5种致泻大肠埃希氏菌实时荧光定量PCR快速检测技术
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摘要
将5种致泻大肠埃希氏菌的毒力基因保守序列进行设计并合成了引物和探针,建立的多重实时荧光PCR4种试剂盒可以同时检测5种致泻大肠埃希氏菌。结果表明:4种试剂盒的12对毒力基因通过5种致泻大肠埃希氏菌的标准储备菌株均得到验证;并且12对毒力基因只对对应的致泻大肠埃希氏菌特异性起作用,对常见的食源性致病菌都无扩增曲线;escV、bfpB、stx1、ipaH和invE基因的检出限是10CFU/mL,aggR基因的检出限是10~2 CFU/mL,astA和pic基因的检出限是10~3 CFU/mL,而stx_2A、ipaH和stIb基因菌液浓度<10CFU/mL时,均可观察到明显的"S"型扩增曲线,且线形较好;并且对抽查和委托检验的肉类、奶和动物腹泻物以及人工污染样品等40份样品进行检测,共检出11份阳性样本,与GB 4789.6—2016标准检测结果相一致,表明建立的4种试剂盒方法具有良好的实用性。
Primers,and probes were designed for the conserved sequences of five virulence genes involved in Escherichia coli diarrhea mentioned in the new standard of GB 4789.6—2016 national food safety standard.Four kits of multiple real-time fluorescence PCR were established to detect five diarrhea-causing E.coli simultaneously.The results showed that 12 pairs of virulence genes of the four kits were verified by the standard reserve strains of the five diarrhea-causing E.coli strains.In addition,these 12 pairs of virulence genes only play a role in the specific role of corresponding diarrhea-causing E.coli,and have no amplification curve for common foodborne pathogens.The detection limit of escV,bfpB,stx1,ipaH and invEgenes is 10 CFU/mL,and that of aggR gene is 10~2 CFU/mL.The detection limit of astA and Pic genes was 10~3 CFU/mL.When the concentration of stx_2 A,ipaH and stIb genes was less than 10 CFU/mL,a significant"S"type amplification curve could be observed,and the line shape was better.In addition,40 samples including meat, milk,animal diarrhea,and some artificial contamination samples were tested,and a total of 11 positive samples were found.Consistent with the test results of the new standard 4789.6—2016,The results showed that the four kit methods were simple,specific,sensitive and practical.
Primers,and probes were designed for the conserved sequences of five virulence genes involved in Escherichia coli diarrhea mentioned in the new standard of GB 4789.6—2016 national food safety standard.Four kits of multiple real-time fluorescence PCR were established to detect five diarrhea-causing E.coli simultaneously.The results showed that 12 pairs of virulence genes of the four kits were verified by the standard reserve strains of the five diarrhea-causing E.coli strains.In addition,these 12 pairs of virulence genes only play a role in the specific role of corresponding diarrhea-causing E.coli,and have no amplification curve for common foodborne pathogens.The detection limit of escV,bfpB,stx1,ipaH and invEgenes is 10 CFU/mL,and that of aggR gene is 10~2 CFU/mL.The detection limit of astA and Pic genes was 10~3 CFU/mL.When the concentration of stx_2 A,ipaH and stIb genes was less than 10 CFU/mL,a significant"S"type amplification curve could be observed,and the line shape was better.In addition,40 samples including meat, milk,animal diarrhea,and some artificial contamination samples were tested,and a total of 11 positive samples were found.Consistent with the test results of the new standard 4789.6—2016,The results showed that the four kit methods were simple,specific,sensitive and practical.
引文
[1]燕勇,高文洁,吉季梅.一株极易误检为侵袭性大肠埃希菌O124:K72的无丙二酸柠檬酸杆菌[J].中国卫生检验杂志,2007,17(5):945-946.
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[7]陈小玲.与O1群霍乱弧菌多价血清交叉凝集的肠道产毒性大肠埃希氏菌的鉴定[J].医学动物防制,2015(11):1 289-1 290.
[8]姚栋,张如胜,欧新华.多重PCR快速检测3种致泻性大肠埃希菌方法的建立[J].国际检验医学,2015(8):1 032-1 033.
[9]李红,陈松,周汉洪,等.致泻性大肠埃希菌2种检测方法的比较[J].现代预防医学,2015,42(20):3 777-3 779.
[10]劳希,梁炳健,林静梅.多重实时荧光PCR检测食品中致泻性大肠埃希菌[J].中国城乡企业卫生,2017(12):28-31.
[11]GUION C E,OCHOA T J,WALKER C M,et al.Detection of diarrheagenic Escherichia coli by use of meltingcurve analysis and real-time multiplex PCR[J].Journal of Clinical Microbiology,2008,46(5):1 752-1 757.
[12]WEST D M,SPRINGINGS K A,CASSAR C,et al.Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan PCR assays[J].Veterinary Microbiology,2007,122(3):323-331.
[13]YANG Ji-rong,WU Fang-tzy,TSAI Jin-lai,et al.Comparison between O serotyping method and multiplex real-time PCRto identify diarrheagenic Escherichia coli in Taiwan[J].J Clin Microbiol,2007,45(11):3 620-3 625.
[14]PERSSON S,OLSEN K E P,SCHEUTZ F,et al.A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory[J].Clin Microbiol Infect,2007,13(5):516-524.
[15]金玉娟,陈应坚,刘渠,等.多重实时荧光PCR检测三种致泻性大肠埃希菌和志贺菌方法的建立[J].中国卫生检验,2013(1):103-108.
[16]金玉娟,刘渠,陈应坚,等.四种致泻性大肠埃希氏菌实验室检测研究进展[J].中国热带医学,2011,11(1):116-118.
