鸭三种病毒性疾病多重PCR检测方法的建立及初步应用

Multiplex PCR Assay for Simultaneous Detection of Three Duck Viral Diseases

为了一次性检测临床样品中是否存在鸭新城疫病毒(NDV)、鸭瘟病毒(DPV)或鸭坦布苏病毒(DTMUV)感染,本研究根据GenBank已登录的3种病原的保守基因序列,分别设计了3对特异性引物,通过优化扩增条件,建立了可同时检测NDV、DPV和DTMUV的多重PCR检测方法。特异性检验表明,该多重PCR方法可同时扩增出NDV(510bp)、DPV(400bp)、DTMUV(300bp)的特异片段,对其他鸭常感染病毒扩增结果均为阴性;敏感性测定表明,该多重PCR技术最低能检出1.02×10~4拷贝·μL^(-1) NDV、3.50×10~3拷贝·μL^(-1) DPV和1.17×10~4拷贝·μL^(-1) DTMUV。采用建立的多重PCR方法对250份临床样品进行检测结果显示,其检测结果与该3种病毒的常规单一PCR检测结果符合率为100%。以上结果表明,本研究建立的多重PCR检测方法具有快速、特异性强、敏感性高等优点,适用于临床样品中上述3种病原的检测。

To simultaneously detect the presence of Newcastle disease virus(NDV),duck plague virus(DPV)and/or duck Tembusu virus(DTMUV)in ducks,3 pairs of specific primers based on the conserved regions of the genomes of these viruses deposited in Gen Bank database were used for the development of a multiplex PCR assay method.The PCR reaction conditions were optimized,and specificity and sensitivity of the assay determined.The results showed that the new method could amplify the specific gene fragments of NDV at 510 bp,DPV at 400 bp,and DTMUV at 300 bp simultaneously without adulterations.The detection sensitivity of the assay was 1.02×10 4 copies·μL-1 on NDV,3.50×10 3 copies·μL-1 on DPV,and 1.17×10 4 copies·μL-1 on DTMUV.The detection results were completely agreeable with those obtained by the conventional PCR method on 250 clinical samples.It appeared that the currently established methodology to simultaneously detect the 3 target viruses was rapid and convenient with high specificity and sensitivity.