基于实时荧光环介导等温扩增快速检测鸡肉中的大肠杆菌O157:H
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摘要
以大肠杆菌O157:H7的VT2基因序列设计特异性引物,利用Midori Green新型核酸荧光染料,建立鸡肉中大肠杆菌O157:H7实时荧光定量环介导等温扩增(Rti-LAMP)检测方法。纯培养大肠杆菌O157:H7检测灵敏度达3.5 CFU/反应,需时45 min。模拟大肠杆菌O157:H7污染鸡肉样品,经37℃增菌4 h,用Whirl-pak无菌袋过滤离心得沉淀,提取样品DNA模板用于Rti-LAMP反应,检测大肠杆菌O157:H7灵敏度达140 CFU/g,整个检测流程约7 h。采用蒙脱石封闭的活性炭前处理污染鸡肉样品,不经增菌过程,结果表明:该Rti-LAMP检测方法灵敏度达12 CFU/g,整个检测耗时约4.5 h。市购鸡肉样品51份,以大肠杆菌国标检测法为对照,研究基于Rti-LAMP联合增菌或封闭活性炭预处理而不经增菌的两种方法,对比检测临床鸡肉样品大肠杆菌O157:H7污染率。结果:国标法和增菌的Rti-LAMP两种方法均检测到同一鸡肉样品为大肠杆菌O157:H7阳性,经封闭活性炭预处理而未经增菌的Rti-LAMP则检测到8份鸡肉样品大肠杆菌O157:H7阳性。研究表明,封闭活性炭预处理的Rti-LAMP检测方法较国标法更灵敏、快速、简便、特异地检测鸡肉中大肠杆菌O157:H7污染。
In this study, a real-time loop amplified DNA assay system(Rti-LAMP) was developed for the rapid detection of Escherichia coli(E. coli)O157:H7 on the chicken. It showed that the lowest level of 3.5 CFU/reaction for pure E. coli culture could be detected and finished in 45 minutes by the Rti-LAMP targeting the VT2 gene using Midori green as nucleic acid dye. Furthermore, seeding 25 g portions of chicken with various numbers of E. coli CFU, followed enrichment culture at 37 ℃ for 4 h, then filtration through Whirl-pak bag and pelleting the bacterial cells by centrifugation at 13 000 g. The resulting pellets were suspended in saline and processed for cell lysis and DNA purification. 2 μL purified DNA samples was incorporated into 25 μL Rti-LAMP reactions at 65 ℃ for 60 min. The lowest level 140 CFU/g of E. coli consistently detected by the Rti-LAMP assay. The entire assay could be completed in 7 h. Moreover, the samples were pretreated with activated carbon coated with bentonite and without enrichment for Rti-LAMP reactions. The results showed that the sensitivity of the sample treated with coated activated carbon was 12 CFU/g and finished in 4.5 h.Fifty-one chicken samples purchased from market were detected by the Rti-LAMP assay with enrichment, the Rti-LAMP with bentonite coated activated carbon(BCAC) pretreated but without enrichment, as the control of E. coli O157:H7 culture. The same one sample was E. coli O157:H7 positive by the Rti-LAMP assay with enrichment and E. coli culture,and other 7 samples(total 8 samples) were also E. coli O157:H7 positive detected by the Rti-LAMP pretreated with BCAC. These data suggested that the Rti-LAMP combined with BCAC pretreatment was more sensitive, rapid, simple and specific than that of culture for detection of E. coli O157:H7 retrieved from Chicken.