[17]朱惠芳,黄巧萍.1株与志贺氏菌生化相似、抗原相同的大肠埃希氏菌[J].实用检验医师,2014,6(4):256-258.
[18]YOUMANS B P,AJAMI N J,JIANG Zhi-dong,et al.Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli(ETEC)Heat Labile and Heat Stable Toxin Genes in Travelers'Diarrhea Samples[J].American Journal of Tropical Medicine&Hygiene,2014,90(1):124-32.
[19]FIALHO O B,DE SOUZA E M,DE B D C,et al.Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol[J].Journal of Clinical Laboratory Analysis,2013,27(2):155-161.
[20]赵爱兰,熊衍文,白雪梅,等.鉴定五类致泻性大肠埃希菌和志贺菌的多重PCR方法[J].疾病监测,2011,26(1):65-67.
[21]张红宇,孟祥晨,姜博,等.Taqman三重实时PCR快速检测原料乳中致泻性大肠埃希氏菌[J].东北农业大学学报,2010,41(10):108-115.
[22]王娉,袁飞,杨海荣,等.奶液模拟标本中单增李斯特菌real-time PCR检测方法的建立[J].卫生研究,2011,40(6):765-768.
[23]席美丽,只帅,王小璞,等.食源性大肠杆菌的PCR检测[J].西北农业学报,2011,20(12):188-191.
[24]舒畅,姜琛璐,钟慈平,等.三种食源性致病菌多重PCR检测方法的建立[J].食品工业科技,2014,35(12):49-54.
[2]燕勇,吉季梅,张建平.致泻大肠埃希菌实验室检测工作的探讨与分析[J].中国卫生检验,2007,17(10):1 895-1 896.
[3]陈芳,陈云,杨林,等.双重PCR检测猪产肠毒素性大肠埃希菌耐热肠毒素基因[J].黑龙江畜牧兽医,2010(11):123-124.
[4]丁琦.八种不同血清型肠出血性大肠埃希氏菌多重PCR检测方法的建立及应用[D].广州:华南理工大学,2017.
[5]张铁男,李继昌,鲁成武,等.3种致泻性大肠埃希氏菌多重PCR检测方法的研究[J].中国兽医,2007,43(9):17-19.
[6]秦小玄,朱朝敏.致泻性大肠杆菌的流行及耐药现状[J].儿科药学,2008,14(2):61-64.
[7]陈小玲.与O1群霍乱弧菌多价血清交叉凝集的肠道产毒性大肠埃希氏菌的鉴定[J].医学动物防制,2015(11):1 289-1 290.
[8]姚栋,张如胜,欧新华.多重PCR快速检测3种致泻性大肠埃希菌方法的建立[J].国际检验医学,2015(8):1 032-1 033.
[9]李红,陈松,周汉洪,等.致泻性大肠埃希菌2种检测方法的比较[J].现代预防医学,2015,42(20):3 777-3 779.
[10]劳希,梁炳健,林静梅.多重实时荧光PCR检测食品中致泻性大肠埃希菌[J].中国城乡企业卫生,2017(12):28-31.
[11]GUION C E,OCHOA T J,WALKER C M,et al.Detection of diarrheagenic Escherichia coli by use of meltingcurve analysis and real-time multiplex PCR[J].Journal of Clinical Microbiology,2008,46(5):1 752-1 757.
[12]WEST D M,SPRINGINGS K A,CASSAR C,et al.Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan PCR assays[J].Veterinary Microbiology,2007,122(3):323-331.
[13]YANG Ji-rong,WU Fang-tzy,TSAI Jin-lai,et al.Comparison between O serotyping method and multiplex real-time PCRto identify diarrheagenic Escherichia coli in Taiwan[J].J Clin Microbiol,2007,45(11):3 620-3 625.
[14]PERSSON S,OLSEN K E P,SCHEUTZ F,et al.A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory[J].Clin Microbiol Infect,2007,13(5):516-524.
[15]金玉娟,陈应坚,刘渠,等.多重实时荧光PCR检测三种致泻性大肠埃希菌和志贺菌方法的建立[J].中国卫生检验,2013(1):103-108.
[16]金玉娟,刘渠,陈应坚,等.四种致泻性大肠埃希氏菌实验室检测研究进展[J].中国热带医学,2011,11(1):116-118.
[17]朱惠芳,黄巧萍.1株与志贺氏菌生化相似、抗原相同的大肠埃希氏菌[J].实用检验医师,2014,6(4):256-258.
[18]YOUMANS B P,AJAMI N J,JIANG Zhi-dong,et al.Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli(ETEC)Heat Labile and Heat Stable Toxin Genes in Travelers'Diarrhea Samples[J].American Journal of Tropical Medicine&Hygiene,2014,90(1):124-32.
[19]FIALHO O B,DE SOUZA E M,DE B D C,et al.Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol[J].Journal of Clinical Laboratory Analysis,2013,27(2):155-161.
[20]赵爱兰,熊衍文,白雪梅,等.鉴定五类致泻性大肠埃希菌和志贺菌的多重PCR方法[J].疾病监测,2011,26(1):65-67.
[21]张红宇,孟祥晨,姜博,等.Taqman三重实时PCR快速检测原料乳中致泻性大肠埃希氏菌[J].东北农业大学学报,2010,41(10):108-115.
[22]王娉,袁飞,杨海荣,等.奶液模拟标本中单增李斯特菌real-time PCR检测方法的建立[J].卫生研究,2011,40(6):765-768.
[23]席美丽,只帅,王小璞,等.食源性大肠杆菌的PCR检测[J].西北农业学报,2011,20(12):188-191.
[24]舒畅,姜琛璐,钟慈平,等.三种食源性致病菌多重PCR检测方法的建立[J].食品工业科技,2014,35(12):49-54.