多重PCR同时检测食品中4种细菌与常见霉菌
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摘要
建立一种同时检测食品中金黄色葡萄球菌、单核细胞增生李斯特氏菌、沙门氏菌、大肠杆菌O157:H7以及霉菌的多重聚合酶链式反应(polymerase chain reaction,PCR)检测方法。根据金黄色葡萄球菌的耐热核酸酶基因(nuc)、沙门氏菌的侵袭正调节蛋白基因(hilA)、大肠杆菌O157:H7鞭毛基因(flic)、单核细胞增生李斯特氏菌的毒力调控蛋白基因(prfA)及霉菌18S rRNA基因V5区分别设计5对特异性引物,并对PCR扩增反应体系和扩增条件进行优化,确定获得特异性良好的PCR扩增产物。而后在37℃对人工污染致病菌的香肠、面包和豆腐进行增菌培养,5种致病微生物在20 h内的检出限均可达到100 CFU/25 g。本实验建立的多重PCR检测方法适用于食品中4种细菌与常见霉菌的同时检测,相较于传统检测方法,具有快速、简便、特异性高的优点。
In this study, a multiplex polymerase chain reaction(PCR) method was developed to simultaneously detect Staphylococcus aureus, Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and mold in foods. Speciesspecific primers were designed targeting the heat-resistant nuclease gene(nuc) in S. aureus, the invader upregulation protein gene(hilA) in Salmonella, the flagellum gene(flic) in E. coli O157:H7, the virulence regulation protein gene(prfA) in L. monocytogenes and the V5 region of the 18 S rRNA gene in mold, and the PCR reaction system and conditions were optimized for highly specific amplification. Sausage, bread and tofu artificially contaminated with the five foodborne pathogens were enriched at 37 ℃.?The?detection?limits?of?the?five?food-borne?pathogens?within?20?hours?all?reached?100 CFU/25 g. The multiplex PCR method established in this study is suitable for rapid detection of common pathogenic organisms in foods. Compared with traditional detection methods, it has the advantages of rapidity, simplicity and high specificity?and?thus,?can?be?used?to?simultaneously?detect?bacteria?and?mold?in?foods.
In this study, a multiplex polymerase chain reaction(PCR) method was developed to simultaneously detect Staphylococcus aureus, Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and mold in foods. Speciesspecific primers were designed targeting the heat-resistant nuclease gene(nuc) in S. aureus, the invader upregulation protein gene(hilA) in Salmonella, the flagellum gene(flic) in E. coli O157:H7, the virulence regulation protein gene(prfA) in L. monocytogenes and the V5 region of the 18 S rRNA gene in mold, and the PCR reaction system and conditions were optimized for highly specific amplification. Sausage, bread and tofu artificially contaminated with the five foodborne pathogens were enriched at 37 ℃.?The?detection?limits?of?the?five?food-borne?pathogens?within?20?hours?all?reached?100 CFU/25 g. The multiplex PCR method established in this study is suitable for rapid detection of common pathogenic organisms in foods. Compared with traditional detection methods, it has the advantages of rapidity, simplicity and high specificity?and?thus,?can?be?used?to?simultaneously?detect?bacteria?and?mold?in?foods.
引文
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[11]闫琳,王晓英,郭云昌,等.增菌-PCR法与传统方法检测禽肉中沙门氏菌的比较研究[J].卫生研究,2011,40(3):348-351;354.
[12]钱红玫,胡文忠,冯可,等.食源性致病菌快速检测的前增菌培养的研究进展[J].食品工业科技,2016,37(13):360-364.DOI:10.13386/j.issn1002-0306.2016.13.066.
[13]顾双,陈俊霖,王向阳.传统霉菌发酵食品中的霉菌DNA扩增[J].食品科学,2017,38(4):83-86.DOI:10.7506/spkx1002-6630-201704014.
[14]敖特根巴雅尔,乌兰其其格,陆继爽,等.干肉制品中霉菌PCR鉴定方法的建立[J].畜牧与饲料科学,2016,37(3):49-51;54.DOI:10.16003/j.cnki.issn1672-5190.2016.03.040.
