集在线荧光分析的连续流动反转录-聚合酶链式反应快速扩增检测食源致病性轮状病毒  

Rapid Amplification and Detection of Foodborne Pathogenic Rotavirus by Continuous-flow Reverse Transcription-Polymerase Chain Reaction Integrated with Online Fluorescence Analysis

作　　者：章春笋[1] 李彧媛[1] 王海英[1] 

机构地区：[1]华南师范大学激光生命科学教育部重点实验室暨激光生命科学研究所,广州510631

出　　处：《分析化学》2011年第5期645-651,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.61072030;30700155);国家自然科学基金-广东联合基金重点项目(No.U0931005);科技部国家重点基础研究973计划项目(Nos.2010CB732602:2011CB910402);教育部"长江学者与创新团队计划"创新团队项目(No.IRT0829)资助项目

摘　　要：采用含RNA反转录(Reverse transcription,RT)和在线荧光分析的连续流动聚合酶链式反应(Poly-merase chain reaction,PCR)微流控技术检测食源致病性轮状病毒。此RT-PCR微流控装置以加热铜块组成恒温带,以聚四氟乙烯毛细管微通道为反应体系构建而成。采用循环水冷却退火区,此装置能获得低至31℃的退火温度,而且温度控制及其稳定性良好,因而能满足不同PCR的要求。为了充分体现PCR微流控技术的优越性,在线荧光分析被用于检测PCR产物。当流速为7.5 mm/s时,轮状病毒RNA的cDNA扩增和在线荧光分析能在约12 min完成(扩增约10 min,在线分析约2 min)。在此集成化的RT-PCR微流控装置上,1 h可完成轮状病毒RNA的RT-PCR以及其扩增产物的在线荧光分析,样品检出限达到6.4×104 copies/μL。A continuous-flow polymerase chain reaction（PCR） microfluidics integrated with reverse transcription（RT） of RNA and online fluorescence analysis has been introduced,which has been successfully applied for fast amplification and identification of foodborne pathogenic Rotavirus.On this continuous-flow RT-PCR device,the isothermal heating copper blocks provides the temperatures for RT-PCR,and the polytetrafluoroethylene（PTFE） capillary was used as the RT-PCR reaction channel.By using the cycling water to cool the annealing zone,the annealing temperature as low as 31 ℃ could be obtained for the presented device,where good temperature control and stability was also achieved.Therefore,this device can meet different PCR requirements.To make the best of the advantages of microfluidic PCR,the online fluorescence analysis of amplification products has been performed.When the flow rate of 7.5 mm/s was used,the amplification of cDNA synthesized from Rotavirus RNA and then the online fluorescence analysis of amplification products could be completed in about 12 min（about 10 min for amplification and 2 min for analysis）.By using this microfluidic device,the RT-PCR of Rotavirus RNA and the online fluorescence analysis of its products can be accomplished within 1 h or so,and as well the detection limit of the RNA concentration is 6.4×104 copies/μL.

关 键 词：连续流动反转录-聚合酶链式反应 在线荧光分析 微流控技术 食源致病性轮状病毒 

分 类 号：TS207.4[轻工技术与工程—食品科学]