利用RT-LAMP可视化检测技术检测H1亚型禽流感病毒及N1、N2亚型的分型

Visual Detection of H1 Subtype and Identification of N1,N2 Subtype of Avian Influenza Virus by Reverse Transcription Loop-mediated Isothermal Amplification Assay

为直接根据颜色变化进行可视化检测H1亚型、N1亚型、N2亚型禽流感病毒(AIV),根据环介导等温扩增技术(LAMP),建立针对H1亚型AIV及特异性鉴定N1、N2亚型的逆转录环介导等温扩增(RT-LAMP)检测方法。根据GenBank中的AIV基因序列,设计了三套分别针对H1亚型AIV-HA基因及N1、N2亚型AIV-NA基因的特异性简并引物,并优化反应条件和体系。结果表明建立的检测方法对其它亚型AIV及禽呼吸道病原体无交叉扩增反应并能特异性地检测N1、N2亚型AIV,灵敏度优于传统的RT-PCR方法。整个反应在常规水浴中50min就可完成,反应结束后不需打开反应管盖,可根据反应液的颜色变化对结果直接进行判定。120份临床样品用建立的RT-LAMP方法检测到14份H1N1亚型AIV、8份H1N2亚型AIV,结果与病毒分离结果相符。本研究建立的三种RT-LAMP可视化检测技术特异、灵敏、快速、操作和结果判定简便,适合在基层进行H1亚型AIV的快速检测及N1、N2亚型AIV的分型。

In order to visually detect H1, N1 and N2 subtype of avian influenza virus （AIV）, three reverse transcription loop-mediated isothermal amplification （RT-LAMP） assays were developed. According to the sequ subt mlze ences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 ype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were finished. Fourteen specimens of HIN1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field.