环介导等温扩增联合横向流动试纸条可视化检测HPV16及HPV58方法的建立

Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect HPV16 and HPV58

目的将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合应用,建立一种快速、可视化的HPV16、HPV58检测方法。方法运用LAMP在线引物设计软件,针对HPV16E6基因及HPV58L1基因保守区域设计引物及探针。采用生物素标记LF,荧光基团FAM和淬灭基团BHQ-1标记探针,LFD进行目视检测,建立HPV16和HPV58的LAMP-LFD检测方法,并对其特异性、灵敏度及临床实用性进行验证。将临床样品使用该方法的检测结果与实时LAMP扩增及qPCR结果相比较,计算符合率。结果优化的LAMP最佳反应温度为63℃,最佳反应时间为50min。LFD检测仅需5min,故完成检测耗时仅55min;建立的LAMP-LFD方法能特异性检出HPV16、HPV58,其他5种常见高危型HPV呈阴性反应;该方法针对HPV16和HPV58的检测灵敏度均为100拷贝/μl,50份临床样品检测结果与实时LAMP和qPCR检测的符合率达100%。结论建立的HPV16、HPV58LAMP-LFD检测体系快速、简便,可视化。该方法对设备的要求低,仅需恒温孵育器即可完成,结果易观察,适用于现场HPV检测,以及经济落后和资源匮乏地区的HPV筛查。

Objective To establish a method for rapid and visual detection of HPV16 and HPV58 involving a combination of loop-mediated isothermal amplification(LAMP)and a lateral flow dipstick(LFD)assay. Methods The online design software Primer Explorer V5(https://primerexplorerj.p/)was used to design specific primers and probes for the conserved regions of the HPV16 E6 and HPV58 L1 genes.Biotin-labeled loop primers,FAM-labeled probes,and BHQ1-labeled probes were prepared.LAMP-LFD to detect HPV16 and HPV58 was established.The specificity and sensitivity of the method were tested.Clinical samples were used to verify the validity of the method by comparing it to real-time LAMP and qPCR,and the rate of concordance was calculated. Results A LAMP-LFD reaction system was successfully constructed.The reaction temperature was 63 degrees and the reaction time was 50 minutes when LAMP-LFD was optimized.The detection of LFD only takes 5 minutes,so it takes only 55 minutes to complete the detection.LAMP-LFD can specifically detect HPV16 and HPV58,which was confirmed by the negative reaction to five other types of common highrisk HPV.The sensitivity of LAMP-LFD for HPV16 and HPV58 was 100 copies/μl.Results from 50 clinical samples tested using LAMP-LFD were consistent with real-time LAMP and qPCR,and the rate of concordance was 100%. Conclusion LAMP-LFD that was established to detect HPV16 and HPV58 is fast,simple,and yields visually apparent results.The method requires less equipment and fewer skills than conventional LAMP or qPCR.It is extremely suitable for point-of-care testing and HPV screening in primary hospitals and resource-poor areas.