犬瘟热病毒野毒株与疫苗株鉴别检测RT-LAMP方法的建立

Establishment of canine distemper virus specific RT-LAMP protocol for differentiation of infected and vaccinated animals

为建立一种鉴别犬瘟热病毒(CDV)野毒株与疫苗株的反转录-环介导等温扩增方法(RT-LAMP),本研究通过比对野毒株与疫苗株H基因设计特异性引物,对反应体系中的Mg2+、Betaine、Bst DNA Polymerase、dNTP和反应温度等条件分别进行优化,建立用于鉴别检测CDV野毒株与疫苗株的RT-LAMP。建立的RT-LAMP方法检测CDV野毒株时,在65℃水浴锅中反应40 min即可完成。该方法具有高度特异性,对犬细小病毒、犬腺病毒、狂犬病毒、犬冠状病毒无交叉反应,敏感度可达40 copies/μL,是常规RT-PCR方法的100倍。

Canine distemper virus （CDV） causes several fatal epidemics in canids. To develop a differential detection for CDV in nature infection and vaccinated animals, the reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay was established based on a set of six specific primers targeting the H gene of CDV. Under the optimized reaction conditions, the amplification was completed for 40 min in a constant temperature water bath at 65 ℃. The detection results shown that the RT-LAMP assay was able to efficiently distinguish wild type CDV isolates from the vaccine strains, with a detection limit of 40 copies of CDV, and no cross-reactions with canine adenovirus, canine parvovirus, rabies virus, and canine coronavirus. The rapid and easy-to-performed RT-LAMP facilitates differential detection between wild type CDV and vaccine strain for both laboratory and clinical diagnosis practice,