表面等离子共振技术结合滚环扩增法检测丙型肝炎病毒  

Surface plasmon resonance technology combined with rolling circle amplification for detection of hepatitis C virus

作　　者：季明辉[1,2] 胡贵方[1] 郑义[3] 顾大勇[2] 龙军[4] 鲁卫平[5] 何建安[2] 谈书勤[1] 史蕾[2] 刘春晓[2] 赵纯中[2] 徐云庆[2] 徐华[6] 欧青叶[6] 孙秋香[7] 滕娟[8] 

机构地区：[1]南方医科大学公共卫生与热带医学学院,广州515000 [2]深圳出入境检验检疫局疾病预防控制研究室,深圳518045 [3]清华大学深圳研究生院生物医药研究中心,深圳市基因与抗体治疗重点实验室,深圳518000 [4]南方医科大学珠江医院,广州515000 [5]第三军医大学大坪医院,重庆400042 [6]南通大学公共卫生学院职业卫生与环境毒理学教研室,南通226019 [7]中山大学公共卫生学院,广州515000 [8]海南出入境检验检疫局国际旅行卫生保健中心,海口570311

出　　处：《上海交通大学学报（医学版）》2012年第6期693-698,705,共7页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(30972827;81171667);国家质检总局项目(2010IK212);深港创新圈计划联合资助项目(ZYB200907090128A);深圳市科技计划切块项目(HZ0907004);重庆市科技攻关课题(2011AC5033)~~

摘　　要：目的研究滚环扩增(RCA)技术结合特异性表面等离子共振(SPR)金膜芯片检测丙型肝炎病毒(HCV)的方法。方法根据丙型肝炎x-tail区域的特异检测序列,设计并合成针对RCA法检测HCV的探针和引物,分成3组(实验组、阴性样品组和阳性样品组)进行RCA实验,验证RCA法检测HCV的特异性。将模板浓度按10倍梯度稀释,评价SPR技术结合RCA法检测的检测限。在普通金膜芯片的基础上进行表面化学处理,形成高特异性核酸芯片,用抗蛋白实验验证芯片抗蛋白能力。采用双通道SPR仪对RCA反应及信号放大反应进行实时观测。运用SPR技术结合RCA法检测63例临床血液样品,并与Real-Time PCR法比较,评价其灵敏度和特异度。结果 SPR技术结合RCA法对HCV阳性标准品的最低检测浓度为1 pmol/L,低于Real-TimePCR法的检测限(0.1 nmol/L)。SPR芯片具备良好的抗蛋白质非特异性吸附能力。双通道SPR仪对RCA芯片系统的检测信噪比为100,实现了对HCV的检测。对临床样品的检测灵敏度为90.0%(27/30),特异度为84.8%(28/33)(χ2=8.10,P=0.004)。结论 SPR技术结合RCA法将生物传感技术及原位扩增技术相结合,实现了快速、非标记和实时检测HCV。Objective To develop rolling circle amplification（RCA） method combined with specific surface plasmon resonance（SPR） nucleic acid gold-chip for the detection of hepatitis C virus（HCV）.Methods According to the specific test sequence of HCV x-tail region,probes and primers for detecting HCV with RCA method were designed and synthesized,and were divided into test group,negative sample group and positive sample group for RCA experiments to detect HCV.The template concentration was diluted into ten gradients,and the detection limit of SPR combined with RCA method was evaluated.Based on the ordinary gold chip,through the surface chemical processing,the nucleic acid chip with high specificity was obtained,and the anti-protein capacity of protein chip was verified by anti-protein experiment.Real-time detection of RCA reaction and signal magnification reaction was conducted with double channel SPR apparatus.Sixty-three blood samples collected from clinics were detected by SPR combined with RCA method,comparisons were made with Real-Time PCR,and the sensitivity and specificity were evaluated.Results The minimum detection concentration of SPR combined with RCA method in HCV testing was 1 pmol/L,which was lower than the detection limit of Real-Time PCR（0.1 nmol/L）.SPR chip had the favorable anti-protein absorptive capacity.The signal-to-noise ratio of double channel SPR apparatus in detection of RCA chip system was 100,which achieved the detection of HCV.The sensitivity of SPR combined with RCA method in detection of clinical samples was 90.0%（27/30）,and the specificity was 84.8%（28/33）（χ2=8.10,P=0.004）.Conclusion SPR combined with RCA method combines biological sensing technology with in situ amplification technology,which may detect HCV in a fast,label-free and real-time way.

关 键 词：表面等离子共振 丙型肝炎 滚环扩增 

分 类 号：R440[医药卫生—诊断学]