基于试纸条核酸提取和重组聚合酶扩增技术的HPV 16型DNA纸基电化学发光快速检测方法

Rapid detection of HPV 16DNA by paper-based electrochemiluminescence based on test strip nucleic acid extraction and recombinant polymerase amplification

目的建立一种人乳头瘤病毒(HPV)高危16亚型快速、灵敏、特异的检测方法。方法采用前期构建的试纸条法提取样本HPV DNA,并以其为模板,使用电化学发光试剂(酯化钌)标记上游引物,生物素标记下游引物,进行重组聚合酶恒温扩增,通过亲和素化的磁微球将生物素标记的扩增产物,在磁场的作用下富集,通过纸基电极-电化学发光检测技术,获得有酯化钌标记的待测物的浓度信息。结果酯化钌与上游引物以20∶1的摩尔比可以获得更高的标记效率和发光强度,该法最低检出限为50copies/mL,比实时荧光PCR的灵敏度高16倍;批内检测相对标准偏差低于5%,单个样本从核酸提取到获得扩增结果所需时间小于1h,具有较好的特异性、灵敏度和重复性。结论该法检测快速、灵敏、特异、操作简单,具有一定的临床应用价值。

Objective To develop a rapid,sensitive and specific method for the detection of human papillomavirus 16 DNA.Methods HPV DNA was extracted from the sample by the paper strip method which was developed in the previous studies.Using the extracted HPV DNA as template,the upstream primers were labeled with electrochemiluminescence reagent(Ru-NHS ester)and the downstream primers were labeled with biotin.Then the mixture was amplified at constant temperature by recombinant polymerase.The biotin labeled amplification products were enriched under the action of magnetic field through avidin-magnetic microspheres.The concentration information of HPV DNA labeling with Ru-NHS ester was obtained by the paper-based electrochemiluminescence detection technology.Results When the molar ratio of Ru-NHS ester to amino modified primer was 20∶1,the labeling rate of Ru labeled primer and the electrochemiluminescence intensity were better than 40∶1,which was enough to meet the needs of subsequent electrochemiluminescence detection.The detection limit of the developed method was 50 copies/mL,which was 16 times higher than the RT-PCR method.The relative standard deviations were less than 5% for within-batch precision.Furthermore,the time required for each sample from nucleic acid extraction to amplification was less than one hour.The specificity,sensitivity and repeatability were all good.Conclusion The developed method,which based on test strip nucleic acid extraction,recombinant polymerase amplification and paper-based electrochemiluminescence,is rapid,sensitive,specific and simple,and has certain application prospect in clinical diagnosis.