Establishment Of One-step Reverse-transcription Pan-PRRSV Differential FRET-qPCR And Its Application

Porcine reproductive and respiratory syndrome(PRRS),first recognised in the USA in 1987,is characterised by respiratory disease in young pigs and severe reproductive failure in sows,including abortion,stillbirths and weak piglets.PRRSV has caused immense economic losses in the pig industry and is considered to be one of the most important infectious diseases of pigs in the world.The disease is caused by porcine reproductive and respiratory syndrome virus.According to the differences in antigenicity,there are currently two recognized PRRSV genotypes:the European type represented by Lelystad virus(LV strain)and the American type represented by ATCC VR-2332(VR strain)strain.PRRSV was first isolated from aborted fetuses by GUO Baoqing in China in 1996.In 2006,a highly pathogenic virus strain(HP-PRRSV)was identified in pigs in southern China.At present,the main way for the prevention and control of PRRSV in China is to vaccinate with the modified-live vaccine,including JXA1-R,TJM-F92 and Ingelvac.Clinically,it is difficult to accurately diagnose the disease caused by PRRSV,due to the potentials of coinfections by highly pathogenic strain,classical strain and vaccine strains of PRRSV.At present,the main PRRSV detection methods are viral isolation,serology and molecular biology diagnostic technology.Viral isolation is time consuming,not suitable for timely detection;serological method shows a low specificity.In comparson,molecular biological diagnostic technology demonstrates greater sensitivity and specificity,but can't detect and differentiate multiple PRRSV strains simultaneously.Therefore,this study was to establish a rapid sensitive and can detect differentiate multiple PRRSV strains method,and successfully applied to pigs PRRSV testing of farms and slaughterhouses in China.1.Establishment of a highly pathenogenic strain,pathenogenic strain and vaccine strains,one-step reverse-transcription FRET-qPCR methodIn this study,we designed a pair of specific primers and probes based on Nsp2 gene,and established a one-step reverse transcription FRET-qPCR method to detect and differentiate highly pathogenic PRRSV strain,classical PRRSV strain and vaccine strains(PCR products:197 bp).The method established in this study was used to detect the standard strains prepared by PRRSV and the results showed that the sensitivity was 1 copy/reaction.In addition,different dilutions of highly pathogenic strain(HP-BB)and vaccine strain(JXA1-R)nucleic acids were mixed with SPF pig blood samples,experiment with spiking test to verify the sensitivity of the method,the results show that one-step reverse transcription FRET-qPCR method has highly sensitivity in this study.By analyzing the melting curve of five PRRSV strains,it was found that there was a difference in Tm between PRRSV strains,which could be divided into five groups,indicating that this method was highly specific.In conclusion,the method established in this study has highly sensitivity and specificity,which can simultaneously detect and distinguish 5 common highly pathogenic PRRSV strain,classical strain and vaccine strains in China.2.Application of multiple PRRSV strains,one-step reverse-transcription FRET-PCR methodThe one-step reverse transcription FRET-qPCR established in this study was used to detect the clinical samples of healthy swine in 10 provinces of China.The results showed that PRRSV positive rate was 3.3%(28/846)in clinical samples from 7 regions.The positivity was found to be highest in assayed lung specimens while the mixed infections of vaccine strains and wild virus strains were also identified and confirmed by DNA sequencing.In conclusion,the established one-step reverse transcription FRET-qPCR method was highly senstitive,being able to detect a single copy Nsp2 gene per reaction.The established method can detect and differentiate 5 types of PRRSV strain and discriminate them based on high-resolution melting curve analysis.The established method in this study provides a valuable tool for clinical monitoring and prevention and control of PRRS.