Establishment Of Molecular Identification Method Of Prrsv Infection And Immunity

Porcine reproductive and respiratory syndrome virus(PRRSV)mainly causes reproductive failure(such as pregnant sows miscarriage,stillbirth,mummified),piglets breathing difficulty and high mortality,with characteristics of fast spread,wide prevalence and highly contagious.Especially the highly pathogenic porcine peproductive and respiratory syndrom spread almost all over the country,and caused serious economic losses in pig industry.Because the PRRS exhibited very similar clinical symptoms as other diseases causing porcine reproductive disorders,the genes of new epidemic strains mutated with virulence increasing,and especially the mixed infection of PRRSV with other pathogens such as porcine circo virus,classical swine fever virus emerged,these resulted in complex and atypical pathological changes,which brought new difficulty in clinical diagnosis and disease prevention.In order to effectively control PRRS,pigs are required to vaccinate with PRRSV vaccines in our country.Due to instable immune effect of PRRSV inactivated vaccine,now PRRSV attenuated vaccine was used extensively.But most of the pigs inoculated with PRRSV attenuated vaccine will occur a certain period of viremia,carry the virus,or even discharge virus.Once pigs infect natural wild virus,it will be difficult to distinguish natural infection from vaccination,and not conducive to clinical diagnosis and control of PRRSV.Therefore,it is necessary to establish a new method for differential diagnosis of PRRSV natural infection and vaccination.RT-PCR method is a quick detection biotechnology,which can detect nucleic acid of virus in the blood,semen and tissue samples.Different genotypes of PRRSV strains can be distinguished by RT-PCR with specific primers.In this paper,we first established two conventional RT-PCR methods,and then detected samples from 2011 to 2014 by the method we established.The results are as follows:1 The establishment of conventional RT-PCR methodBoth of ORF5 gene and Nsp2 gene of PRRSV are highly variable regions in the genome.In this study,two conventional RT-PCR were built targetting GP5 and Nsp2 genes.Two pairs of primers for GP5(PI,P2)and five pairs of primers for Nsp2(N1～N5)were designed,and GP5 and Nsp2 genes were cloned.After screening and optimizing,the best primers were chosen(P2 and N3),and the specificity and sensitivity of conventional RT-PCR methods were tested.The results showed that conventional RT-PCR established in this paper only amplified PRRSV strains,and could not amplify CSFV,PPV,TGEV,PRV,FMDV,PCV2 and SIV.The sensitivity result of conventional RT-PCR about DNA duplexes was(GP5):700 copies.The sensitivity test results of the RT-PCR about genome(GP5):the dilution was 106.The sensitivity result of the RT-PCR about DNA duplexes was(Nsp2):80 copies.The sensitivity test results of the RT-PCR about genome(Nsp2):the dilution was 107.The results showed that conventional RT-PCR established in this paper had good specificity and high sensitivity.2 The clinical application of conventional RT-PCR method329 batches of blood samples,clinical samples and other materials from farms in Jiangsu from 2011 to 2014 were detected by the conventional RT-PCR methods we established.After positive samples were sequenced,we built the infected virus sequence library.Then we compared the viral sequences of all samples by primer P2 with the corresponding sequences of the vaccine used in these farms by DNAstar software.Then we analysed homology of the viral sequences with the sequences of the vaccine used in four farms.It was found the RT-PCR method established in this study can distinguish natural PRRSV infection from vaccine immune.In addition to this,the RT-PCR method by primer N3 could also distinguish classic PRRSV from high pathogenic PRRSV.