双重环介导等温检测大肠杆菌和副溶血弧菌方法建立
发布时间：2019-04-03 19:19
【摘要】：本研究针对大肠杆菌rfbE基因和副溶血弧菌pR72H基因分别设计了LAMP特异性引物,利用LAMP技术扩增基因组DNA,建立了大肠杆菌和副溶血弧菌的快速LAMP检测技术,并初步应用于食物样品的检测,主要研究结果如下。1.建立并优化了大肠杆菌LAMP反应条件,并对引物特异性进行了研究。结果表明大肠杆菌最佳反应条件为：25μL反应体系中包含10μmol/L内引物FIP-E和BIP-E各3.0μL,缺省外引物F3-E和B3-E,10μmol/L dNTP 2.0μL,10×buffer 2.5μL, 100mmol/LMg2+0.5μL,DNA模版2.0μL,BstDNA聚合酶16U,双蒸水10μL补足25μL,反应温度65℃,反应时间60min,75℃保温10min终止反应。引物特异性实验表明,该引物能扩增5株供试大肠杆菌,其他供试菌为阴性。在此条件下LAMP反应对大肠杆菌的检出限为100fg/反应。2建立并优化了副溶血弧菌LAMP反应条件,并对引物特异性进行了研究。结果表明副溶血弧菌最佳反应条件为：25μL反应体系中包含10μmol/L内引物FIP-V和BIP-V各3.0μL,缺省外引物F3-V和B3-V,10μmol/L dNTP 2.0μL,10×buffer 2.5μL, 100mmol/L Mg2+ 0.5μL, DNA模版2.0gL,BstDNA聚合酶16U,双蒸水10μL补足25gL,反应温度65℃,反应时间60min,75℃保温10min终止反应。引物特异性实验表明,该引物能扩增3株供试副溶血弧菌,其他供试菌为阴性。在此条件下LAMP反应对副溶血弧菌的检出限为100fg/反应。3.大肠杆菌和副溶血弧菌的双重LAMP检测方法的建立经过优化实验确定了大肠杆菌和副溶血弧菌的双重LAMP反应的最佳条件为：25μL反应体系中包含大肠杆菌内引物FIP-E和BIP-E各3.0μL,副溶血弧菌内引物FIP-V和BIP-V各3.0μL,缺省外引物F3-E和B3-E、F3-V和B3-V,10mmol/L dNTP 2.0μL, 10×buffer 2.5μL, 100mmol/L Mg2+0.5μL,大肠杆菌DNA模板2.0μL,副溶血弧菌DNA模板2.0μL,BstDNA聚合酶16U,双蒸水2.0pL补足25μL,反应温度65℃,反应时间60min,75℃保温10min结束反应。此条件下双重LAMP反应检测大肠杆菌和副溶血弧菌的灵敏度均达到100fg/反应,是传统PCR的灵敏度的100倍。在交互实验中,对人工污染大肠杆菌和副溶血弧菌的食物样品进行检测,结果表明双重LAMP反应可以特异性检测出供试样品中含有大肠杆菌或者副溶血弧菌的样品。本研究建立了大肠杆菌和副溶血弧菌的双重LAMP检测方法,并应用于人为污染病原菌的食品样品的检测,能达到同时快速检测两种病原菌的目的。
[Abstract]:In this study, specific LAMP primers were designed for the rfbE gene of Escherichia coli and pR72H gene of Vibrio parahaemolyticus, and a rapid LAMP technique for the detection of E. coli and Vibrio parahaemolyticus was established by using LAMP technique to amplify genomic DNA,. It has been applied to the detection of food samples. The main results are as follows. The LAMP reaction conditions of Escherichia coli were established and optimized, and the specificity of primers was studied. The results showed that the optimal reaction conditions of Escherichia coli were as follows: 10 渭 mol / L internal primer FIP-E and 3.0 渭 L BIP-E, default external primer F 3 渭 E and B 3 渭 E, 10 渭 mol / L dNTP 2.0 渭 L, 10 脳 buffer 2.5 渭 L, 100mmol/LMg2 0.5 渭 L, respectively. DNA template 2.0 渭 L, BstDNA polymerase 16U, disteaming water 10 渭 L supplemented with 25 渭 L, reaction temperature 65 鈩，
本文编号：2453488
[Abstract]:In this study, specific LAMP primers were designed for the rfbE gene of Escherichia coli and pR72H gene of Vibrio parahaemolyticus, and a rapid LAMP technique for the detection of E. coli and Vibrio parahaemolyticus was established by using LAMP technique to amplify genomic DNA,. It has been applied to the detection of food samples. The main results are as follows. The LAMP reaction conditions of Escherichia coli were established and optimized, and the specificity of primers was studied. The results showed that the optimal reaction conditions of Escherichia coli were as follows: 10 渭 mol / L internal primer FIP-E and 3.0 渭 L BIP-E, default external primer F 3 渭 E and B 3 渭 E, 10 渭 mol / L dNTP 2.0 渭 L, 10 脳 buffer 2.5 渭 L, 100mmol/LMg2 0.5 渭 L, respectively. DNA template 2.0 渭 L, BstDNA polymerase 16U, disteaming water 10 渭 L supplemented with 25 渭 L, reaction temperature 65 鈩，
本文编号：2453488
本文链接：https://www.wllwen.com/yixuelunwen/yufangyixuelunwen/2453488.html
