滚环扩增结合DNA纳米传感器超灵敏检测HIV整合酶方法的建立

Establishment of the ultra-sensitive detection method for HIV integrase by rolling circle amplification combined with DNA nanosensor

目的以H I V整合酶(integrase,I N)为特异性靶标,利用滚环扩增(rollingcircle amplification,RCA)技术结合DNA纳米传感器建立一种HIV IN等温扩增检测方法(rolling-circle-enhanced-INactivity-detection,REIAD)。方法提取纯化的重组HIV IN或包装HIV颗粒,以HIV IN活性为靶标,设计IN识别的附着位点(attachment site,att)长末端重复序列(long terminal repeat,LTR)和双链DNA环,与DNA纳米传感器检测技术结合,将LTR固定于载玻片表面并催化其末端插入双链DNA环生成一个有切口的DNA环,进行界面RCA扩增和荧光标记探针检测,建立REIAD。通过检测梯度稀释HIV IN和包装HIV颗粒,评价检测方法的敏感性,确定检测限;通过梯度稀释小鼠白血病病毒(murine leukemia virus,MLV)和HIV IN抑制剂雷特格韦作用,评价检测方法的特异性。通过对梯度稀释HIV IN中加入HEK293全细胞提取物及10%胎牛血清细胞培养基检测,评价患者血浆样本检测的可行性。结果REIAD方法在37℃、整合50min、滚环扩增30min条件下完成梯度稀释HIV IN及包装HIV颗粒的检测,具有良好的敏感性,重组HIV IN和包装HIV颗粒的最低检出限可达15fmol和2TU/μl,与MLV无交叉反应,具有良好的特异性。结论本研究建立的REIAD检测方法具有敏感性高、特异性好,操作简便,平台要求低等优点,有望为HIV早期检测提供一种新的方法。

Objective A novel isothermal amplification assay(REIAD)for human immunodeficiency virus(HIV)integrase(IN)was developed using RCA and DNA nanosensors.Method Purified recombinant HIV IN or packaged HIV particles were extracted,and the LTR terminal sequence of att site and double-stranded DNA ring identified by IN were designed with the activity of HIV IN as the target,and then combined with DNA nanosensor detection technology.The LTR sequence was fixed on the surface of the slide and catalyzed to insert the end of the double-stranded DNA ring to produce a DNA ring with incision for interface RCA amplification and fluorescent labeling probe detection,and the HIV integrase isothermal amplification assay(REIAD)was established.The sensitivity of the detection method was evaluated and the detection limit was determined by detecting the gradient dilution of HIV IN and packaging HIV particles.The specificity of the assay was evaluated by detecting the effects of gradient dilution mouse leukemia virus(MLV)and HIV IN inhibitor raltegravir.By adding HEK293 whole cell extract and 10%fetal bovine serum cell culture medium to gradient diluted HIV IN,the feasibility of detecting plasma samples from patients was evaluated.Result The detection of graded diluted HIV IN and packaged HIV particles was completed by REIAD method at 37℃,integration for 50 minutes,and rolling ring amplification for 30 minutes.The minimum detection limits of recombinant HIV IN and packaged HIV particles were up to 15 fmol and 2 TU/μl,and there was no cross-reaction with MLV.It has good specificity.Conclusion The REIAD detection method established in this study has high sensitivity,good specificity,simple operation and low platform requirements,and is expected to provide a new detection method for the early detection of HIV virus.