四种食源性致病菌多重PCR检测技术的建立
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- 英文论文题名：Establishment of multiplex PCR detection method for four foodborne pathogens
- 论文作者：冯可 ; 胡文忠 ; 萨仁高娃 ; 姜爱丽 ; 郑诗雨 ; 司琦
- 英文论文作者：Feng ke ; Hu Wenzhong ; Sarengaowa ; Jiang Aili ; Zheng Shiyu ; Si Qi ; College of Life Science and Biotechnology ; Dalian University of Technology ; 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：大连理工大学生命科学与技术学院;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：多重PCR ; 快速检测 ; 食源性致病菌
- 英文论文关键词：multiplex PCR ; rapid detection ; 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：121k
- 原文格式：O
- 会议级别：全国
摘要
本实验目的为建立一种能同时检测副溶血性弧菌、金黄色葡萄球菌、单增李斯特菌、大肠杆菌O157:H7四种食源性致病菌的多重PCR检测方法。分别对四种目标菌设计特异性引物,优化多重PCR反应体系中的主要影响因素(退火温度、引物浓度、Mg~(2+)浓度、dNTP浓度及酶浓度)。确定多重PCR反应体系及反应条件为10×PCR buffer 2.5μL,MgCl_2(25mmol/L)浓度为2.5mmol/L,dNTP(2.5mmol/L)浓度为0.35mmol/L,单增李斯特菌引物浓度为0.75mmol/L,大肠杆菌O157:H7引物浓度为0.40mmol/L;副溶血性弧菌引物浓度为0.75mmol/L,金黄色葡萄球菌引物浓度为0.40mmol/L。病原菌DNA模板各1μL,Taq酶(5U/μL)活力为1.5 U,加ddH_2O补充至25μL;反应程序为:95℃预变性3min,94℃变性30s,62℃退火30s,72℃延伸30s,进行32个循环,最后72℃终延伸10min,4℃保存。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,检出限为1.2×10~5 cfu/mL。建立了一个简捷、迅速,能同步检测食品中单增李斯特菌、大肠杆菌O157:H7、副溶血性弧菌和金黄色葡萄球菌的多重PCR方法。
The purpose of this study was to establish a multiplex PCR method for Listeria monocytogenes,Staphylococcus aureus,Vibrio parahemolyticus and E.coli 0157:H7.Four pairs of specific primers are designed,the major factors of multiplex PCR,such as annealing temperature,primer concentration,Mg~(2+) concentration,dNTP concentration and enzyme activity were optimized to develop detection method of mPCR.As a result,the multiplex PCR reaction system was determined to be 25μL,2.5μL 10 × PCR buffer,the concentration of MgCl_2(25mmol/L) was 2.50mmol/L,the concentration of dNTP(2.5mmol/L) was 0.35mmol/L,the activity of DNA polymerase was 0.3U.The concentration of each primer were as follows;L.monocytogenes was 0.75mmol/L,E.coli 0157:H7 was 0.40mmol/L,V.parahemolyticus was 0.75mmol/L,S.aureus was0.40mmol/L.DNA template of each foodborne pathogens was 1.0μL.Pre-degenerated at 95℃ for 5min,degenerated at 94℃,for 30 s,annealing at 62 ℃ for 30 s,extension at 72℃ for 30 s,32cycles,the final extension at 72℃ for 10 min.It shows that this method has specificity through the primer specific experiment and the reaction specific experiment.The sensitivity of the multiplex PCR method was 1.2 × 10~5cfu/mL.A simple,rapid and simultaneous detection method for L.monocytogenes,E.coli 0157:H7,V.parahaemolyticus and S.aureus were established.
The purpose of this study was to establish a multiplex PCR method for Listeria monocytogenes,Staphylococcus aureus,Vibrio parahemolyticus and E.coli 0157:H7.Four pairs of specific primers are designed,the major factors of multiplex PCR,such as annealing temperature,primer concentration,Mg~(2+) concentration,dNTP concentration and enzyme activity were optimized to develop detection method of mPCR.As a result,the multiplex PCR reaction system was determined to be 25μL,2.5μL 10 × PCR buffer,the concentration of MgCl_2(25mmol/L) was 2.50mmol/L,the concentration of dNTP(2.5mmol/L) was 0.35mmol/L,the activity of DNA polymerase was 0.3U.The concentration of each primer were as follows;L.monocytogenes was 0.75mmol/L,E.coli 0157:H7 was 0.40mmol/L,V.parahemolyticus was 0.75mmol/L,S.aureus was0.40mmol/L.DNA template of each foodborne pathogens was 1.0μL.Pre-degenerated at 95℃ for 5min,degenerated at 94℃,for 30 s,annealing at 62 ℃ for 30 s,extension at 72℃ for 30 s,32cycles,the final extension at 72℃ for 10 min.It shows that this method has specificity through the primer specific experiment and the reaction specific experiment.The sensitivity of the multiplex PCR method was 1.2 × 10~5cfu/mL.A simple,rapid and simultaneous detection method for L.monocytogenes,E.coli 0157:H7,V.parahaemolyticus and S.aureus were established.
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