鸭源新城疫病毒、鸭瘟病毒和鸭圆环病毒三重PCR检测方法的建立

Development of a Trilex PCR Assay for Detection of Newcastle Disease Virus,Plague Virus and Circovirus in Ducks

为了建立能同时检测鸭源新城疫病毒(DuNDV)、鸭瘟病毒(DPV)和鸭圆环病毒(DuCV)的方法,根据DuNDV、DPV和DuCV基因的保守序列设计了3对特异性引物,预特异扩增DuNDV片段大小为823bp,DPV为576bp和DuCV为338bp。经过反应条件的优化,建立了DuNDV、DPV和DuCV的三重PCR检测方法。该方法特异性好,对鸭的其他病原检测结果为阴性;敏感性高,DuNDV、DPV和DuCV的核酸最低检出限分别为2.59×103、1.12×103、4.67×102拷贝/μL。对180份病料进行检测,结果DuCV阳性10份,阳性率为5.6%;DuNDV阳性1份,阳性率为0.56%。结果表明,建立的多重PCR方法可以应用于DuNDV、DPV和DuCV的临床检测和流行病学调查。

To develop a rapid triplex PCR assay for detection of Newcastal disease virus, plague virus and circovirus in ducks, three pairs of specific primers were designed according to the sequences of DuNDV gene, DPV gene and DuCV gene in GenBank, of which one specifically amplified 823 bp gene fragment of duck paramyxo virus, the others amplified 576 bp gene fragment of duck plague virus and 338 bp gene fragment of duck circovirus. And the reaction conditions were optimized to develop a duplex PCR assay for detection of Newcastal disease virus, plague virus and circovirus in ducks. The method has high specificity and sensitivity. As little as 2.59 X 10^3 copies of NDV, 1.12 X 10^3 copies/L DPV and 4.67 X 10^2 copiesof DuCV DNA or cDNA could be detected. Furthemore,the established assay was successfully used to detect 180 clinical samples, of which 10 samples were positive for DuCV and lsample for NDV. These results suggest that the multiplex PCR assay was suitable for rapid detection and epidemic surveillance of NDV, DPV and DuCV.