基于大肠埃希菌O157∶H7的荧光定量冻干检测试剂盒的研制  

Development of Fluorescence Quantitative Lyophilized Detection Kit Based on Escherichia coli O157∶H7

作　　者：粟元 朱龙佼 曹继娟 刘建龙 许文涛[1] SU Yuan;ZHU Long-jiao;CAO Ji-juan;LIU Jian-long;XU Wen-tao(Key Laboratory of Safety Assessment of Genetically Modified Organism(Food Safety),Ministry of Agriculture and Rural Affairs,College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083;Dalian Minzu University,Dalian 116600;Hebei Elisa Biotechnology Co.,Ltd.,Zhuozhou 072750)

机构地区：[1]中国农业大学营养与健康系食品精准营养与质量控制教育部重点实验室,北京100083 [2]大连民族大学,大连116600 [3]河北伊莱莎生物技术有限公司,涿州072750

出　　处：《生物技术通报》2022年第3期264-275,共12页Biotechnology Bulletin

基　　金：国家自然科学基金面上项目(31871875);山东省重点研发计划(2019JZZY011014)。

摘　　要：大肠埃希菌O157∶H7(Escherichia coli O157∶H7,E.coli O157∶H7)作为一种食源性致病菌,其分布广泛、危害性大。为了研制一种基于E.coli O157∶H7的既能实现快速、简单和灵敏检测,又能够实现常温储存及运输的试剂盒,本研究利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,qPCR)技术,结合真空冷冻干燥技术,研制了保留核酸检测性能、易于常温储存及运输、减少气溶胶污染的E.coli O157∶H7荧光定量冻干检测试剂盒。研制的试剂盒采用冻干技术保留了核酸扩增试剂的检测性能,复水后,在Taq DNA聚合酶的作用下通过循环扩增实时监测荧光信号的积累,实现荧光定量检测,所提出的方法在40 min内可检测到2.1 copies/μL的eaeA基因。该技术为E.coli O157∶H7的检测提供良好的研究基础和技术参考,填补了市场灵敏度高、便于储存的E.coli O157∶H7检测试剂盒的缺乏。Escherichia coli O157∶H7,as a food-borne pathogen,is widely distributed and causes great harms.To develop a kit based on E.coli O157∶H7 that can not only achieve rapid,simple and sensitive detection,but also achieve room temperature storage and transportation,this study utilized real-time fluorescence quantitative polymerase chain reaction(qPCR),combinig with vacuum lyophilizing technology,and developed an E.coli O157∶H7 fluorescence quantitative lyophilizing detection kit.This kit retains nucleic acid detection performance,and is easy to store and transport at room temperature,and reduces aerosol pollution.The developed kit adopts lyophilizing technology to retain the detection performance of nucleic acid amplification reagents.After rehydration,the accumulation of fluorescent signals is monitored in real time through cyclic amplification under the action of Taq DNA polymerase to achieve quantitative fluorescence detection.By the proposed method 2.1 copies/μL of eaeA gene can be detected within 40 min.This technology provides a fine research foundation and technical reference for the detection of E.coli O157∶H7,and fills the lack of detection kits with high sensitivity and easy storage in the market.

关 键 词：E.coli O157∶H7 核酸 荧光定量PCR 冻干保护剂 试剂盒 

分 类 号：TS207.4[轻工技术与工程—食品科学]