食源性致病菌生物芯片-RCA检测新方法  

A New Method for Detecting Food-borne Pathogenic Bacteria by Biochip-RCA

作　　者：李同祥[1] 黄天姿[1] 孙会刚[1] 汤薇[1] 田林[2] 李文[1] 张传丽[1] 王春燕[1] LI Tongxiang;HUANG Tianzi;SUN Huigang;TANG Wei;TIAN Lin;LI Wen;ZHANG Chuanli;WANG Chunyan(School of Food (Biology)Engineering,Xuzhou Institute of Technology,Xuzhou 221018,China;School of Chemical Engineering and Technology,Xuzhou Institute of Technology,Xuzhou 221018,China)

机构地区：[1]徐州工程学院食品(生物)工程学院,江苏徐州221018 [2]徐州工程学院化学化工学院,江苏徐州221018

出　　处：《徐州工程学院学报（自然科学版）》2019年第2期26-34,共9页Journal of Xuzhou Institute of Technology(Natural Sciences Edition)

基　　金：江苏省重点研发计划项目(BE2016648);江苏省高校自然科学研究重大项目(16KJA210001)

摘　　要：将生物芯片技术和滚环扩增(rolling circle amplification,RCA)技术结合,建立一种在片RCA检测食源致病性菌新方法.依据待检测致病菌的基因组序列设计捕获探针(capture probe,CP)、检测探针(detect probe,DP)和滚环探针(rolling circle probe,RCP),其中捕获探针、检测探针与待检测致病菌基因组序互补,RCP不含任何待检菌基因组DNA序列,CP的5′末端修饰氨基,将其点样制备成CP微阵列,DP、RCP与待检菌gDNA混合后变性,将其与CP微阵列杂交并连接, RCA扩增反应体系中加入链亲和素进行在片扩增反应,分析检测结果.研究表明,该方法能灵敏、特异性地检测单一和混合靶标分子,以金黄色葡萄球菌为检测对象,其最低检测限可到达50 fg/μL,其线性范围是400 ~50 fg/μL, R 2 达到0.97,证实了该方法的可行性和可靠性,为食源性致病菌检测提供了新的技术方法.A novel method for detecting food-borne pathogenic bacteria was developed by combining biochip with rolling circle amplification (RCA) technology.The capture probes (CP),detection probe (DP) and rolling circle probe (RCP) were designed according to the genome sequence of the pathogenic bacteria to be detected.CPs and DPs were designed to match sequences of the "target" pathogenic bacteria,but RCP does not contain any sequence of it and then chemically synthesized.The 5'end of CP is modified with amino groups.The CP microarray was fabricated.DP and RCP were denatured after mixing with gDNA of the "target" bacteria.They were hybridized with the CP microarray and performed ligation reactions.Streptavidin was added to the RCA amplification reaction system to perform RCA on chip.The chip was scanned and the results were analyzed.The results show that this method can detect single and mixed "target" molecules with high sensitivity and specificity.It can quantitatively detect the gDNA of pathogenic bacteria.The minimum detection limit can reach 50 fg/μL.Its linear range is 400~50 fg/μL and R2 reaches 0.97.The feasibility and reliability of this method was confirmed.The established method has the characteristics of high sensitivity,high signal-to-noise ratio,good specificity and no dependence on antibodies.It provides a new method and development direction for the detection of foodborne pathogenic bacteria.

关 键 词：生物芯片 滚环扩增 致病菌 检测探针 

分 类 号：Q789[生物学—分子生物学]