Epizootic Research Center, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan.
J Virol Methods. 2011 Dec;178(1-2):239-42. doi: 10.1016/j.jviromet.2011.07.015. Epub 2011 Aug 30.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to the detection of equine influenza virus (EIV). Because equine influenza is caused currently by EIV of the H3H8 subtype, the RT-LAMP primer set was designed to target the hemagglutinin gene of this subtype. The detection limit of the RT-LAMP assay was a virus dilution of 10(-5); which was 10(3) times more sensitive than the Espline Influenza A&B-N test and 10 times more sensitive than a reverse transcription polymerase chain reaction (RT-PCR) assay. The specificity of the RT-LAMP assay was examined by using several equine pathogens and nasal swabs collected from horses with fever in 2010 after EIV was eradicated in Japan. No cross-reactions were observed. Using 100 nasal swabs collected from horses with fever during an EIV outbreak in 2007, the RT-LAMP assay detected EIV in 52 samples, whereas the Espline test and the RT-PCR assay detected EIV in only 17 and 41 samples, respectively. These results indicate that the RT-LAMP assay is specific for EIV and more sensitive than the Espline test and the RT-PCR assay. Because it provides high sensitivity and ease of manipulation without the need for a thermal cycler or gel electrophoresis, the RT-LAMP assay should be applicable for laboratory diagnosis of EIV.
反转录环介导等温扩增（RT-LAMP）被应用于检测马流感病毒（EIV）。由于马流感目前是由 H3N8 亚型的 EIV 引起的，因此 RT-LAMP 引物组被设计为针对该亚型的血凝素基因。RT-LAMP 检测的检测限为 10(-5)的病毒稀释度；比 Espline 流感 A&B-N 检测灵敏 103 倍，比逆转录聚合酶链反应（RT-PCR）检测灵敏 10 倍。通过使用几种马病原体和 2010 年在日本根除 EIV 后从发烧马采集的鼻拭子，对 RT-LAMP 检测的特异性进行了检查。未观察到交叉反应。使用在 2007 年 EIV 爆发期间从发烧马采集的 100 个鼻拭子，RT-LAMP 检测到 52 个样本中的 EIV，而 Espline 检测和 RT-PCR 检测仅在 17 和 41 个样本中检测到 EIV。这些结果表明，RT-LAMP 检测对 EIV 具有特异性，比 Espline 检测和 RT-PCR 检测更灵敏。由于它提供了高灵敏度和易于操作，而不需要热循环仪或凝胶电泳，因此 RT-LAMP 检测法应该适用于 EIV 的实验室诊断。
