Establishment And Application Of RT-PCR For Differential Diagnosis Of Highly Pathogenic PRRSV

Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a infectious diseases caused by porcine reproductive and respiratory syndrome virus (PRRSV).It cause reproductive disorder in sows and respiratory diseases in piglets.PRRSV consists of two genotypes,the European genotype and the American genotype.Since the first strain CH-1a was isolated in 1996,prevalent strains in our country are all American genotype.After 2006, highly pathogenic porcine reproductive and respiratory syndrome variant strains (HP-PRRSV) began to epid,which characterized by "high fever".The HP-PRRSV was variant strain with 90bp nucleotide deletion in nonstructural protein2(NSP2) by genome sequencing.It is essential to establish a method to diagnose and distinguish the two PRRSV strains.According to multiple sequence comparison of highly pathogenic PRRSV strain and classical PRRSV strain Nsp2 gene published in GenBank,a pair of primer that contains deletion region was designed. For the classical strains and the highly pathogenic strain of PRRSV,a fragment of 576bp and 486bp could be respectively amplified. According to the different fragments of RT-PCR, the classical strains and the highly pathogenic strain of PRRSV can be distinguished by once RT-PCR. A specific, sensitive, reproducible antidiastole method was established,which can quickly distinguish the two different strains of PRRSV.The detection of classical swine fever virus (CSFV), porcine parvovirus virus (PPV), porcine pseudorabies virus (PRV), porcine circovirus typeâ…¡(PCV2) by this method was negative, which indicated that the method is specificity. The sensitivity test of the RT-PCR revealed that the detection threshold of the PCR was 10-5 and 10-4 of viral cDNA concentration for highly pathogenic strain and classical strains. And the 3 times repetition test of the same sample indicated that the RT-PCR was reproducible. Detected 38 suspected materials that collected from various regions of Hebei Province by this method,the result shows 20 samples were positive while 16 samples were highly pathogenic PRRSV. The result of comparison test were conformity,which indicated that the detection method was high accuracy.The porcine reproduetive and respiratory syndrome virus isolated from the 4 positive sample all emerge typical cytopathic (CPE) in Marc-145, identified and named QHD1, TS01, TS002, TS55.By cloning and sequencing the partial Nsp2 gene of the 4 isolated strain,it is found that they had the same deletion compared with highly pathogenic strain. They were all 30 amino acids non-consecutive deletion in 481 and 532~560, wich indicated that the strains are variant strains. Throug homology and phylogenetic analysis of major epidemic PRRSV strains,it is highly homologous with the highly pathogenic strains that isolated recent years.This provid science evidence for effective diagnosis of PRRS and enriched the epidemiology data of PRRSV.