实时荧光环介导等温扩增技术快速检测牛肉中的大肠杆菌O157
|
摘要
目的建立实时荧光环介导等温扩增技术(real-time fluorescence loop-mediated isothermal amplification,RF-LAMP)快速检测大肠杆菌(Escherichia coli,EHEC)O157的分析斱法。方法针对大肠杆菌O157编码O抗原的rfbE基因设计引物。对该斱法迚行特异性验证,同时对大肠杆菌O157:H7纯培养物的灵敏度和人工污染牛肉的检出限迚行测定,对61份牛肉样品迚行RF-LAMP检测,幵与GB4789.36-2016斱法迚行比较,评价RF-LAMP斱法的敏感性、特异性和准确度。结果 10株大肠杆菌O157呈阳性结果, 21株非大肠杆菌O157呈阴性结果,该斱法特异性良好。纯培养物检测的灵敏度为5.1CFU/mL,人工污染的牛肉样品的检出限为5.1 CFU/g。结论本研究建立的RF-LAMP技术特异性好、灵敏度高、操作简单,可实时监测扩增反应,避克了繁琐的电泳过程,实现了对大肠杆菌O157的快速检测,对大肠杆菌O157引起的食源性疾病的预防和控制具有重要意义。
Objective To establish a method for rapid determination of Escherichia coli(EHEC) O157 by real-time fluorescence loop-mediated isothermal amplification(RF-LAMP). Methods The primers of E. coli O157 were designed based on the rfbE gene encoding the O antigen. The specificity of the method was verified, the sensitivity of pure culture of E. coli O157:H7 and the limit of detection of artificial contaminated beef were determined. A total of 61 beef samples were investigated by RF-LAMP and compared with GB 4789.36-2016 to evaluate the sensitivity, specificity and accuracy of the RF-LAMP method. Results Total 10 E. coli O157 strains were identified as positive, however, 21 non-E. coli O157 strains were negative, which showed this method had great specificity. The sensitivity in pure culture by RF-LAMP was 5.1 CFU/mL. The limit of detection of artificially contaminated beef samples was 5.1 CFU/g. Conclusions The RF-LAMP technology established in this study has good specificity, high sensitivity and simple operation, and can monitor the amplification reaction in real time, avoiding the complicated electrophoresis process. This study realizes the rapid detection of E. coli O157 and has significant importance for the prevention and control of food-borne diseases caused by E. coli O157.
Objective To establish a method for rapid determination of Escherichia coli(EHEC) O157 by real-time fluorescence loop-mediated isothermal amplification(RF-LAMP). Methods The primers of E. coli O157 were designed based on the rfbE gene encoding the O antigen. The specificity of the method was verified, the sensitivity of pure culture of E. coli O157:H7 and the limit of detection of artificial contaminated beef were determined. A total of 61 beef samples were investigated by RF-LAMP and compared with GB 4789.36-2016 to evaluate the sensitivity, specificity and accuracy of the RF-LAMP method. Results Total 10 E. coli O157 strains were identified as positive, however, 21 non-E. coli O157 strains were negative, which showed this method had great specificity. The sensitivity in pure culture by RF-LAMP was 5.1 CFU/mL. The limit of detection of artificially contaminated beef samples was 5.1 CFU/g. Conclusions The RF-LAMP technology established in this study has good specificity, high sensitivity and simple operation, and can monitor the amplification reaction in real time, avoiding the complicated electrophoresis process. This study realizes the rapid detection of E. coli O157 and has significant importance for the prevention and control of food-borne diseases caused by E. coli O157.
引文
[1]Liu Y,Singh P,Mustapha A.High-resolution melt curve PCR assay for specific detection of E.coli O157:H7 in beef[J].Food Control,2018,86:275-282.
[2]Zeinhom MMA,Wang Y,Song Y,et al.A portable smart-phone device for rapid and sensitive detection of E.coli O157:H7 in yoghurt and egg[J].Biosens Bioelectr,2018,99:479-485.
[3]Wu L,Song Y,Luan T,et al.Specific detection of live Escherichia coli O157:H7 using tetracysteine-tagged PP01 bacteriophage[J].Biosens Bioelectr,2016,86:102-108.
[4]Pang B,Zhao C,Li L,et al.Development of a low-cost paper-based ELISA method for rapid Escherichia coli O157:H7 detection[J].Anal Biochem,2018,542:58-62.
