| Porcine reproductive and respiratory syndrome virus(PRRSV)is one of the most important pathogens,which causing a highly harmful immunosuppressive respiratory-borne disease that has had a huge impact on pork production worldwide.PRRSV is an RNA virus.The most dangerous is gene mutations and recombination,which have led to the emergence of new variant strains.Nowdays,the clinical epidemic strains mainly belong to American-type PRRSV which including highly pathogenic mutant strains(HP-PRRSV),classic strains(C-PRRSV),and NADC30-like strains(NL-PRRSV).Thus it is difficulty to make correct diagnosis and to control this disease.At present,there are many detection methods for the virus,but there are not many studies on the differential diagnosis methods of the prevalent strains.AS there lacks the corresponding commercial detection kits at present.GP5 protein is an important immunoprotective protein of PRRSV,and GP5 gene mutation can cause changes in viral antigenicity or virulence.Strengthening the monitoring of GP5 gene mutations in PRRSV epidemic strains and establishing methods for detecting epidemic strains is of great significance for disease prevention and control.The main research contents are as following:1.Porcine Reproductive and Respiratory Syndrome Virus Detection and GP5 Variation Analysis in some areas of China in 2018Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)is an important pathogen that causes significant economic losses to the global pig industry.In order to study the molecular epidemiological characteristics of PRRSV in China,this study collected lung tissue samples from 258 clinically in 11 provinces and cities in China,including Jiangsu,Zhejiang,Shandong,Anhui,Shanghai,and Fujian in 2018.142 were positive for PRRSV,the positive rate was 55%.Randomly,sequencing and analyzing the ORF5 of 43 PRRSV strains.The results were as follows:the 43 sequenced strains in this study were the American type and could be divided into 3 gene subtypes,of which 26 were NADC30-like strains,accounting for up to 60.5%.In some PRRSV strains,GP5 protein A epitope presented V29?A29 mutation,and B epitope presented H38?K38 mutation.The number of GP5 protein glycosylation sites is increasing and complicated.The main glycosylation sites are located at:N30,N33,N34,N35,N44 and N51,indicating that PRRSV epidemic strains in China have been undergoing genetic mutations.The study provides a reliable theoretical basis for preventing and controlling the disease.2.Establishment and Application of SYBR Green ? Fluorescent Quantitative PCR Method for Porcine Reproductive and Respiratory Syndrome VirusIn this study,a pair of primers was designed based on the porcine reproductive and respiratory syndrome virus(PRRSV)ORF7 gene sequence,and a quantitative real-time PCR method with SYBR Green I dye was established.The method is highly specific to PRRSV,which having no cross-reaction with porcine epidemic diarrhea(PEDV),swine fever virus(CSFV),Seneca virus(SVV),encephalomyocarditis virus(EMCV),pseudorabies virus(PRV)and porcine circovirus 2 Type(PCV2).By using different titer PRRSV measurement,the critical for the viral diagnosis was determined as following:when CT?32 and showing the obvious exponential amplification curve,it was judged as PRRSV positive,CT?32 or no CT value was judged as PRRSV negative.The detection limit of the virus with this method is 100.5TCID50 of the virus,which being higher than that with the national standard of conventional RT-PCR(GB/T18090-2008).The intra-and inter-assay coefficient of variation is less than 1%.The 15 lung samples and 60 serum samples of PRRSV in the PRRS V-infected piglets were positive,and 8 lung samples and 25 serum samples from the negative piglets were negative.The 245 lung tissue samples from the clinical piglets were detected by using this method and conventional RT-PCR.The results showed that the positive rate of PRRSV detected with the SYBR Green I real-time PCR was 67.35%,which was higher than that with conventional RT-PCR(57.14%).It indicated that this SYBR Green I real-time PCR can be used for detection of PRRSV in clinical sample of pigs.3.Establishment and Application of TaqMan Fluorescence Quantitative PCR Detection Methods for Two Subtypes of PRRSVThe study designed primers and TaqMan probes for HP-PRRSV and NL-PRRSV NSP2 respectively by comparing HP-PRRSV,NL-PRRSV and classic PRRSV(C-PRRSV)genome sequences.This study has established a dual TaqMan fluorescent quantitative PCR method that can detect HP-PRRSV and NL-PRRSV.When the red amplification curve shows obvious exponential amplification and CT?32,it is judged as HP-PRRSV positive;CT? 34 or no CT is judged as HP-PRRSV negative;when 32 |
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