高致病性猪繁殖与呼吸综合征病毒双重液相基因芯片检测方法的建立

为了实现快速检测猪繁殖与呼吸综合征病毒(PRRSV)并同步鉴别高致病性PRRSV毒株(HPPRRSV),根据PRRSV囊膜蛋白GP2基因保守序列和HP-PRRSV特有的Nsp2基因区保守序列设计特异扩增引物和杂交探针,通过双重一步法RT-PCR不对称扩增和双重微球杂交反应,建立了双重液相基因芯片方法。对12株PRRSV以及其他12种猪病原体的检测显示,该法能特异性检测12株PRRSV毒株并准确鉴别其中7株HP-PRRSV,与其他病原体无交叉反应;对PRRSV、HP-PRRSV病毒液的检测低限均小于每个反应0.5TCID_(50);其组内、组间检测变异系数均10%;检测55份疑似临床样品并与商品化荧光RTPCR试剂盒比较检测结果,符合率达98.2%(54/55)。研究结果为适应临床快速检测PRRSV提供了一种新的分子生物学检测方法。

Development of a Duplex xMAP Assay for Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

A duplex xMAP bead-based assay was developed for rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV)and differentiation of highly pathogenic strains (HP-PRRSV).The xMAP assay could simultaneously detect two target sequences,including the GP2 envelope protein gene of PRRS and the Nsp2 gene conserved region specific for HP-PRRSV strains.The assay detected 12 PRRSV strains used in this study and identified 7 strains as HP-PRRSV,without cross-reactions with 12 non-tar-get swine pathogens.The lower detection limit of the assay on PRRSV,HP-PRRSV strains was deter-mined as below 0.5 TCID50 per reaction,respectively.The inter-assay and intra-assay variances (CV%) were both lower than 10%.The assay was applied to test 55 suspected field samples and compared with a commercialized single-plex real-time RT-PCR method.The detection results of the two methods were 98.2% (54/55 )in consistency.The study provided a novel molecular method for rapid diagnosis of PRRSV infection.