Complete Genome Sequence Analysis Of PPV6 Anhui Strain And Establishment Of The QPCR Detection Method

Porcine Parvovirus is one of the main pathogens causing reproductive disorders in pregnant sows,which often causes abortion,infertility,stillbirth,mummified fetus and other symptoms in sows.The virus can also cause diarrhea,dermatitis and other symptoms of newborn piglets,bringing huge economic losses to the world pig industry.PPV is often mixed with respiratory viruses to aggravate its infection hazards.Therefore,it is very important to strengthen the detection and prevention of PPV.PPV6 was first discovered in china in 2014.However,there are few researches on this type.In this study,the whole genome of a PPV6 positive sample obtained from Anhui region was cloned and sequenced,and the obtained NS1 and Cap gene sequences were bioinformatics analyzed.Finally,a rapid detection method for PPV6 SYBR Green I fluorescence quantitative PCR was established.The specific research contents are as follows:Ten pairs of specific primers were designed to amplify the whole genome sequence of the samples tested positive for PPV6 by PCR and named PPV6 AH strain.Sequence analysis was carried out between PPV6 AH strain and other PPV6 reference strains.The results showed that the nucleotide homology between PPV6 AH strain and reference strain was 97.1% ~ 99.5%.The phylogenetic tree shows that AH strain has the same evolutionary origin with BJ,BJ2,SC and JS strains.NS1 gene and Cap gene have 3 and4 nucleotide site mutations respectively.The amino acid of Cap protein mutated at positions 402 and 404.This study reported for the first time that PPV6 carried infection in pigs in Anhui province.the results of this study provide some references for molecular genetic evolution of PPV6 in Anhui province.The NS1 and Cap protein sequences of PPV6 AH strain were analyzed by bioinformatics software.The results showed that NS1 protein belongs to hydrophobically unstable protein,with 5 transmembrane domains,? helix as the main body,98 potential phosphorylation sites,7 B cell antigen epitopes,2 CTL epitopes and 10 Th epitopes.Cap protein is a hydrophilic and unstable protein with irregular helix as the main body,with233 potential phosphorylation sites,5 B cell antigen epitopes,2 CTL epitopes and 1 Th epitope.This experiment successfully predicted the basic biological characteristics of NS1 and Cap proteins of PPV6 AH strain,which laid a certain foundation for revealing the pathogenic mechanism of PPV6 and the subsequent epitope vaccine development.The constructed recombinant plasmid p MD19T-PPV6-Cap was used as the positive standard plasmid.The SYBR Green I fluorescence quantitative PCR amplification was carried out using the recombinant plasmid diluted 10 times as the template.The sensitivity,specificity and repeatability were verified by optimizing the reaction conditions.The results showed that the method had a good linear relationship in the range of4.78×108~4.78×101 copies/?L,the minimum detection amount of virus was 47.8 copies,and the sensitivity was 1000 times higher than that of conventional PCR electrophoresis.The method has no cross reaction with pathogens such as PPV7 and PCV2.The coefficient of variation of intra-group and inter-group repeatability experiments are all less than 1%,with good repeatability.The detection results of clinical samples show that the positive detection rate of the established fluorescence quantitative PCR is 7.78% higher than that of the traditional PCR.PPV6 SYBR Green I fluorescence quantitative PCR detection method successfully established in this experiment can be used for rapid detection of ppv6 infection in clinic.