基于反转录环介导等温扩增技术检测大肠杆菌O157
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摘要
建立反转录环介导等温扩增(Reverse transcriptase loop-mediated isothermal amplification RT-LAMP)方法特异性检测大肠杆菌O157。针对大肠杆菌O157的特异性保守rfb E基因设计多组引物,通过对引物筛选和反应条件优化,建立了检测大肠杆菌O157的实时荧光RT-LAMP方法。利用大肠杆菌O157及其人工污染脱脂乳样品,研究了方法的特异性和灵敏度,并与r RTPCR(Real-time fluorescent quantitative reverse transcription polymerase chain reaction)方法的灵敏度进行比较。结果显示,等温65℃条件下,30 min内能完成RT-LAMP扩增反应。所建立的实时荧光RT-LAMP方法特异性强,除了2株大肠杆菌O157以外其余菌株均未产生特异性扩增反应。同时该方法灵敏度高,对人工污染脱脂乳样品检测灵敏度能达到20 CFU/g,比r RT-PCR方法高10倍。建立的实时荧光RT-LAMP方法将为大肠杆菌O157现场快速检测提供有力手段。
A reverse transcriptase loop-mediated isothermal amplification(RT-LAMP)method was developed to specifically detect Escherichia coli O157. A number of primers were designed for specific conservative rfb E gene in E. coli O157. A real-time fluorescent RT-LAMP for detecting E. coli O157 was established by optimizing primer selection and reaction conditions. The specificity and sensitivity of the method were verified by using E. coli O157 and its artificial contaminated skim milk samples,and compared with the sensitivity of r RT-PCR(real-time fluorescent quantitative reverse transcription polymerase chain). The RT-LAMP reaction was completed within 30 min under the condition of isothermal temperature of 65℃. RT-LAMP was highly specific since there was no specific amplification except two strains of E. coli O157. In the detection of artificial contaminated skim milk samples,the sensitivity reached 20 CFU/g,10 times of that by the r RT-PCR,meaning this method was highly sensitive. The established real-time fluorescent RT-LAMP will provide a powerful means for rapid detection of E. coli O157 on site.
A reverse transcriptase loop-mediated isothermal amplification(RT-LAMP)method was developed to specifically detect Escherichia coli O157. A number of primers were designed for specific conservative rfb E gene in E. coli O157. A real-time fluorescent RT-LAMP for detecting E. coli O157 was established by optimizing primer selection and reaction conditions. The specificity and sensitivity of the method were verified by using E. coli O157 and its artificial contaminated skim milk samples,and compared with the sensitivity of r RT-PCR(real-time fluorescent quantitative reverse transcription polymerase chain). The RT-LAMP reaction was completed within 30 min under the condition of isothermal temperature of 65℃. RT-LAMP was highly specific since there was no specific amplification except two strains of E. coli O157. In the detection of artificial contaminated skim milk samples,the sensitivity reached 20 CFU/g,10 times of that by the r RT-PCR,meaning this method was highly sensitive. The established real-time fluorescent RT-LAMP will provide a powerful means for rapid detection of E. coli O157 on site.
引文
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[15]Inomata A,Kishda I N,Momoda T,et al.Development andevaluation of a reverse transcription loop-mediated isothermalamplification assay for rapid and high-sensitive detection ofCryptosporidium in water samples[J].Water Science andTechnology,2009,60(8):2166-2172.
[16]Zhang GD,Brown EW,Narjol GE.Comparison of Real-time PCR,reverse transcriptase real-time PCR,loop-mediated isothermalamplification,and the FDA conventional microbiological methodfor the detection of Salmonella spp.in produce[J].Appl EnvironMicrobiology,2011,77(18):6495-6501.
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[20]刘道亮,胡连霞,赵占民,等.改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157:H7[J].微生物学通报,2011,38(3):430-435.
[21]张雪寒,何孔旺,叶青,等.快速检测大肠杆菌O157的LAMP方法的建立与评价[J].中国兽医学报,2013,33(7):1027-1031.
[22]Yukiko HK,Noriko K,Kayoko O,et al.Detection of vero toxigenicEscherichia coli O157 and O26 in food by plating methods andLAMP method A collaborative study[J].International Journal ofFood Microbiology,2008,122(12):156-161.
[23]张雪寒,何孔旺,叶青,等.快速检测志贺毒素2基因的LAMP方法的建立[J].江苏农业学报,2013,29(2):455-457.
[24]Wang F,Jiang L,Yang QR,et al.Rapid and specific detection ofEscherichia coli serogroups O26,O45,O103,O111,O121,O145,and O157 in ground beef,beef trim,and produce by loop-mediatedisothermal amplification[J].Appl Environ Microbiology,2012,78(8):2727-2736.
[25]Techathuvanan C,D’Souza DH.Reverse-transcriptase loopmediated isothermal amplification as a rapid screening/monitoringtool for Salmonella Enterica detection in liquid whole eggs[J].Journal of Food Science,2012,77(4):200-205.
[26]Fan FX,Wang SJ,Lou J.Establishment of RT-LAMP assay todetect Salmonella Typhi in blood[J].Disease Surveillance,2012,27(4):325-329.
