反转录环介导等温扩增甲型流行性感冒病毒

Detection of influenza A virus by reverse transcription-loop mediated isothermal amplification

目的通过反转录环介导等温扩增技术（RT‐LAMP）检测甲型流行性感冒（以下简称为甲流）病毒，建立一种快速、简便的可视化核酸检测技术。方法针对甲流病毒血凝素基因区段设计RT‐LAMP引物。通过检测不同流感病毒验证RT‐LAMP的特异性，通过检测倍比稀释的RNA样本验证RT‐LAMP的灵敏度。用羟基萘酚蓝（HNB）染料对产物进行可视性检测，以凝胶电泳进行结果验证。最后用RT‐LAM P检测临床样本，并比较与荧光定量PCR的一致性。结果 RT‐LAM P的引物特异性好，可准确扩增甲流病毒。通过对稀释模板的扩增，得出RT‐LAM P的灵敏度为2．5＆#215;103拷贝／mL。另外，RT‐LAMP扩增结果可通过HNB的颜色改变进行判定，与凝胶电泳的效果一致。对临床样本检测后，RT‐LAMP和荧光定量PCR的一致性好。结论 RT‐LAMP可快速、简便地检测甲流病毒。

Objective To detect influenza A virus by reverse transcription‐loop mediated isothermal amplification （RT‐LAMP） assay to established a rapid ,simple and visualization nucleic acid detection method .Methods The RT‐LAMP primers were designed in accordance with the hemagglutinin gene of influenza A virus .Then ,the specificity of the primers was evaluated by detection of different influenza viruses ,and the sensitivity was confirmed by testing multiple diluted RNA samples . Hydroxynaphthol blue （HNB） was used for visually evaluation and gel electrophoresis was used for validation .Clinical samples were detected by RT‐LAMP assay .Its consistency with fluorescent quantitative polymerase chain reaction （PCR） methods was compared .Results The primers of RT‐LAMP assay had high specificity . This technique could amplify influenza A virus accurately .The detection limit of RT‐LAMP assay was 2 .5 ＆#215; 103 copies/mL by detection of multiple diluted RNA samples .In addition ,the results of RT‐LAM P assay could be visually inspected using HNB by color change ,and the results was in accordance with that of gel electrophoresis . RT‐LAMP assay was in consistence with fluorescent quantitative PCR when clinically applied .Conclusions RT‐LAMP assay is a rapid ,specific ,sensitive and simple method to detect influenza A virus .