H10亚型禽流感病毒RT-LAMP可视化检测方法的建立

Development of a visual detection of reverse transcription loop-mediated isothermal amplification assay for H10 subtype avian influenza virus

根据GenBank中H10亚型AIV的HA基因序列,选取特异性序列,设计了6条LAMP引物,用于H10亚型AIV的检测,优化反应条件,建立了H10亚型AIV RT-LAMP可视化检测方法,对该法进行特异性和敏感性验证。结果显示:该法特异性强,只与H10亚型AIV反应,与其他亚型AIV和常见的禽类疾病无交叉反应;灵敏度高,检测下限为100拷贝/μL模板;仪器简单,只需一个可控温的水浴锅在1h内即可完成全部扩增反应;结果可视化,肉眼直接观察结果。结果表明:本试验建立的H10亚型AIV检测方法简便、快速,尤其适合于养殖场和基层工作站的检测,为该病的诊断和防控提供一种有效的方法。

To develop a rapid assay for detection of H10 subtype avian influenza（AIV）, six LAMP primers were designed according to the specific sequences of HA gene H10 subtype AIV in Gen- Bank. The reaction conditions were optimized and the reverse transcription loop-mediated isother- mal amplification （RT-LAMP） assay was established. The specificity and sensitivity of assay were evaluated. There was no cross-reactivity to the other subtypes AIV and pathogens of avian, the detection limit of the assay was 100 copies//~L of template,indicating a good specificity and sensi- tivity. The amplification could be finished by a controlled temperature water bath within lh,and the presence of H10 subtype of AIV could be detected and visualized by naked eyes. In conclu- sion, the LAMP assay is a simple, rapid and can identify H10 subtype avian influenza virus visually.