[5]丁浩,苏战强,夏利宁,等.克疫磁珠富集法对牛源大肠杆菌O157:H7分离鉴定的影响[J].中国畜牧兽医,2017,44(4):1189-1194.Ding H,Su ZQ,Xia LN,et al.Influence of immunomagnetic enrichment method for isolation and identification of E.coli O157:H7 from bovine[J].China Anim Husband Veter Med,2017,44(4):1189-1194.
[6]Zhou Y,Karwe MV,Matthews KR.Differences in inactivation of Escherichia coli O157:H7 strains in ground beef following repeated high pressure processing treatments and cold storage[J].Food Microbiol,2016,58:7-12.
[7]Song C,Li J,Liu J,et al.Simple sensitive rapid detection of Escherichia coli O157:H7 in food samples by label-free immunofluorescence strip sensor[J].Talanta,2016,156-157:42-47.
[8]Ju W,Moyne AL,Marco ML.RNA-based detection does not accurately enumerate living Escherichia coli O157:H7 cells on plants[J].Front Microbiol,2016,7:223.
[9]Zhu CJ,Zhao GY,Dou WC.Core-shell red silica nanoparticles based immunochromatographic assay for detection of Escherichia coli O157:H7[J].Anal Chim Acta,2018,1038:97-104.
[10]Li QR,Yang YX,Hu F,et al.Rapid detection of Escherichia coli O157:H7 by a fluorescent microsphere-based immunochromatographic assay and immunomagnetic separation[J].Anal Biochem,2018,564-565:32-39.
[11]Wang C,Peng J,Liu DF,et al.Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M-1 and Escherichia coli O157:H7 in milk[J].J Dairy Sci,2018,101(10):8767-8777.
[12]王力均,许恒毅,熊勇华,等.荧光定量PCR在大肠杆菌O157:H7检测中的应用研究迚展[J].食品科学,2013,34(11):358-362.Wang LJ,Xu HY,Xiong YH,et al.Application of real-time fluorescent quantitative polymerase chain reaction in detection of Escherichia coli O157:H7[J].Food Sci,2013,34(11):358-362.
[13]Shridhar PB,Noll LW,Shi X,et al.Multiplex quantitative PCR assays for the detection and quantification of the six major non-O157 Escherichia coli serogroups in cattle feces[J].J Food Protect,2016,79(1):66-74.
[14]Zhou B,Liang T,Zhan Z,et al.Rapid and simultaneous quantification of viable Escherichia coli O157:H7 and Salmonella spp.in milk through multiplex real-time PCR[J].J Dairy Sci,2017,100(11):8804-8813.
[15]Yang Y,Yang Q,Ma X,et al.A novel developed method based on single primer isothermal amplification for rapid detection of Alicyclobacillus acidoterrestris in apple juice[J].Food Control,2016,75:187-195.
[16]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucl Acids Res,2000,28(12):e63.
[17]Chen X,Ma L,Qiang S,et al.Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L.in chickpeas[J].Sci Reports,2016,6:25688.
[18]Wang Z,Yang Q,Zhang Y,et al.Saltatory rolling circle amplification(SRCA):A novel nucleic acid isothermal amplification technique applied for rapid detection of Shigella spp.in vegetable salad[J].Food Anal Methods,2017,11(2):504-513.
[19]马桂芬,张蕴哲,付博宇,等.实时荧光LAMP技术快速检测变形杆菌[J].食品研究与开収,2016,37(11):127-132.Ma GF,Zhang YZ,Fu BY,et al.Real-time fluorescence LAMP assay for rapid detection of proteus[J].Food Res and Dev,2017,37(11):504-513.
[20]GB 4789.36-2016食品安全国家标准食品微生物学检验大肠埃希氏菌O157:H7/NM检验[S].GB 4789.36-2016 National food safety standard-Food microbiological analysis-Determination of Escherichia coli O157:H7/NM[S].
[21]ISO 16140:2003/Amd 1:2011.Microbiology of food and animal feeding stuffs-Protocol for the validation of alternative methods[S].
[22]Li BG,Liu HL,Wang WM.Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E-coli[J].BMC Microbiol,2017,17(1):215.
[2]Zeinhom MMA,Wang Y,Song Y,et al.A portable smart-phone device for rapid and sensitive detection of E.coli O157:H7 in yoghurt and egg[J].Biosens Bioelectr,2018,99:479-485.
[3]Wu L,Song Y,Luan T,et al.Specific detection of live Escherichia coli O157:H7 using tetracysteine-tagged PP01 bacteriophage[J].Biosens Bioelectr,2016,86:102-108.
