Establishment Of A Dual Fluorescence RT-PCR For Discrimination Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome And Vacine Strain JXA1-R

Porcine reproductive and respiratory syndrome(PRRS)is an acute,highly contagious disease clinically characterized by sow reproductive disorders and a variety of day-old pigs(especially pigs)respiratory symptoms,the pathogens is porcine reproductive and respiratory syndrome virus(PRRSV).Piglets of the infected sows generally have low survival rate after weaning,grown in poor condition if resistance.The major epidemic strain in China is the classic PRRSV,in 2006 highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)appeared.At present,vaccination is the main measures to prevent the HP-PRRSV.The vaccine includes JXA1-R,HuN4-F112 and TJM-F92,among them,the JXA1-R is the most widely used.This study successfully established a dual fluorescence RT-PCR for discrimination of HP-PRRSV and vaccine strain JXA1-R.The main research content is as follows:1.Establishment of fluorescence RT-PCR for detection of HP-PRRSVAccording to the comparison of whole genome sequence between HP-PRRSV and classic PRRSV in GenBank,designed a pair of primers and a probe at HP-PRRSV nsp2,established a fluorescence RT-PCR for detection of HP-PRRSV.The sensitivity of the mothod was 4.43copies/?L,R2 of standard curve was 0.998;Evaluated the repeatability with three different dilution standard plasmid,demonstrated that the method had good repeatability,the variation coefficient within group was 0.39%-0.96%,the variation coefficient between group was 1.1%-2.6%.The method was applied to detect several viruses,such as classical PRRSV,HP-PRRSV,PCV,PEDV,PRV,TGEV,only HP-PRRSV could be detected,showed that the method had high specificity.The detection sensitivity of the method was 1000 times of ordinary RT-PCR.2.Establishment of fluorescence RT-PCR for detection of JXA1-RAccording to the comparison of whole genome sequence between HP-PRRSV and JXA1-R,designed a pair of primers and a probe,established a fluorescence RT-PCR for detection of JXA1-R.The sensitivity of the mothod was 8.25copies/?L,R2 was 0.999;The method had good repeatability,the variation coefficient within group was 0.42%-1.9%,the variation coefficient between group was 0.68%-2.74%;The method was applied to detect several viruses,such as classical PRRSV,HP-PRRSV,PCV,PEDV,PRV,TGEV.There were no cross reaction,showed that the method had high specificity.The detection sensitivity of the method was 1000 times of ordinary RT-PCR.3.Establishment of dual fluorescence RT-PCR for discrimination of HP-PRRSV and JXA1-R.On the basis of the single fluorescent RT-PCR,after optimization of the detection conditions,established a dual fluorescence RT-PCR for discrimination of HP-PRRSV and JXA1-R.The minimal detection limits of HP-PRRSV and JXA1-R were 4.43 copies/?L and 4.12 copies/?L;The maximum variation coefficient of HP-PRRSV within group was 0.70%,the maximum variation coefficient between group was 2.8%,the maximum variation coefficient of JXA1-R within group was 0.62%,the maximum variation coefficient between group was 3.0%,no more than 5%,showed that the method had good repeatability;The method was applied to detect several viruses,such as classical PRRSV,PCV,PEDV,PRV,TGEV,etc.There were no specific reaction,showed that the method had high specificity.200 blood samples were tested by the established method.The fluorescence RT-PCR for detection of HP-PRRSV detected 32 positive,positive rate is 16%(32/200);The fluorescence RT-PCR for detection of JXA1-R detected 9 positive,positive rate is 4.5%(9/200);200 blood samples were tested by the dual fluorescence RT-PCR method,the results are consistent with the single fluorescent RT-PCR method,showed the established method had great value in clinical application.