美洲型猪繁殖与呼吸综合征病毒荧光RT-PCR检测方法的建立

Development of real-time RT-PCR for the detection of North American type porcine reproductive and respiratory syndrome virus

参照美洲型猪繁殖与呼吸综合征病毒(PRRSV)M基因序列,设计引物和探针,并建立其荧光RT-PCR检测方法,以检测严重危害我国养猪业的此型PRRSV。该法能检测到1 TCID50的PRRSV。用此法检测猪瘟病毒、乙脑病毒、伪狂犬病毒、马动脉炎病毒、圆环病毒Ⅱ型和MARC145细胞的RNA,结果均为阴性。对126份临床样品进行检测应用,检测结果(7份阳性,119份阴性)与病毒分离完全符合。此法中的阳性对照为体外转录的dsRNA,具有高度的稳定性,解决了此类检测方法所用的RNA阳性对照所普遍存在的易于降解的难题。

A real-time RT-PCR assay for the detection of North American type porcine reproductive and respiratory syndrome virus （PPRSV） was developed using a pair of primers derived from the published sequences of the Matrix gene of PPRSV. This assay could detect 1 TCID50 of the virus with a high specificity. No products detected against RNAs of hog cholera virus, Japanese encephalitis B virus, porcine pseudorabies virus, equine arteritis virus, porcine circovirus virus Ⅱ and MARC 145 cell. Testing on 126 clinical samples detected 7 positive virus infection, which was consistent with results of virus isolation. This assay used an in vitro transcribed dsRNA RNA as positive control, which provides a reliable alternative over conventional and less stable single stranded RNA control.