[4]Pang B,Zhao C,Li L,et al.Development of a low-cost paper-based ELISA method for rapid Escherichia coli O157:H7 detection[J].Anal Biochem,2018,542:58-62.
[5]丁浩,苏战强,夏利宁,等.克疫磁珠富集法对牛源大肠杆菌O157:H7分离鉴定的影响[J].中国畜牧兽医,2017,44(4):1189-1194.Ding H,Su ZQ,Xia LN,et al.Influence of immunomagnetic enrichment method for isolation and identification of E.coli O157:H7 from bovine[J].China Anim Husband Veter Med,2017,44(4):1189-1194.
[6]Zhou Y,Karwe MV,Matthews KR.Differences in inactivation of Escherichia coli O157:H7 strains in ground beef following repeated high pressure processing treatments and cold storage[J].Food Microbiol,2016,58:7-12.
[7]Song C,Li J,Liu J,et al.Simple sensitive rapid detection of Escherichia coli O157:H7 in food samples by label-free immunofluorescence strip sensor[J].Talanta,2016,156-157:42-47.
[8]Ju W,Moyne AL,Marco ML.RNA-based detection does not accurately enumerate living Escherichia coli O157:H7 cells on plants[J].Front Microbiol,2016,7:223.
[9]Zhu CJ,Zhao GY,Dou WC.Core-shell red silica nanoparticles based immunochromatographic assay for detection of Escherichia coli O157:H7[J].Anal Chim Acta,2018,1038:97-104.
[10]Li QR,Yang YX,Hu F,et al.Rapid detection of Escherichia coli O157:H7 by a fluorescent microsphere-based immunochromatographic assay and immunomagnetic separation[J].Anal Biochem,2018,564-565:32-39.
[11]Wang C,Peng J,Liu DF,et al.Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M-1 and Escherichia coli O157:H7 in milk[J].J Dairy Sci,2018,101(10):8767-8777.
[12]王力均,许恒毅,熊勇华,等.荧光定量PCR在大肠杆菌O157:H7检测中的应用研究迚展[J].食品科学,2013,34(11):358-362.Wang LJ,Xu HY,Xiong YH,et al.Application of real-time fluorescent quantitative polymerase chain reaction in detection of Escherichia coli O157:H7[J].Food Sci,2013,34(11):358-362.
[13]Shridhar PB,Noll LW,Shi X,et al.Multiplex quantitative PCR assays for the detection and quantification of the six major non-O157 Escherichia coli serogroups in cattle feces[J].J Food Protect,2016,79(1):66-74.
[14]Zhou B,Liang T,Zhan Z,et al.Rapid and simultaneous quantification of viable Escherichia coli O157:H7 and Salmonella spp.in milk through multiplex real-time PCR[J].J Dairy Sci,2017,100(11):8804-8813.
[15]Yang Y,Yang Q,Ma X,et al.A novel developed method based on single primer isothermal amplification for rapid detection of Alicyclobacillus acidoterrestris in apple juice[J].Food Control,2016,75:187-195.
[16]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucl Acids Res,2000,28(12):e63.
[17]Chen X,Ma L,Qiang S,et al.Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L.in chickpeas[J].Sci Reports,2016,6:25688.
[18]Wang Z,Yang Q,Zhang Y,et al.Saltatory rolling circle amplification(SRCA):A novel nucleic acid isothermal amplification technique applied for rapid detection of Shigella spp.in vegetable salad[J].Food Anal Methods,2017,11(2):504-513.
[19]马桂芬,张蕴哲,付博宇,等.实时荧光LAMP技术快速检测变形杆菌[J].食品研究与开収,2016,37(11):127-132.Ma GF,Zhang YZ,Fu BY,et al.Real-time fluorescence LAMP assay for rapid detection of proteus[J].Food Res and Dev,2017,37(11):504-513.
[20]GB 4789.36-2016食品安全国家标准食品微生物学检验大肠埃希氏菌O157:H7/NM检验[S].GB 4789.36-2016 National food safety standard-Food microbiological analysis-Determination of Escherichia coli O157:H7/NM[S].
[21]ISO 16140:2003/Amd 1:2011.Microbiology of food and animal feeding stuffs-Protocol for the validation of alternative methods[S].
[22]Li BG,Liu HL,Wang WM.Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E-coli[J].BMC Microbiol,2017,17(1):215.