基于颜色判定的逆转录环介导恒温扩增技术检测HIV-1病毒

Visual detection of HIV-1 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye

目的建立一种基于颜色判定的用于人类免疫缺陷病毒I型（HIV—1）检测的逆转录环介导恒温扩增（RT-LAMP）技术。方法根据HIV-1gag基因保守区的序列设计RT-LAMP引物，利用已建立好的基于羟基萘酚蓝（HNB）颜色判定的RT-LAMP体系验证其灵敏度与特异性，并对实时荧光逆转录PCR（qRT-PCR）确认的临床样本进行一致性对比。结果FiT-LAMP引物特异性高，检出限为1000个拷贝RNA，对43份临床样本检测的灵敏度和特异性分别为94．6％～100％。结论基于颜色判定的环介导恒温扩增方法摆脱了对昂贵仪器的依赖，有望应用于HIV-1感染的现场筛选，为快速检测HIV-1病毒提供了新方法和新思路。

Objective A reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay was developed for rapid visual detection of HIV-1. Methods RT-LAMP primers were designed according to conservative sequences of HIV-1 gag gene, and their sensitivity and specificity were evaluated by the established RT-LAMP protocol with the addition of the hydroxynaphthol blue （ HNB ） dye prior to amplification. The performanee of RT-LAMP on clinical samples was compared with real-time reverse transcription PCR（qRT-PCR）. Results The RT-LAMP assay showed a high specificity, and its detection limit was 1000 copies RNA per tube. The sensitivity and specificity of this method using 43 clinical samples were 94.6% and 100% ,respectively,in comparison with those of qRT-PCR. Conclusion RT-LAMP assay using hydroxynaphthol blue dye does not need expensive instruments, and offer an alternative for the rapid detection of HIV-1 with the potential to be applied in field diagnosis.