逆转录环介导等温扩增技术检测HIV-1方法的建立及颜色判定法的改良

Establishment of method and modification of colorimetric judgment on HIV-1 virus detection by reverse transcription loop-mediated isothermal amplification

目的 基于逆转录环介导等温扩增（RT-LAMP）技术建立可用于现场检测HIV-1的方法.方法 构建实时荧光检测法时,先测试其特异性和灵敏度,并对其定量检测进行探索,然后用35份HIV-1阳性样本对该方法进行评定；在颜色判定法上,先对羟基萘酚蓝（HNB）进行改良,并验证其实际显色效果,然后选取效果明显的样本进行显色灵敏度测试和35份阳性样本显色评定.结果 实时荧光法对HIV-1有很好的检测特异性,灵敏度可达每反应管10 ～ 100拷贝数RNA.该方法对所有阳性临床样本均100％ （35/35）检出,无一漏检,但其定量检测只局限于不低于每反应管103拷贝数.通过改良得到3种易分辨的HNB改良型显色剂,显色灵敏度等同于琼脂糖凝胶电泳级别,对35份HIV-1阳性样本的显色判定也无一漏检.结论 基于RT-LAMP技术的现场实时荧光法和改良型颜色判定法可以为真正实现现场快速、准确和灵敏检测HIV-1提供帮助.

Objective To establish the reverse transcription loop-mediated isothermal amplification （RT-LAMP） methods for on-site HIV-1 detection.Methods As for the real-time fluorescent RT-LAMP,we firstly tested the specificity and sensitivity,then explored its quantitative determination,and finally applied the method to the detection of 35 HIV-1 positive samples.For colorimetric judgment,after choosing different ameliorants to modify Hydroxynaphthol blue （HNB）,we tested their real effects on coloration,and then picked out the modified dyes with obvious color change to test the sensitivity and the detection of the 35 HIV-1-positive samples.Results The real-time fluorescent RT-LAMP showed great specificity of HIV-1,and the sensitivity to detect HIV-1 RNA was between 10 and 100 copies per reaction.On testing 35 HIV-1-positive samples,the method could reach 100 percent detection rate.However,for the quantitative determination,the quantitative relation was not observed regarding the HIV-1 RNA of below 103 copies per reaction.Three modified HNB dyes with clear color variation between the reaction tubes of the negative and the positive were got in the study,and their sensitivities equaled to the level of agarose gel electrophoresis.Similarly,100％ （35/35） detection rate was reached when the colorimetric RT-LAMP with the modified dyes was applied to detect 35 HIV-1-positive samples.Conclusion The established real-time fluorescence method and the modified color judgment of RT-LAMP could be helpful for truly achieving rapid,accurate,and sensitive on-site detection of HIV-1.