新城疫病毒实时荧光RT-LAMP检测方法的建立

Establishment of Real-time Fluorescent Reverse Transcription Loop-Mediated Isothermal Amplification for Newcastle Disease Virus Detection

新城疫是影响家禽养殖业的重大动物疫病,建立现场快速分子学检测方法是其防控关键。借助热裂解法抽提病毒RNA,在筛选出最佳核酸染料Calcein的基础上,建立新城疫病毒(NDV)实时荧光反转录环介导等温扩增(rRT-LAMP)检测法,并对该法进行特异性、敏感性及准确性分析。试验结果显示,本试验所建立的NDV-rRT-LAMP方法可有效鉴别NDV,其敏感性可检测至50个基因拷贝,与国家标准实时荧光定量RT-PCR检测法一致;该法特异性强,与AIV、IBV等常见禽呼吸道传染病病原无交叉反应。小规模田间试验显示,该法用于NDV的普查监测,结果可靠。结果表明,建立的NDV-rRT-LAMP检测法可满足NDV现场快速分子检测需求。

Newcastle disease virus（NDV）is a major causative agent and a rapid and sensitive molecular biological diagnosis is crucial to prevent and control Newcastle disease.NDV real-time fluorescent reverse transcription loop-mediated isothermal amplification（NDV-rRT-LAMP）was established by means of heat treatment of the samples.The sensitivity,specificity and repeatability of this method were evaluated.The results showed the sensitivity of this method was equal to the NDV real-time RT-PCR assay,and the detection limit was fifty gene copy per reaction.This method had no cross-reactivity with other avian disease agents such as AIV,IBV et al.Small-scale tests for field samples indicated this method was reliable for survey monitoring of N5 on site.