LAMP实时浊度法快速检测大肠杆菌方法的建立与优化研究
|
摘要
根据大肠杆菌的特异性基因(malB),设计与筛查环介导等温扩增(LAMP)的引物,通过对甜菜碱用量、环引物的浓度、反应温度、dNTPs用量等反应条件的优化,得到较佳的LAMP反应体系,建立了LAMP实时浊度法快速检测大肠杆菌;在较优条件下,先后对9株非大肠杆菌菌株进行特异性检测和5株大肠杆菌的进行同源性检测。此外,结合课题组自行研制的多通道浊度仪,对LAMP扩增产物进行实时浊度监测。结果表明:LAMP实时浊度法检测大肠杆菌的灵敏度达16.010 ng/L,与实时荧光定量PCR的检测限相当。因此,建立与优化LAMP实时浊度法可为食品中大肠杆菌的现场快速检测提供了有力手段。
According to the specific gene(malB) of Escherichia coli(E.coli), the primers of loop mediated isothermal amplification(LAMP) were designed and screened, and the optimum LAMP reaction system was obtained by optimizing the reaction conditions such as betaine dosage, concentration of loop primer, reaction temperature and dNTPs dosage. Then the optimal method was established for rapid detection of E.coli by LAMP real-time turbidity method. In the optimal conditions, 9 strains of non-Escherichia coli strains were tested for specificity and 5 strains of Escherichia coli were tested for homology. Moreover, using the multichannel turbidity device developed by the research group, the produce of LAMP could be real-time monitored. The results showed that the sensitivity of E.coli was 16.010 ng/L, in line with the detection limit of real-time fluorescence quantitative PCR. Therefore, the optimization and establishment of real-time turbidity LAMP method could provide a powerful approach to rapid field-detection of E.coli in food.
According to the specific gene(malB) of Escherichia coli(E.coli), the primers of loop mediated isothermal amplification(LAMP) were designed and screened, and the optimum LAMP reaction system was obtained by optimizing the reaction conditions such as betaine dosage, concentration of loop primer, reaction temperature and dNTPs dosage. Then the optimal method was established for rapid detection of E.coli by LAMP real-time turbidity method. In the optimal conditions, 9 strains of non-Escherichia coli strains were tested for specificity and 5 strains of Escherichia coli were tested for homology. Moreover, using the multichannel turbidity device developed by the research group, the produce of LAMP could be real-time monitored. The results showed that the sensitivity of E.coli was 16.010 ng/L, in line with the detection limit of real-time fluorescence quantitative PCR. Therefore, the optimization and establishment of real-time turbidity LAMP method could provide a powerful approach to rapid field-detection of E.coli in food.
引文
[1]何晓华,顿玉慧,卢力,等.环介导等温扩增技术在肠杆菌科致病菌检测中的研究进展[J].食品科学,2014,35(19):312-317.HE Xiaohua,DUN Yuhui,LU Li,et al.Recent Progress in Application of Loop-Mediated Isothermal Amplification Method for Detection of Enterobacteriaceae Pathogens[J].Food Science,2014,35(19):312-317.
[2]樊杰,伏小平.O157:H7型大肠杆菌荧光定量PCR检测方法的建立[J].中国兽医科学,2008,38(1):15-19.FAN Jie,FU Xiaoping.Establishment of Fluorescent Quantitative PCR Assay for Detection of Escherichia coli O157:H7[J].Chinese Veterinary Science,2008,38(1):15-19.
[3]韩小姣,黄正.自来水消毒对内毒素及微生物处理效果分析[J].中国公共卫生,2016,32(2):215-217.HAN Xiaojiao,HUANG Zheng.Effect of Disinfection Treatment on Endotoxin and Microorganism in Drinking Water in Wuhan City[J].Chinese Journal of Public Health,2016,32(2):215-217.
[4]GABRIEL A A.Combinations of Selected Physical and Chemical Hurdles to Inactivate Escherichia coli O157:H7 in Apple and Orange Juices[J].Food Control,2015,50:722-728.
[5]马凯,白羽,陈尔凝,等.鲜猪肉中沙门和金葡菌荧光定量PCR检测[J].食品研究与开发,2015,36(17):117-122.MA Kai,BAI Yu,CHEN Erning,et al.Real-Time PCR Assay for Salmonella and Staphylococcus aureus in Pork[J].Food Research and Development,2015,36(17):117-122.
[6]许一平.多重PCR检测沙门菌、大肠杆菌和金黄色葡萄球菌的研究[D].武汉:华中农业大学,2006.XU Yiping.Study on the Detection of Salmonella spp.,Escherichia coli and Staphylococcus aureus by Multiplex PCR[D].Wuhan:Huazhong Agricultural University,2006.
