Advances on Rapid Detection of Foodborne Pathogens and the Application
-
摘要: 随着食品工业的发展，食品安全成为人们日益关注的公共健康问题。在影响食品安全的因素中，病原微生物是最主要的因素之一。因此，建立食源性致病菌的快速检测技术对确保食品安全和保障人类健康意义重大。传统的食源性致病菌检测方法，如微生物培养法和菌落技术法耗时费力，远不能满足食品安全快速检测的要求。目前已报道多种食源性致病菌的快速检测方法，如免疫学技术、分子生物学技术、生物传感器技术等。本文综述了国内外食源性致病菌的快速检测技术及其应用研究进展，比较分析各项检测技术的特点，为新的食源性致病菌检测技术的开发提供参考。Abstract: With the development of food industry, product safety is becoming an increasingly important public health issue. Among the factors that affect food safety, illnesses caused by pathogenic microorganisms is of paramount concern. To prevent or curtail the undesirable incidence from happening, establishment of rapid detection methodologies on foodborne pathogens is one of the indispensable measures. Conventional bacterial detection methods, such as culture and colony counting, are time consuming, laborious and inadequate to meet the demand of a modern society. In recent years, various rapid detection methods have been developed, which apply the technologies involving immunological, molecular biotechnical and/or bio-sensing approaches. This article reviews the methodologies currently available in China and abroad, and compares the pros and cons of the associated technologies and applications.
-
表 2 分子生物学技术在检测食源性致病菌中的应用
Table 2. Nucleic acid-based methods for foodborne pathogen detection
检测方法 检测的致病菌 检测限度 检测食品 检测时间 参考文献 mPCR 大肠杆菌O157:H7，单增李斯特菌，金黄色葡萄球菌，沙门氏菌 103 CFU·mL-1 人工污染的猪肉 - [30] 沙门氏菌，大肠杆菌O157:H7，单增李斯特菌 沙门氏菌，大肠杆菌O157:H7为10 CFU·g-1，单增李斯特菌为102 CFU·g-1 人工污染的牛肉 18 h [55] RT-qPCR 沙门氏菌， 103 CFU·g-1 熟火腿 - [39] 金黄色葡萄球菌，沙门氏菌，志贺氏菌 金黄色葡萄球菌为9.6 CFU·g-1，沙门氏菌为2.0 CFU·g-1，志贺氏菌为6.8CFU·g-1 鲜猪肉 ＜8 h [13] 大肠杆菌O157:H7 78 pg·管-1 - ＜30 min [56] LAMP 沙门氏菌 0.05 ng·mL-1 DNA 生鸡蛋，禽肉，水产品等 ＜40 min [57] NASBA 副溶血性弧菌 5.1×102CFU·mL-1 - - [48] 基因芯片技术 大肠杆菌，沙门氏菌，金黄色葡萄球菌等多种细菌 10 CFU·mL-1 人工污染的牛奶和肉 3h [58] 
下载: 导出CSV
表 3 生物传感器检测技术在检测食源性致病菌中的应用
Table 3. Biosensor-based methods for foodborne pathogen detection
下载: 导出CSV
表 4 常见食源性致病菌检测技术的比较
Table 4. Comparison on common technologies applied for foodborne pathogen detection
种类 名称 特点 不足 免疫学检测技术 ELISA 特异、稳定、费用低、易于操作；可进行大量样品的同时检测；能够检测毒素。 影响因素多；不能同时对多种成分进行分析；对试剂的选择性高；存在假阴性。 IMS 特异性强、灵敏度高、分离速度快。 样品需求量大；必须筛选到致病菌的特异性抗原靶标；可能存在假阴性。 IFT 特异性强；检测时间短。 灵敏度偏低，具有主观因素；技术程序比较复杂。 IGLT 操作简单；易于携带；无需专业人员和其他仪器；反应迅速；成本低廉；适合在基层及大批量样品的现场筛查。 灵敏度低。 LA 特异性强、操作简便、快速、经济、判断直观，适合基层使用。 灵敏度较低。 分子生物学检测技术 mPCR 特异性、灵敏度较高；一次可同时检测多种致病菌。 会受PCR抑制物影响；扩增效率低；引物设计较难；无法区分死菌和活菌。 RT-qPCR 特异性强，敏感性高；不需扩增后处理；实时监测扩增产物。 成本高；会受PCR抑制物影响；做多重qPCR较难；无法区分死菌和活菌；需要专业人员操作。 LAMP 特异性强，敏感性高；容易操作；成本低。 引物设计难；扩增序列不能超过300 bp；容易交叉污染；假阳性高。 NASBA 特异性、灵敏度较高；反应迅速，操作简单；成本低。 不能进行大通量检测；样品制备复杂；样品必须是活的微生物。 基因芯片技术 特异性强，敏感性高；能实现对食品中的致病菌高通量和并行检测；操作简便快速。 成本高；需要专业人员操作；芯片制备和杂交过程耗时。 生物传感器检测技术 只需极微量的检测样本；灵敏度高，重复性好。 成本高。 代谢学检测技术 ATP生物发光技术 检测时间特别短；采用该方法制作的检测仪体积小，携带方便。 检测试剂贵；反应易受到各类因素影响。
下载: 导出CSV
