猪繁殖与呼吸综合征病毒荧光定量PCR检测方法的建立  

Quantitative TaqMan real-time PCR for detection of porcine reproductive and respiratory syndrome virus

作　　者：徐辉 方立[1,2] 陈伟杰 赵灵燕 倪柏锋 方维焕[2] 

机构地区：[1]浙江省畜牧兽医局,浙江杭州310020 [2]浙江大学动物预防医学研究所,浙江省重点实验室,浙江杭州310029

出　　处：《浙江农业学报》2008年第5期328-332,共5页Acta Agriculturae Zhejiangensis

基　　金：浙江省科技厅项目(2005C22032)

摘　　要：针对猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)N基因保守序列设计引物与TaqMan探针,在建立常规PCR的基础上,设计并优化了TaqMan荧光定量PCR方法用于PRRSV核酸的检测。应用新建立的荧光定量PCR方法检测其它主要猪病病原,无任何非特异性反应;针对PRRSV阳性克隆质粒的检测灵敏度可以达到1.2×101copy/ml,比常规PCR高100倍;通过对48份临床疑似样本的检测表明有29份样本为PRRSV阳性,与巢式PCR检测结果一致,而常规PCR只能检出其中19份阳性样本,灵敏度优于常规PCR方法。结果表明,试验建立的PRRSV TaqMan荧光定量PCR方法具有良好的特异性、敏感性和重复性,可为PRRSV临床诊断提供一个更为有效的方法。A TaqMan-based real-time PCR assay was developed to rapidly detect the porcine reproductive and respiratory syndrome virus （PRRSV）. Primers and probe specific to the conserved region of the PRRSV N gene were selected, and the reactive system and conditions were optimized to improve the sensitivity, specificity and repetition of the assay. The results showed that the real-time PCR assay was specific and there were no cross reactions with other common porcine viruses. The sensitivity of the assay was 1.2 × 10^1 copy/ml of plasmid coding partial PRRSV N gene, which is 100 times higher than conventional PCR. It took no more than three hours from viral RNA extraction to complete the real-time PCR, and the assay was simple and had good repeats. Among tissue samples of 48 clinical diseased pigs,29 were confirmed PRRSV infected by realtime PCR, in which only 19 were confirmed by conventional PCR. This TaqMan-based real-time PCR assay is a specific, sensitive and quick tool suitable for early and quick detection of PRRSV in clinical labs.

关 键 词：猪繁殖与呼吸综合征病毒 荧光定量PCR TAQMAN探针 

分 类 号：S828[农业科学—畜牧学]