两种食源性致病菌双重荧光定量PCR检测技术的建立

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• 英文论文题名：Establishment of dual fluorescence quantitative PCR detection technology of two kinds of food borne pathogens
• 论文作者：司琦 ; 胡文忠 ; 冯可 ; 姜爱丽 ; 赵蕾
• 英文论文作者：Si Qi ; Hu Wenzhong ; Feng ke ; Jiang Aili ; Zhao Lei ; College of Life Science ; Dalian Minzu University ; Research Center of Engineering and Technology for Rapid Detection of Foodborne Microorganisms and Control
• 年：2016
• 作者机构：大连民族大学生命科学学院辽宁省食源性病原微生物快速检测与控制工程中心;
• 论文关键词：双重荧光定量PCR ; 大肠杆菌O157 ; H7 ; 单核增生李斯特菌 ; 食源性致病菌
• 英文论文关键词：dual fluorescence quantitative PCR ; escherichia coli O157 ; H7 ; listeria monocytogenes ; foodborne pathogen
• 会议召开时间：2016-11-09
• 会议录名称：中国食品科学技术学会第十三届年会论文摘要集
• 英文会议录名称：Abstracts of the 13th Annual Meeting of CIFST
• 语种：中文
• 分类号：TS207.4
• 学会代码：ZGSP
• 会议名称：中国食品科学技术学会第十三届年会
• 会议地点：中国北京
• 主办单位：中国食品科学技术学会
• 学会名称：中国食品科学技术学会
• 页数：2
• 文件大小：119k
• 原文格式：O
• 会议级别：全国

摘要

本实验的目的在于建立检测单核增生李斯特菌和大肠杆菌O157:H7的双重荧光定量PCR方法。实验根据单核增生李斯特菌Ina基因和大肠杆菌0157:H7的Wzy基因设计两对特异性引物。对双重荧光定量PCR反应体系中的引物添加量、退火温度等指标进行了优化,并确定适宜的双重荧光定量PCR反应体系及反应条件。最终确定其反应体系为(40μL反应体系),10×PCR buffer 5μL,MgCl_2(25mmol/L)4μL,dNTP(2.5mmol/L)4μL,大肠杆菌O157:H7(10μmol/L)上下引物1μL,探针2μL;单增李斯特菌引物(10μmol/L)2μL,探针2μL,DNA模板各3μL,Taq酶0.6μL,加无菌水补充至40μL。反应程序为:95℃预变性2min;94℃变性30s,52.7℃退火30s,72℃延伸20s,40个循环,4℃永久保温。通过引物特异性试验及灵敏性实验,证明该方法具有较好的特异性,大肠杆菌O157:H7和单增李斯特菌的最低检出限分别为1.95×10~4CFU/mL和1.87×10~4CFU/mL。最终建立了简便、快速、灵敏性高且能同步检测食品中大肠杆菌O157:H7和单核细胞增生性李斯特菌的双重荧光定量PCR方法。
The purpose of this study is to establish a dual fluorescence quantitative PCR method for Listeria monocytogenes and Escherichia coli O157:H7.Two pairs of specific primers were designed according to the Ina gene of Listeria monocytogenes and the Wzy gene of the gene of Escherichia coli O157:H7.Then,the double fluorescence quantitative PCR reaction system was optimized,and the annealing temperature and other indicators were optimized,and the suitable dual fluorescence quantitative PCR reaction system and reaction conditions were determined.Eventually the reaction system;40 μL reaction system;10 ×PCR buffer 5 μL,MgCl_2(25mmol/L) 4μL,dNTP(2.5mmol/l) 4μL,Escherichia coli O157:H7 up and down primer(10μmol/L) lμL,probe 2μL;Listeria monocytogenes up and down primers(10μmol/L) 2μL,2μL of probe,DNA template,3μL,Taq enzyme 0.6 μL and ddH_2O supplement to 40 μL.The reaction procedure is:an initial denaturation at 95℃ for2 min followed by 40 cycles of amplification at 94℃ for 30 s,annealing at 52.7℃ for 30s;extension at 72℃ for 20s;4℃ permanent insulation.Then,the specificity test and sensitivity test were used to prove that the method has good specificity,the detection limits of the Escherichia coli 0157:H7 and Listeria monocytogenes were 1.95× 10~4CFU/mL and 1.87 × 10~4CFU/mL,respectively.Finally,a simple,rapid and sensitive method was established for the simultaneous detection of Escherichia coli0157:H7 and Listeria monocytogenes in food with double fluorescence quantitative PCR method.

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