多重连接探针扩增技术检测食源性致病菌  

Detection of foodborne pathogenic bacteria using multiplex ligation-dependent probe amplification

作　　者：何名扬 王鸣秋 刘艳 李诗瑶 付文雯 郭雅晴 周陶鸿 张莉 彭青枝 HE Ming-yang;WANG Ming-qiu;LIU Yan;LI Shi-yao;FU Wen-wen;GUO Ya-qing;ZHOU Tao-hong;ZHANG Li;PENG Qing-zhi(Hubei Provincial Institute for Food Supervision and Test/Key Laboratory of Detection Technology of Focus Chemical Hazards in Animal-derived Food for State Market Regulation/Hubei Provincial Engineering and Technology Research Center for Food Quality and Safety Test,Wuhan 430075,China)

机构地区：[1]湖北省食品质量安全监督检验研究院/国家市场监管重点实验室(动物源性食品中重点化学危害物检测技术)/湖北省食品质量安全检测工程技术研究中心,武汉430075

出　　处：《湖北农业科学》2024年第11期168-174,222,共8页Hubei Agricultural Sciences

基　　金：湖北省重点研发计划项目(2020BCA091);湖北省食品质量安全监督检验研究院联合创新项目(HBQT-LH202102)。

摘　　要：基于多重连接探针扩增技术(MLPA),建立了一种能够同时检测食品中11种常见食源性致病菌的方法。通过设计并合成特异性MLPA探针,与高温变性后的标准菌株DNA进行杂交、连接、PCR扩增反应,利用毛细管电泳法分析PCR扩增产物。结果表明,利用11对探针分别对单一致病菌核酸样品进行检测,每种待测物均为单一峰,未出现杂峰,说明11对探针之间不存在交叉影响,探针特异性良好;利用11对探针同时对混合致病菌核酸样品进行多重检测,每种待测物均可得到与预期大小一致的扩增峰,扩增峰之间互不干扰,空白对照中没有扩增出任何目的扩增峰,说明该体系能同时检测多种食源性致病菌。该技术检测结果特异性强,可检出的最低致病菌污染量为1.5×10^(5)CFU/mL,作为对传统微生物检测技术的补充,MLPA技术可应用于食源性致病菌的早期筛查以及食品微生物安全风险的监控。A method for simultaneous detection of 11 common foodborne pathogenic bacteria in food was established based on the multiplex ligation-dependent probe amplification(MLPA).By designing and synthesizing specific MLPA probes,hybridization,ligation,and PCR amplification reactions with standard strain DNA after high-temperature denaturation were performed.The size and presence of PCR amplification products were analyzed by capillary electrophoresis.The results showed that using 11 pairs of probes to detect single bacterial nucleic acid samples,each analyte had a single peak and no impurity peaks appeared,indicating that there was no cross influence between the 11 pairs of probes and the specificity of the probes was good;using 11 pairs of probes for simultaneous multiplex detection of mixed pathogenic nucleic acid samples,each analyte could obtain amplification peaks of the same size as expected,and the amplification peaks did not interfere with each other.No target amplification peak was amplified in the blank control,indicating that the system could simultaneously detect multiple foodborne pathogenic bacteria.The detection results of this technology were highly specific,with a minimum detectable contamination level of 1.5×10^(5) CFU/mL.As a supplement to traditional microbiological detection techniques,MLPA technology could be applied for early screening of foodborne pathogenic bacteria and monitoring of food microbiological safety risks.

关 键 词：食源性致病菌 多重连接探针扩增技术 食品检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]