[15]HANSON K E,SLECHTA E S,KILLPACK J A,et al.Preclinical assessment of a fully automated multiplex PCR panel for detection of central nervous system pathogens[J].Journal of Clinical Microbiology,2016,54(3):785-787.DOI:10.1128/JCM.02850-15.
[16]XU Y G,LIU Z M,ZHANG B Q,et al.Development of a novel target-enriched multiplex PCR(Tem-PCR)assay for simultaneous detection of five foodborne pathogens[J].Food Control,2016,64:54-59.DOI:10.1016/j.foodcont.2015.12.022.
[17]TIAN D,SUO Y,ZHANG Z,et al.A multiplex RT-PCR Assay for S.aureus,L.monocytogenes,and Salmonella spp.detection in raw milk with pre-enrichment[J].Frontiers in Microbiology,2017,8:989-1000.DOI:10.3389/fmicb.2017.00989.
[18]刘红玉,李岩,崔洪斌.肉中4种致病菌的PCR快速检测方法的建立[J].食品科学,2011,32(6):213-216.
[19]ZHOU G,WHONG W Z,ONG T,et al.Development of a fungusspecific PCR assay for detecting low-level fungi in an indoor environment[J].Molecular&Cellular Probes,2000,14(6):339-348.DOI:10.1006/mcpr.2000.0324.
[20]WENCHAO Y P D.Multiplex PCR primer design for simultaneous detection of multiple pathogens[J].Methods in Molecular Biology,2015,1275:91-101.DOI:10.1007/978-1-4939-2365-6_6.
[21]KORBIE D J,MATTICK J S.Touchdown PCR for increased specificity and sensitivity in PCR amplification[J].Nature Protocols,2008,3(9):1452-1456.DOI:10.1038/nprot.2008.133.
[22]KUMAR S,BALAKRISHNA K,BATRA H V.Detection of Salmonella enterica serovar Typhi(S.Typhi)by selective amplification of invA,viaB,fliC-d and prt genes by polymerase chain reaction in mutiplex format[J].Letters in Applied Microbiology,2006,42(2):149-154.DOI:10.1111/j.1472-765X.2005.01813.
[23]LEE N,KWON K Y,OH S K,et al.A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7,Bacillus cereus,Vibrio parahaemolyticus,Salmonella spp.,Listeria monocytogenes,and Staphylococcus aureus in Korean ready-to-eat food[J].Foodborne Pathogens&Disease,2014,11(7):574-580.DOI:10.1089/fpd.2013.1638.
[24]吴科明,黄飞,李叶青,等.多重PCR在食源性微生物快速检验中的应用与评价[J].中国卫生检验杂志,2017,27(11):1557-1559.
[25]ZHOU B L,XIAO J W,LIU S F,et al.Simultaneous detection of six food-borne pathogens by multiplex PCR with;a GeXPanalyzer[J].Food Control,2013,32(1):198-204.DOI:10.1016/j.foodcont.2012.11.044.
[26]ZHANG D,ZHANG H,YANG L,et al.Simultaneous detection of Listeria monocytogenes,Staphylococcus aureus,Salmonella enterica and Escherichia coli O157:H7 in food samples using multiplex PCRmethod[J].Journal of Food Safety,2009,29(3):348-363.DOI:10.1111/j.1745-4565.2009.00161.
[27]胡冰雪,舒沿沿,潘道东,等.荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立[J].食品科学,2016,37(20):209-214.DOI:10.7506/spkx1002-6630-201620036.
[28]冯可,胡文忠,姜爱丽,等.鲜切果蔬中4种病原微生物多重PCR检测技术[J].食品科学,2018,39(6):276-283.DOI:10.7506/spkx1002-6630-201806043.
[29]李博,陈福生,王小红,等.多重PCR检测食品中的金黄色葡萄球菌、志贺菌和沙门菌[J].卫生研究,2008,37(4):438-442.DOI:10.3969/j.issn.1000-8020.2008.04.013.
[30]KAWASAKI S,HORIKOSHI N,OKADA Y,et al.Multiplex PCR for simultaneous detection of Salmonella spp.,Listeria monocytogenes,and Escherichia coli O157:H7 in meat samples[J].Journal of Food Protection,2005,68(3):551-556.DOI:10.4315/0362-028X-68.3.551-556.
