Establishment Of A Differential Method Of Highly Pathogenic Strain And Classical Strain Of PRRSV By SYBR Green-Ⅰ Real-time PCR Assay

Porcine reproductive and respiratory syndrome virus is classified in the family Arteriviridae, order Nidovirales, containing a single positive stranded RNA genome. PRRSV consists of two major genotypes, the European genotype and the North American genotype in terms of their different sequences. Prevalent PRRSV strains are all North Amercian genotype, since 1996 PRRSV (CH-1a strain) was isolated in our country. PRRS is an important infectious disease in swine herds and a compulsory immunized infectious disease listed by veterinary executive department. The large-scale of hightly pathogenic porcine reproductive and respiratory syndrome broken out in the south of China in 2006, which characterized by high fever, eye conjunctivitis, cough, ashma, neurological symptoms and high mortality. The infectious disease gives a negative impact to our national economy. The study has proved that highly pathogenic PRRSV belongs to the North American genotype and the Nsp2 gene sequence is deleted 90nt by the analysis of genomic sequences. At present, the pathogens of prelavent PRRSV belong to two strains. Both of them have similar symptoms and they are hard to be differentiated. In terms of above conditions, it is necessary to develop a differential diagnostic method in order to detect two kinds of strains.This study is aimed to develop a duplex real-time PCR assay in order to differential diagnostic detect both highly pathogenic and classical strains of PRRSV. All genomic sequences of the North American genotype PRRSV available in GenBank were aligned. The universal primers based on N gene sequence were designed and the amplifications of highly pathogenic and classical PRRSV can be acquired. The differential primers were designed according to the different sequences of Nsp2 gene between highly pathogenic and classical PRRSV. The amplification of highly pathogenic PRRSV can be acquired by the differential primers, while the amplification of classical PRRSV cannot be gained. A duplex SYBR Green-I real-time PCR assay was developed to distinguish between highly pathogenic and classical PRRSV with specific, sensitive and repeatable characteristics. The method can distinguish them by different Tm values of their products. The specific analyses using cDNA of JEV, CSFV, FMDV and PRCV as the templates were negative while amplifications of highly pathogenic strain and classical strain of PRRSV were positive. The sensitivity was 10 TCID50/mL. Inter- and intra-assay variables of CV were less than 0.3%. The results of the whole blood samples from 3, 5, 7, 10, 14, 21d pigs infected-experimentally with PRRSV were positive while the 28d samples were negative.Duplex real-time PCR assay is a qualitative method. The quatitative real-time PCR assay is developed in order to satisfy quantitative detection for some samples. According to Nsp2 gene of highly pathogenic PRRSV, the primers were designed and a 843bp PCR fragment was cloned. Then recombinant plasmids were determined by spectrophotometric reading and converted to molecular copies by the formula. The plasmid standards were prepared by serially diluting the recombinant plasmid. The SYBR Green-I real-time PCR assay was established, and its coeffcient correlation(R2) of the standard curve was 0.997. The specific test shows that the amplifications of JEV, CSFV, FMDV and classic PRRSV are negative. The sensitivity of real-time PCR is 10 copies/μL. The CVs were 1.57%, 1.82%, 0.76% of intra-assay of 1000, 100, 10 copies/μL standards; the CVs of inter-assays were 2.83%, 3.28% and 2.27%. The results of study illustrated that highly pathogenic PRRSV SYBR Green-I real-time PCR assay is specific, sensitive and repeatable. The results of detected samples from pig infected-experientally were the same as those of North American genotype PRRSV real-time PCR assay.The study has developed a duplex SYBR Green-I real-time PCR assay for highly pathogenic and classical PRRSV and a quantitative real-time PCR for highly pathogenic PRRSV. The former is to distinguish between highly pathogenic and classical PRRSV, and the latter can detect highly pathogenic PRRSV. Both are prepared for developing the differential diagnosis kit.