磁珠提取技术在副溶血弧菌检测中的应用  

Application of Magnetic Bead Extraction Technology in Detection of Vibrio parahaemolyticus

作　　者：肖雨诗 曹强 黄蓉 程雷[3] 刘娜 刘欢[2] 吴立冬[2] XIAO Yushi;CAO Qiang;HUANG Rong;CHENG Lei;LIU Na;LIU Huan;WU Lidong(College of Fisheries and life, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Control of Quality and Safety for Aquatic Products, Ministry of Agriculture and Rural Affairs, Chinese Academy of Fishery Sciences, Beijing 100141, China;Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing 100048, China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院农业农村部水产品质量安全控制重点实验室,北京100141 [3]北京工商大学北京市食品添加剂工程技术研究中心,北京100048

出　　处：《食品科学技术学报》2021年第6期165-174,共10页Journal of Food Science and Technology

基　　金：中央级公益性科研院所基本科研业务费专项资金项目—国际合作项目(2020GH09);科技部国家重点研发计划项目(2017YFC1600706)。

摘　　要：副溶血弧菌(Vibrio parahaemolyticus)是一种常见的食源性致病菌,为解决副溶血弧菌检测步骤复杂且周期长的问题,研究开发了一种磁珠提取与PCR联用技术快速检测食品中的副溶血弧菌。首先通过碱性共沉淀法完成Fe_(3)O_(4)磁珠的制备,随后在室温条件下通过水解正硅酸乙酯完成Fe_(3)O_(4)@SiO_(2)磁珠制备。探讨了正硅酸乙酯添加量对Fe_(3)O_(4)@SiO_(2)磁珠粒径和磁性的影响。以DNA提取能力为目标,已知浓度DNA为提取对象,分析了Fe_(3)O_(4)@SiO_(2)磁珠粒径和提取体系pH值对Fe_(3)O_(4)@SiO_(2)磁珠与DNA结合能力的影响。结果表明,Fe_(3)O_(4)@SiO_(2)磁珠结合副溶血弧菌DNA的较佳粒径为105.89 nm、Fe_(3)O_(4)@SiO_(2)磁珠的SiO_(2)层为25 nm,此条件下Fe_(3)O_(4)@SiO_(2)磁珠表面光滑、分散性良好、饱和磁化强度高,在磁场的作用下10 s即可完成副溶血弧菌DNA与杂质的分离,副溶血弧菌DNA的提取率高达80%(提取体系pH值为5.0)。采用优化后的Fe_(3)O_(4)@SiO_(2)磁珠进行DNA提取,结合PCR联用技术,对92份市售水产样品(32份对虾、25份牡蛎、35份贻贝)进行副溶血弧菌筛查。结果表明,Fe_(3)O_(4)@SiO_(2)磁珠提取的DNA均可直接用于PCR检测,被测水产样品中有17份样本的副溶血弧菌检测结果为阳性。此技术与国标检测方法(GB 4789.7—2013)相比,检测时间由一周缩短至4 h,提高了副溶血弧菌的检测效率。磁珠提取结合PCR联用技术可检测不同食品中的多种微生物菌种,此研究为食品中的微生物快速检测提供了一种实用新方法。Vibrio parahaemolyticus is a common food borne pathogen.In order to solve the problems of complex steps and long detection period of Vibrio parahaemolyticus,a magnetic bead extraction and PCR combined technique was developed for rapid detection of Vibrio parahaemolyticus in food.Firstly,Fe_(3)O_(4) magnetic beads were prepared by alkaline coprecipitation method,and then Fe_(3)O_(4)@SiO_(2) magnetic beads were prepared by hydrolysis of tetraethy orthosilicate at room temperature.The effects of tetraethyl orthosilicate addition on the particle size and magnetic properties of Fe_(3)O_(4)@SiO_(2) magnetic beads were discussed.The effects of particle size of Fe_(3)O_(4)@SiO_(2) magnetic beads and pH value of extraction system on the binding ability of Fe_(3)O_(4)@SiO_(2) magnetic beads to DNA were analyzed.The results showed that the optimal particle size of Fe_(3)O_(4)@SiO_(2) magnetic beads combined with Vibrio parahaemolyticus DNA was 105.89 nm,and the SiO_(2) layer of Fe_(3)O_(4)@SiO_(2) magnetic beads was 25 nm.Under these conditions,Fe_(3)O_(4)@SiO_(2) magnetic beads had smooth surface,good dispersion and high saturation magnetization.The separation of Vibrio parahaemolyticus DNA and impurities could be completed in 10 seconds under the action of magnetic field.The extraction rate of Vibrio parahaemolyticus DNA was as high as 80%(pH value of extraction system was 5.0).The optimized Fe_(3)O_(4)@SiO_(2) magnetic beads were used for DNA extraction.Combined with PCR,92 commercial aquatic samples(32 prawns,25 oysters and 35 mussels)were screened for Vibrio parahaemolyticus.The results showed that the DNA extracted by Fe_(3)O_(4)@SiO_(2) magnetic beads could be directly used for PCR detection,and 17 samples were positive for Vibrio parahaemolyticus.Compared with the national standard detection method(GB 4789.7—2013),the detection time was shortened from one week to 4 hours,which improved the detection efficiency of Vibrio parahaemolyticus.Magnetic bead extraction combined with PCR could detect a variety of microbial

关 键 词：副溶血弧菌 磁珠提取 DNA提取 PCR 食品微生物检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]