[31]孙婷婷,赵明,李亚莉,等.普洱茶发酵样品细菌和真菌DNA同时提取方法研究[J].中国农学通报,2011,27(15):249-253.
[32]SATYABRATA B,BIPASA S,OJASVI M,et al.An improved method for high quality metagenomics DNA extraction from human and environmental samples[J].Scientific Reports,2016,6:26775.DOI:10.1038/srep26775-26784.
[2]HU K,RENLY S,EDLUND S,et al.A modeling framework to accelerate food-borne outbreak investigations[J].Food Control,2016,59:53-58.DOI:10.1016/j.foodcont.2015.05.017.
[3]EFSA(European Food Safety Authority).Analysis of the baseline survey on the prevalence of Listeria monocytogenes in certain readytoeat foods in the EU,2010-2011.Part B:analysis of factors related to prevalence and exploring compliance[J].EFSA Journal,2014,12(8):3810.DOI:10.2903/j.efsa.2014.3810.
[4]YANG S,PEI X,WANG G,et al.Prevalence of food-borne pathogens in ready-to-eat meat products in seven different Chinese regions[J].Food Control,2016,65:92-98.DOI:10.1016/j.foodcont.2016.01.009.
[5]TOLOUEE M,ALINEZHAD S,SABERI R,et al.Effect of Matricaria chamomilla L.flower essential oil on the growth and ultrastructure of Aspergillus niger van Tieghem[J].International Journal of Food Microbiology,2010,139(3):127-133.DOI:10.1016/j.ijfoodmicro.2010.03.032.
[6]魏超,代晓航,郭灵安,等.草莓致腐霉菌的鉴定及其产毒能力研究[J].食品安全质量检测学报,2017,8(5):1721-1726.
[7]刘程惠,胡文忠,王艳颖,等.鲜切生菜致腐霉菌的分离鉴定[J].食品工业科技,2016,37(3):135-138.DOI:10.13386/j.issn1002-0306.2016.03.020.
[8]SETTANNI L,CORSETTI A.The use of multiplex PCR to detect and differentiate food-and beverage-associated microorganisms:a review[J].Journal of Microbiological Methods,2007,69(1):1-22.DOI:10.1016/j.mimet.2006.12.008.
[9]WEI C J,ZHONG J L,HU T,et al.Simultaneous detection of Escherichia coli O157:H7,Staphylococcus aureus,and Salmonella,by multiplex PCR in milk[J].Biotech,2018,8(1):76-83.DOI:10.1007/s13205-018-1086-5.
[10]NGUYEN T T,GIAU V V,VO T K.Multiplex PCR for simultaneous identification of E.coli O157:H7,Salmonella spp.and L.monocytogenes in food[J].Biotech,2016,6(2):205-214.DOI:10.1007/s13205-016-0523-6.
[11]闫琳,王晓英,郭云昌,等.增菌-PCR法与传统方法检测禽肉中沙门氏菌的比较研究[J].卫生研究,2011,40(3):348-351;354.
[12]钱红玫,胡文忠,冯可,等.食源性致病菌快速检测的前增菌培养的研究进展[J].食品工业科技,2016,37(13):360-364.DOI:10.13386/j.issn1002-0306.2016.13.066.
[13]顾双,陈俊霖,王向阳.传统霉菌发酵食品中的霉菌DNA扩增[J].食品科学,2017,38(4):83-86.DOI:10.7506/spkx1002-6630-201704014.
[14]敖特根巴雅尔,乌兰其其格,陆继爽,等.干肉制品中霉菌PCR鉴定方法的建立[J].畜牧与饲料科学,2016,37(3):49-51;54.DOI:10.16003/j.cnki.issn1672-5190.2016.03.040.
[15]HANSON K E,SLECHTA E S,KILLPACK J A,et al.Preclinical assessment of a fully automated multiplex PCR panel for detection of central nervous system pathogens[J].Journal of Clinical Microbiology,2016,54(3):785-787.DOI:10.1128/JCM.02850-15.
[16]XU Y G,LIU Z M,ZHANG B Q,et al.Development of a novel target-enriched multiplex PCR(Tem-PCR)assay for simultaneous detection of five foodborne pathogens[J].Food Control,2016,64:54-59.DOI:10.1016/j.foodcont.2015.12.022.
