kobuvirus.A pair primer was designed based on genome of PKoV on GenBank.After optimizing of the reaction system, the SYBR Green I PCR was developed. The standard curve showed a a good linear relationship ftom7.02×101to7.02×109copies/μL, the correlation coefficient r2=0.9975,The method can detect the minimum of7.02×101copies/μL, while theconventional PCR can detect the minimum7.02×103copies/μL。Thefluorescent quantitative PCR is high stability, specifical and therepeatability coefficient is small (<1%), it suited to monitoring of PKoV orepidemic survey and research. The result of the comparison between thefluorescent quantitative PCR and conventional PCR showed improvedsensitivity.