H1N1猪流感病毒环介导等温扩增快速检测方法的建立

目的:建立H1N1猪流感病毒环介导等温扩增(LAMP)快速检测方法。方法:从GenBank中获得H1N1猪流感病毒血凝素(HA)、神经氨酸酶(NA)基因序列,应用DNAStar软件MegAlign程序分析其序列,利用Primer ExplorerV4软件在序列保守区域设计LAMP引物,即外引物和内引物,同时以H1N1猪流感病毒的cDNA作为阳性模板,对试验中的几个反应条件进行优化。结果:LAMP检测方法对H1N1猪流感病毒的灵敏度达到4～6个拷贝,其引物对于H9亚型禽流感病毒、猪瘟病毒和猪圆环病毒均无非特异性扩增,表现出良好的特异性。结论:建立的H1N1猪流感病毒环介导等温扩增快速检测方法灵敏度高、特异性强、重复性好,为快速检测猪流感病毒提供了新方法和新思路。

Development of Rapid Loop-Mediated Isothermal Amplification Assay for Swine Influenza (H1N1) Virus

Objective:To set up loop-mediated isothermal amplification(LAMP) assay for quick detection of H1N14 swine influenza virus(SIV).Methods:The hemagglutinin(HA) and neuraminidase(NA) gene sequences of SIV subtype H1N1 were downloaded from GenBank and analyzed using MegAlign tools of software DNAStar.The outer, inner and loop primers of LAMP were then designed according to their conservative regions using software Primer Explorer V4.The HA and NA fragments of SIV H1N1 were also amplified and cloned to serve as the positive control templates and their LAMP reaction conditions were optimized in this experiment.Results:The results showed that the LAMP assay had a detection limit of 4-6 DNA copies.The primers used in this assay were specific for H1N1 SIV and did not amplify H9 subtype AIV or CSFV or PCV.Conclusion:It is proved mat LAMP method is a reliable and accurate assay for quick detection of H1N1 SIV.This assay offers a new method and idea for detecting SIV.