[7]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-Mediated Isothermal Amplification of DNA[J].Nucleic Acids Research,2000,28(12):e63.
[8]吕恒,张锦海,顾海涛,等.肠出血性大肠杆菌O157:H7的快速可视化检测[J].东南国防医药,2013,15(4):321-324.LüHeng,ZHANG Jinhai,GU Haitao,et al.A Quicker and Visual Method for Detecting Enterohemorrhagic Escherichia coli O157:H7[J].Military Medical Journal of Southeast China,2013,15(4):321-324.
[9]洪艳,贺凡,王晓闻.LAMP检测沙门氏菌方法的建立[J].山西农业科学,2014,42(4):340-342.HONG Yan,HE Fan,WANG Xiaowen.Detection of Salmonella by Loop-Mediated Isothermal Amplification Method[J].Journal of Shanxi Agricultural Sciences,2014,42(4):340-342.
[10]CHEN Z,YANG T,YANG H W,et al.A Portable Multi-Channel Turbidity System for Rapid Detection of Pathogens by Loop-Mediated Isothermal Amplification[J].Journal of Biomedical Nanotechnology,2018,14(1):198-205.
[11]梁玉林,刘秀,丁梦璇,等.环介导等温扩增技术在典型食源性致病菌检测中的应用进展[J].食品研究与开发,2017,38(18):219-224.LIANG Yulin,LIU Xiu,DING Mengxuan,et al.Application of Loop-Mediated Isothermal Amplification in the Detection of Typical Foodborne Pathogens[J].Food Research and Development,2017,38(18):219-224.
[12]PAL V,SAXENA A,SINGH S,et al.Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for Detection of Burkholderia mallei[J].Transboundary and Emerging Diseases,2018,65(1):e32-e39.
[13]YE L,LI Y,ZHAO J,et al.Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Sensitive and Rapid Detection of Listeria monocytogenes[J].Letters in Applied Microbiology,2015,61(1):85-90.
[14]张蕴哲.快速检测肠出血性大肠杆菌O157实时荧光环介导等温扩增技术的建立[D].保定:河北农业大学,2016.ZHANG Yunzhe.Establishment of Real-Time Fluorescence Loop-Mediated Isothermal Amplification Technology for Rapid Detection of Enterohemorrhagic Escherichia coli O157[D].Baoding:Hebei Agricultural University,2016.
[15]YOKOYAMA E,UCHIMURA M,ITO K.Detection of Enteroaggregative Escherichia coli by Loop-Mediated Isothermal Amplification[J].Journal of Food Protection,2010,73(6):1064-1072.
[16]易海华,赵金伟,徐波,等.使用环介导等温扩增技术快速检测食品中肠出血性大肠杆菌O157:H7的初步研究[J].中国食品卫生杂志,2010,22(3):206-213.YI Haihua,ZHAO Jinwei,XU Bo,et al.Rapid Detection of Enterohemorrhagic Escherichia coli O157:H7 in Food by Loop-Mediated Isothermal Amplification Assay[J].Chinese Journal of Food Hygiene,2010,22(3):206-213.
[17]梁磊.环介导等温扩增(LAMP)技术检测牛肉中大肠杆菌O157的研究[D].保定:河北农业大学,2011.LIANG Lei.Study on a Loop Mediated Isothermal Amplification(LAMP)for the Detection of Escherichia coli O157 in Beef[D].Baoding:Hebei Agricultural University,2011.
[18]张小飞,张召军,张广州,等.产肠毒素大肠杆菌快速检测方法的建立和评价[J].中国实验动物学报,2013,21(5):31-35.ZHANG Xiao fei,ZHANG Zhaojun,ZHAN GGuangzhou,et al.Establishment and Evaluation of the Loop-Mediated Isothermal Amplification(LAMP)Assay in Detection of Enterotoxigenic Escherichia coli[J].Acta Laboratorium Animalis Scientia Sinica,2013,21(5):31-35.
[2]樊杰,伏小平.O157:H7型大肠杆菌荧光定量PCR检测方法的建立[J].中国兽医科学,2008,38(1):15-19.FAN Jie,FU Xiaoping.Establishment of Fluorescent Quantitative PCR Assay for Detection of Escherichia coli O157:H7[J].Chinese Veterinary Science,2008,38(1):15-19.
[3]韩小姣,黄正.自来水消毒对内毒素及微生物处理效果分析[J].中国公共卫生,2016,32(2):215-217.HAN Xiaojiao,HUANG Zheng.Effect of Disinfection Treatment on Endotoxin and Microorganism in Drinking Water in Wuhan City[J].Chinese Journal of Public Health,2016,32(2):215-217.
