猪繁殖与呼吸综合征病毒实时荧光定量PCR检测方法的建立及初步应用

Establishment and Preliminary Application of a Real-time PCR for PRRSV Detection

为建立猪繁殖与呼吸综合征病毒(PRRSV)的一种特异、灵敏、量化且耗时短的检测方法,通过设计1对PRRSV ORF7基因的引物,建立Eva Green染料的实时荧光定量PCR(real-time PCR)的检测方法,对该方法的特异性、灵敏性和重复性进行检验,并检测PRRSV疑似感染的临床病料。结果显示,所建立的real-time PCR检测方法标准曲线的线性相关系数R2=0.999,具有良好的线性关系;与传染性胃肠炎病毒(PEDV)、猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)无交叉反应,特异性强;与常规RT-PCR检测方法相比,灵敏度提高100倍;重复试验表明变异系数系数在1%以下,重复性良好。对27份临床样品进行检测,结果比常规PCR多检出3份阳性。说明建立的real-time PCR检测方法在特异性、灵敏度、重复性方面均相比良好,可用于临床PRRSV的检测。

To obtain a specific, sensitive, quick and quantitative detection method of porcine reproductive and respiratory syndrome virus （PRRSV）, specific primers were designed on the basis of PRRSV ORF7 gene, and Eva Green real-time PCR was established. The specificity, sensitivity, repeatability were tested, and clinical samples were used for detecting PRRSV.The results showed that the standard curve of real-time PCR revealed a good linear relationship with the correlation coefficient R2=0.999.The method has high specificity and no cross-reactions with PEDV, CSFV,PRV and PCV2,100 times sensitive than conventional RT-PCR, and good repeatability with the coefficient of variation lower than 1%.This method can be applied to clinical detection of PRRSV because of good specificity, sensitivity and repeatability.