[17]TIAN D,SUO Y,ZHANG Z,et al.A multiplex RT-PCR Assay for S.aureus,L.monocytogenes,and Salmonella spp.detection in raw milk with pre-enrichment[J].Frontiers in Microbiology,2017,8:989-1000.DOI:10.3389/fmicb.2017.00989.
[18]刘红玉,李岩,崔洪斌.肉中4种致病菌的PCR快速检测方法的建立[J].食品科学,2011,32(6):213-216.
[19]ZHOU G,WHONG W Z,ONG T,et al.Development of a fungusspecific PCR assay for detecting low-level fungi in an indoor environment[J].Molecular&Cellular Probes,2000,14(6):339-348.DOI:10.1006/mcpr.2000.0324.
[20]WENCHAO Y P D.Multiplex PCR primer design for simultaneous detection of multiple pathogens[J].Methods in Molecular Biology,2015,1275:91-101.DOI:10.1007/978-1-4939-2365-6_6.
[21]KORBIE D J,MATTICK J S.Touchdown PCR for increased specificity and sensitivity in PCR amplification[J].Nature Protocols,2008,3(9):1452-1456.DOI:10.1038/nprot.2008.133.
[22]KUMAR S,BALAKRISHNA K,BATRA H V.Detection of Salmonella enterica serovar Typhi(S.Typhi)by selective amplification of invA,viaB,fliC-d and prt genes by polymerase chain reaction in mutiplex format[J].Letters in Applied Microbiology,2006,42(2):149-154.DOI:10.1111/j.1472-765X.2005.01813.
[23]LEE N,KWON K Y,OH S K,et al.A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7,Bacillus cereus,Vibrio parahaemolyticus,Salmonella spp.,Listeria monocytogenes,and Staphylococcus aureus in Korean ready-to-eat food[J].Foodborne Pathogens&Disease,2014,11(7):574-580.DOI:10.1089/fpd.2013.1638.
[24]吴科明,黄飞,李叶青,等.多重PCR在食源性微生物快速检验中的应用与评价[J].中国卫生检验杂志,2017,27(11):1557-1559.
[25]ZHOU B L,XIAO J W,LIU S F,et al.Simultaneous detection of six food-borne pathogens by multiplex PCR with;a GeXPanalyzer[J].Food Control,2013,32(1):198-204.DOI:10.1016/j.foodcont.2012.11.044.
[26]ZHANG D,ZHANG H,YANG L,et al.Simultaneous detection of Listeria monocytogenes,Staphylococcus aureus,Salmonella enterica and Escherichia coli O157:H7 in food samples using multiplex PCRmethod[J].Journal of Food Safety,2009,29(3):348-363.DOI:10.1111/j.1745-4565.2009.00161.
[27]胡冰雪,舒沿沿,潘道东,等.荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立[J].食品科学,2016,37(20):209-214.DOI:10.7506/spkx1002-6630-201620036.
[28]冯可,胡文忠,姜爱丽,等.鲜切果蔬中4种病原微生物多重PCR检测技术[J].食品科学,2018,39(6):276-283.DOI:10.7506/spkx1002-6630-201806043.
[29]李博,陈福生,王小红,等.多重PCR检测食品中的金黄色葡萄球菌、志贺菌和沙门菌[J].卫生研究,2008,37(4):438-442.DOI:10.3969/j.issn.1000-8020.2008.04.013.
[30]KAWASAKI S,HORIKOSHI N,OKADA Y,et al.Multiplex PCR for simultaneous detection of Salmonella spp.,Listeria monocytogenes,and Escherichia coli O157:H7 in meat samples[J].Journal of Food Protection,2005,68(3):551-556.DOI:10.4315/0362-028X-68.3.551-556.
[31]孙婷婷,赵明,李亚莉,等.普洱茶发酵样品细菌和真菌DNA同时提取方法研究[J].中国农学通报,2011,27(15):249-253.
[32]SATYABRATA B,BIPASA S,OJASVI M,et al.An improved method for high quality metagenomics DNA extraction from human and environmental samples[J].Scientific Reports,2016,6:26775.DOI:10.1038/srep26775-26784.