[2]Kanki M,Seto K.,Harada T.Comparison of four enrichment brothsfor the detection of non-O157 Shiga-toxin-producing Escherichia coli O91,O103,O111,O119,O121,O145 and O165 from pure cultureand food samples[J].Lett Appl Microbiol,2011,53(2):167-173.
[3]Rayan H,Amandadi M,Sanadgol N.A highly specific and sensitiveloop-mediated isothermal amplification method for the detection ofEscherichia coli O157:H7[J].Microbial Pathogenesis,2016,91:161-165.
[4]金婷婷,马福金,袁耀武.免疫捕获LAMP法快速检测大肠埃希氏O157:H7的研究[J].食品科技,2013,33(8):1027-1031.
[5]Ohtsuka K,Tanaka M,Ohtsuka T.Comparison of detection methodsfor Escherichia coli O157 in Beef Livers and Carcasses[J].Foodborne Pathogens and Diseases,2010,7(12):1563-1567.
[6]Wang DG,Liu F,Huo GC,et al.Development and evaluation ofa loop-mediated isothermal amplification method for detectingEscherichia coli O157 in raw milk[J].Key Laboratory of DairyScience,2009,17(1):55-66.
[7]朱海,吕敬章,范放,等.E.coli O157:H7 LAMP检测方法的建立[J].分子诊断与治疗杂志,2010,2(2):98-101.
[8]Yin FG,Zhu Y,Koutchma T,et al.Inactivation and potentialreactivation of pathogenic Escherichia coli O157:H7 in applejuice following ultraviolet light exposure at three monochromaticwavelengths[J].Food Microbiology,2015,46:329-335.
[9]Wang LJ,Li P,Zhang ZH,et al.Rapid and accurate detectionof viable Escherichia coli O157:H7 in milk using a combinedIMS,sodium deoxycholate,PMA and real-time quantitative PCRprocess[J].Food Control,2014,36(1):119-125.
[10]Gabriel A.Combinations of selected physical and chemical hurdlesto inactivate Escherichia coli O157:H7 in apple and orangejuices[J].Food Control,2015,50:722-728.
[11]党苗苗,李芳菲,费楠,等.肠出血性大肠杆菌O157:H7的分子生物学检测及PCR检测[J].食品安全质量检测学报,2015,6(2):523-527.
[12]张微,姚笛,侯婷婷,等.乳中大肠杆菌O157:H7的荧光定量PCR检测方法的建立[J].中国乳品工业,2015,43(7):52-54.
[13]Notomi T,Okayama H,Masubuchi AH,et al.Loop-mediatedisothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[14]Yu Y,Ma XY,Zhang W.Detection of Staphylococcus aureus in milk using real-time fluorescence loop-mediated isothermalamplification[J].Advance Journal of Food Science andTechnology,2015,8(9):678-684.
[15]Inomata A,Kishda I N,Momoda T,et al.Development andevaluation of a reverse transcription loop-mediated isothermalamplification assay for rapid and high-sensitive detection ofCryptosporidium in water samples[J].Water Science andTechnology,2009,60(8):2166-2172.
[16]Zhang GD,Brown EW,Narjol GE.Comparison of Real-time PCR,reverse transcriptase real-time PCR,loop-mediated isothermalamplification,and the FDA conventional microbiological methodfor the detection of Salmonella spp.in produce[J].Appl EnvironMicrobiology,2011,77(18):6495-6501.
[17]Wang F,Jiang L,Ge BL,et al.Loop-mediated isothermalamplification assays for detecting shigella toxin-producingEscherichia coli in ground beef and human stools[J].Microbiology.2012,50(1):91-97.
[18]Yukiko HK,Jun N,Ikuo G,et al.Surveillance of Shiga toxinproducing Escherichia coli in beef with effective procedures,independent of serotype[J].Foodborne pathogens and Disease,2008,1(5):97-103.
[19]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.
[20]刘道亮,胡连霞,赵占民,等.改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157:H7[J].微生物学通报,2011,38(3):430-435.
[21]张雪寒,何孔旺,叶青,等.快速检测大肠杆菌O157的LAMP方法的建立与评价[J].中国兽医学报,2013,33(7):1027-1031.
[22]Yukiko HK,Noriko K,Kayoko O,et al.Detection of vero toxigenicEscherichia coli O157 and O26 in food by plating methods andLAMP method A collaborative study[J].International Journal ofFood Microbiology,2008,122(12):156-161.
[23]张雪寒,何孔旺,叶青,等.快速检测志贺毒素2基因的LAMP方法的建立[J].江苏农业学报,2013,29(2):455-457.
[24]Wang F,Jiang L,Yang QR,et al.Rapid and specific detection ofEscherichia coli serogroups O26,O45,O103,O111,O121,O145,and O157 in ground beef,beef trim,and produce by loop-mediatedisothermal amplification[J].Appl Environ Microbiology,2012,78(8):2727-2736.
[25]Techathuvanan C,D’Souza DH.Reverse-transcriptase loopmediated isothermal amplification as a rapid screening/monitoringtool for Salmonella Enterica detection in liquid whole eggs[J].Journal of Food Science,2012,77(4):200-205.
[26]Fan FX,Wang SJ,Lou J.Establishment of RT-LAMP assay todetect Salmonella Typhi in blood[J].Disease Surveillance,2012,27(4):325-329.