In this study, a real-time loop amplified DNA assay system(Rti-LAMP) was developed for the rapid detection of Escherichia coli(E. coli)O157:H7 on the chicken. It showed that the lowest level of 3.5 CFU/reaction for pure E. coli culture could be detected and finished in 45 minutes by the Rti-LAMP targeting the VT2 gene using Midori green as nucleic acid dye. Furthermore, seeding 25 g portions of chicken with various numbers of E. coli CFU, followed enrichment culture at 37 ℃ for 4 h, then filtration through Whirl-pak bag and pelleting the bacterial cells by centrifugation at 13 000 g. The resulting pellets were suspended in saline and processed for cell lysis and DNA purification. 2 μL purified DNA samples was incorporated into 25 μL Rti-LAMP reactions at 65 ℃ for 60 min. The lowest level 140 CFU/g of E. coli consistently detected by the Rti-LAMP assay. The entire assay could be completed in 7 h. Moreover, the samples were pretreated with activated carbon coated with bentonite and without enrichment for Rti-LAMP reactions. The results showed that the sensitivity of the sample treated with coated activated carbon was 12 CFU/g and finished in 4.5 h.Fifty-one chicken samples purchased from market were detected by the Rti-LAMP assay with enrichment, the Rti-LAMP with bentonite coated activated carbon(BCAC) pretreated but without enrichment, as the control of E. coli O157:H7 culture. The same one sample was E. coli O157:H7 positive by the Rti-LAMP assay with enrichment and E. coli culture,and other 7 samples(total 8 samples) were also E. coli O157:H7 positive detected by the Rti-LAMP pretreated with BCAC. These data suggested that the Rti-LAMP combined with BCAC pretreatment was more sensitive, rapid, simple and specific than that of culture for detection of E. coli O157:H7 retrieved from Chicken.
引文
[1]赵嘉咏,张白帆,穆玉姣,等.河南省出血性大肠埃希菌O157病原学特征与分子分型研究[J].中国病原生物学杂志, 2016, 11(9):812-814.
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[3]CRAIG S WONG, M D, SRDJAN JELACIC, et al.The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections[J]. New England Journal of Medicine, 2000,342(26):1930-1936.
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[6]赵璐,苏战强,逯晓龙,等.大肠杆菌O157:H7在部分市售鸡肉和鸡蛋中的污染状况调查[J].新疆农业科学, 2014, 51(10):1948-1954.
[7]黄海燕,陈亮,陈萍.双抗体夹心ELISA法检测食品中出血性大肠杆菌O157[J].食品工业科技, 2016,37(11):185-188.
[8]夏灿,蒋蔚,刘迎春,等.肠出血性大肠杆菌O157:H7抗原基因及毒力基因多重荧光定量PCR检测方法的建立[J].畜牧与兽医, 2016, 48(10):13-21.
[9]WU W, ZHAO S M, MAO Y P, et al. A sensitive lateral flow biosensor for Escherichia coli O157:H7detection based on aptamer mediated strand displacement amplification[J]. Analytica Chimica Acta,2015, 861(3):62-68.
[10]NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12):e63.
[11]WANG Y, FANG Y, LIU Y Y, et al. Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis[J].FEMS Microbiology Letters, 2010, 313(1):54-60.
[12]庞心怡,满朝新,赵玥明,等.环介导等温扩增技术快速检测肉中沙门氏菌[J].中国食物与营养,2015, 21(8):12-15.
[13]JEARANAIKOON P, PRAKRANKAMANANT P, LEELAYUWAT C, et al. The evaluation of loop-mediated isothermal amplification-quartz crystal microbalance(LAMP-QCM)biosensor as a real-time measurement of HPV16 DNA[J]. Journal of Virological Methods, 2016, 229(3):8-11.
[14]ZENG J, WEI H Y, ZHANG L, et al. Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loopmediated isothermal amplification[J]. International Journal of Food Microbiology, 2014, 174(9):123-128.
[15]MAYURKUMAR P. BHIMANI, BHARAT B. BHANDERI et al. Loop-mediated Isothermal Amplification assay(LAMP)based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera[J]. Veterinaria Italiana, 2015, 51(2):115-121.
[16]WU G P, CHEN S H, ROBERT E LEVIN. Rapid real-time loop-mediated isothermal amplification combined with coated activated carbon for detection of low numbers of Salmonella enterica from lettuce without enrichment[J]. Food Control, 2015, 56(10):47-52.
[17]LEE J L, ROBERT E LEVIN. Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment[J]. Food Microbiology, 2011, 28(3):562-567.
[18]WU G P, ROBERT E LEVIN. Rapid and sensitive detection of Salmonella enterica ser. enteritis retrieved from lettuce using a Real-time Loop-mediated Amplification Isothermal Assay without enrichment[J]. Food Biotechnology, 2015, 29(3):263-275.
[19]YUKIKO H K, JIRO N, KAYOKO O, et al. Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification[J]. Journal of Medical Microbiology, 2007,56(3):398-406.
[20]ABOLMAATY A, VU C, OLIVER J, et al. Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction[J]. Microbios, 2000, 101(400):181-189.
[21]WU G P, CHEN S H, ROBERT E. LEVIN. Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification[J]. Journal of Microbiological Methods, 2015, 117(10):41-48.
[22]姜彦君,都启晶,赵宏坤.食源性致病菌多重PCR检测方法的建立与应用[J].中国食品学报, 2013,13(10):162-169.
[23]王瑾,林丽萍,郜妍妍,等.实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌[J].食品科学, 2016,37(24):170-174.
[2]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.
[3]CRAIG S WONG, M D, SRDJAN JELACIC, et al.The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections[J]. New England Journal of Medicine, 2000,342(26):1930-1936.
[4]RILEY L W, REMIS R S, HELGERSON S D, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype[J]. New England Journal of Medicine, 1983, 308(12):681-685.
[5]ZHANG S H, ZHU X M, WU Q P, et al. Prevalence and characterization of Escherichia coli O157and O157:H7 in retail fresh raw meat in South China[J]. Annals of Microbiology, 2015, 65(4):1993-1999.
[6]赵璐,苏战强,逯晓龙,等.大肠杆菌O157:H7在部分市售鸡肉和鸡蛋中的污染状况调查[J].新疆农业科学, 2014, 51(10):1948-1954.
[7]黄海燕,陈亮,陈萍.双抗体夹心ELISA法检测食品中出血性大肠杆菌O157[J].食品工业科技, 2016,37(11):185-188.
[8]夏灿,蒋蔚,刘迎春,等.肠出血性大肠杆菌O157:H7抗原基因及毒力基因多重荧光定量PCR检测方法的建立[J].畜牧与兽医, 2016, 48(10):13-21.
[9]WU W, ZHAO S M, MAO Y P, et al. A sensitive lateral flow biosensor for Escherichia coli O157:H7detection based on aptamer mediated strand displacement amplification[J]. Analytica Chimica Acta,2015, 861(3):62-68.
[10]NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12):e63.
[11]WANG Y, FANG Y, LIU Y Y, et al. Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis[J].FEMS Microbiology Letters, 2010, 313(1):54-60.
[12]庞心怡,满朝新,赵玥明,等.环介导等温扩增技术快速检测肉中沙门氏菌[J].中国食物与营养,2015, 21(8):12-15.
[13]JEARANAIKOON P, PRAKRANKAMANANT P, LEELAYUWAT C, et al. The evaluation of loop-mediated isothermal amplification-quartz crystal microbalance(LAMP-QCM)biosensor as a real-time measurement of HPV16 DNA[J]. Journal of Virological Methods, 2016, 229(3):8-11.
[14]ZENG J, WEI H Y, ZHANG L, et al. Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loopmediated isothermal amplification[J]. International Journal of Food Microbiology, 2014, 174(9):123-128.
[15]MAYURKUMAR P. BHIMANI, BHARAT B. BHANDERI et al. Loop-mediated Isothermal Amplification assay(LAMP)based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera[J]. Veterinaria Italiana, 2015, 51(2):115-121.
[16]WU G P, CHEN S H, ROBERT E LEVIN. Rapid real-time loop-mediated isothermal amplification combined with coated activated carbon for detection of low numbers of Salmonella enterica from lettuce without enrichment[J]. Food Control, 2015, 56(10):47-52.
[17]LEE J L, ROBERT E LEVIN. Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment[J]. Food Microbiology, 2011, 28(3):562-567.
[18]WU G P, ROBERT E LEVIN. Rapid and sensitive detection of Salmonella enterica ser. enteritis retrieved from lettuce using a Real-time Loop-mediated Amplification Isothermal Assay without enrichment[J]. Food Biotechnology, 2015, 29(3):263-275.
[19]YUKIKO H K, JIRO N, KAYOKO O, et al. Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification[J]. Journal of Medical Microbiology, 2007,56(3):398-406.
[20]ABOLMAATY A, VU C, OLIVER J, et al. Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction[J]. Microbios, 2000, 101(400):181-189.
[21]WU G P, CHEN S H, ROBERT E. LEVIN. Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification[J]. Journal of Microbiological Methods, 2015, 117(10):41-48.
[22]姜彦君,都启晶,赵宏坤.食源性致病菌多重PCR检测方法的建立与应用[J].中国食品学报, 2013,13(10):162-169.
[23]王瑾,林丽萍,郜妍妍,等.实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌[J].食品科学, 2016,37(24):170-174.