[4]GABRIEL A A.Combinations of Selected Physical and Chemical Hurdles to Inactivate Escherichia coli O157:H7 in Apple and Orange Juices[J].Food Control,2015,50:722-728.
[5]马凯,白羽,陈尔凝,等.鲜猪肉中沙门和金葡菌荧光定量PCR检测[J].食品研究与开发,2015,36(17):117-122.MA Kai,BAI Yu,CHEN Erning,et al.Real-Time PCR Assay for Salmonella and Staphylococcus aureus in Pork[J].Food Research and Development,2015,36(17):117-122.
[6]许一平.多重PCR检测沙门菌、大肠杆菌和金黄色葡萄球菌的研究[D].武汉:华中农业大学,2006.XU Yiping.Study on the Detection of Salmonella spp.,Escherichia coli and Staphylococcus aureus by Multiplex PCR[D].Wuhan:Huazhong Agricultural University,2006.
[7]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-Mediated Isothermal Amplification of DNA[J].Nucleic Acids Research,2000,28(12):e63.
[8]吕恒,张锦海,顾海涛,等.肠出血性大肠杆菌O157:H7的快速可视化检测[J].东南国防医药,2013,15(4):321-324.LüHeng,ZHANG Jinhai,GU Haitao,et al.A Quicker and Visual Method for Detecting Enterohemorrhagic Escherichia coli O157:H7[J].Military Medical Journal of Southeast China,2013,15(4):321-324.
[9]洪艳,贺凡,王晓闻.LAMP检测沙门氏菌方法的建立[J].山西农业科学,2014,42(4):340-342.HONG Yan,HE Fan,WANG Xiaowen.Detection of Salmonella by Loop-Mediated Isothermal Amplification Method[J].Journal of Shanxi Agricultural Sciences,2014,42(4):340-342.
[10]CHEN Z,YANG T,YANG H W,et al.A Portable Multi-Channel Turbidity System for Rapid Detection of Pathogens by Loop-Mediated Isothermal Amplification[J].Journal of Biomedical Nanotechnology,2018,14(1):198-205.
[11]梁玉林,刘秀,丁梦璇,等.环介导等温扩增技术在典型食源性致病菌检测中的应用进展[J].食品研究与开发,2017,38(18):219-224.LIANG Yulin,LIU Xiu,DING Mengxuan,et al.Application of Loop-Mediated Isothermal Amplification in the Detection of Typical Foodborne Pathogens[J].Food Research and Development,2017,38(18):219-224.
[12]PAL V,SAXENA A,SINGH S,et al.Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for Detection of Burkholderia mallei[J].Transboundary and Emerging Diseases,2018,65(1):e32-e39.
[13]YE L,LI Y,ZHAO J,et al.Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Sensitive and Rapid Detection of Listeria monocytogenes[J].Letters in Applied Microbiology,2015,61(1):85-90.
[14]张蕴哲.快速检测肠出血性大肠杆菌O157实时荧光环介导等温扩增技术的建立[D].保定:河北农业大学,2016.ZHANG Yunzhe.Establishment of Real-Time Fluorescence Loop-Mediated Isothermal Amplification Technology for Rapid Detection of Enterohemorrhagic Escherichia coli O157[D].Baoding:Hebei Agricultural University,2016.
[15]YOKOYAMA E,UCHIMURA M,ITO K.Detection of Enteroaggregative Escherichia coli by Loop-Mediated Isothermal Amplification[J].Journal of Food Protection,2010,73(6):1064-1072.
[16]易海华,赵金伟,徐波,等.使用环介导等温扩增技术快速检测食品中肠出血性大肠杆菌O157:H7的初步研究[J].中国食品卫生杂志,2010,22(3):206-213.YI Haihua,ZHAO Jinwei,XU Bo,et al.Rapid Detection of Enterohemorrhagic Escherichia coli O157:H7 in Food by Loop-Mediated Isothermal Amplification Assay[J].Chinese Journal of Food Hygiene,2010,22(3):206-213.
[17]梁磊.环介导等温扩增(LAMP)技术检测牛肉中大肠杆菌O157的研究[D].保定:河北农业大学,2011.LIANG Lei.Study on a Loop Mediated Isothermal Amplification(LAMP)for the Detection of Escherichia coli O157 in Beef[D].Baoding:Hebei Agricultural University,2011.
[18]张小飞,张召军,张广州,等.产肠毒素大肠杆菌快速检测方法的建立和评价[J].中国实验动物学报,2013,21(5):31-35.ZHANG Xiao fei,ZHANG Zhaojun,ZHAN GGuangzhou,et al.Establishment and Evaluation of the Loop-Mediated Isothermal Amplification(LAMP)Assay in Detection of Enterotoxigenic Escherichia coli[J].Acta Laboratorium Animalis Scientia Sinica,2013,21(5):31-35.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |