禽(番鸭)呼肠孤病毒TaqMan探针荧光RT-PCR检测方法的建立及应用

为了建立检测(番鸭)呼肠孤病毒(DRV)感染的方法,本研究根据DRV的σNS基因核苷酸序列设计引物及TaqMan探针,经过反应条件的优化,建立了该病毒的TaqMan探针实时荧光RT-PCR检测方法.用已知浓度的重组质粒为标准品,构建的标准曲线的相关系数达到99%.试验表明,该方法的重复性、稳定性、特异性良好,且灵敏度高,可以检测到1.0×10~2拷贝/μL的标准品,比普通PCR至少高100倍.该检测方法对DRV人工感染番鸭肝脏组织检出率达到100%.本实验所建立的TaqMan探针荧光RT-PCR为的快速检测以及进行分子流行病学的调查提供了新的技术手段.

Establishment and application of TaqMan probe real-time PCR for detection of Muscovy duck reovirus

A real-time PCR assay was developed for detection of duck reovirus (DRV) infection using specific primers and TaqMan probes based on the σNS gene of DRV. The assay could detect 1.0 × 10~2 copies/μL of the standards, which is more than 100-fold sensitive than conventional PCR. This method was used to detect DRV replication in the liver of infected muscovy ducks and achieved 100 % success rate. Taken together, the TaqMan probe RT-PCR provides an effective method for rapid and accurate detection of DRV and for molecular epidemiology study of the virus infection.