-
[1] YASMIN J, AHMED M R, CHO B. Biosensors and their applications in food safety: areview[J]. Journal of Biosystems Engineering, 2016, 41(3):240-254. doi: 10.5307/JBE.2016.41.3.240 [2] Centers for Disease Control and Prevention(CDC). 2015 Food Safety Report[EB/OL]www.cdc.gov/foodnet/index.html. [3] 国家卫生计生委办公厅. 国家卫生计生委办公厅关于2015年全国食物中毒事件情况的通报[EB/OL]. http://www.nhfpc.gov.cn/yjb/s7859/201604/8d34e4c442c54d33909319954c43311c.shtml.2016-04-01/2016-11-28. [4] VELUSAMY V, ARSHAK K, KOROSTYNSKA O, et al. An overview of foodborne pathogen detection: In the perspective of biosensors[J]. Biotechnology Advances, 2010, 28(2):232-254. doi: 10.1016/j.biotechadv.2009.12.004 [5] BOSILEVAC J M, GUERINI M N, KALCHAYANAND N, et al. Prevalence and characterization of salmonellae in commercial ground beef in the United States[J]. Applied & Environmental Microbiology, 2009, 75(7):1892-1900. http://cn.bing.com/academic/profile?id=18ed8e60a31bdb02b8bccc6af0d680cd&encoded=0&v=paper_preview&mkt=zh-cn [6] ZHAO X, LIN C, WANG J, et al. Advances in rapid detection methods for foodborne pathogens[J]. Journal of Microbiology and Biotechnology. 2014, 24(3): 297-312. doi: 10.4014/jmb.1310.10013 [7] 何琳. 环介导等温扩增技术快速检测水产动物病原的研究[D]. 杭州: 浙江大学, 2012. http://cdmd.cnki.com.cn/Article/CDMD-10335-1013186907.htm [8] 江汉湖, 董明盛.食品微生物学[M].北京:中国农业出版社, 2010. [9] KRYSINSKI E P, HEIMSCH R C. Use of enzyme-labeled antibodies to detect Salmonella in foods[J]. Applied & Environmental Microbiology, 1977, 33(4):947-954. http://cn.bing.com/academic/profile?id=0bfb6e90e9c27e2aad11a95223ddb3bf&encoded=0&v=paper_preview&mkt=zh-cn [10] LIU F, LI Y, SONG C, et al. Highly sensitive microplate chemiluminescence enzyme immunoassay for the determination of staphylococcal enterotoxin B based on a pair of specific monoclonal antibodies and its application to various matrices[J]. Analytical Chemistry, 2010, 82(18):7758-7765. doi: 10.1021/ac101666y [11] BRANDÃO D, LIÉBANA S, PIVIDORI M I. Multiplexed detection of foodborne pathogens based on magnetic particles[J]. New Biotechnology, 2015, 32(5):511-520. doi: 10.1016/j.nbt.2015.03.011 [12] PARK S H, AYDIN M, KHATIWARA A, et al. Current and emerging technologies for rapid detection and characterization of Salmonella in poultry and poultry products[J]. Food Microbiology, 2014, 38(4):250-262. http://cn.bing.com/academic/profile?id=3d4735db76d3edbba2fb3643d8b6b740&encoded=0&v=paper_preview&mkt=zh-cn [13] MA K, DENG Y, BAI Y, et al. Rapid and simultaneous detection of Salmonella, Shigella, and Staphylococcus aureus in fresh pork using a multiplex real-time PCR assay based on immunomagnetic separation[J]. Food Control, 2014, 42:87-93. doi: 10.1016/j.foodcont.2014.01.042 [14] GB/T 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]. [15] 覃昱. 应用免疫磁珠分离及LAMP技术快速检测配方奶粉中克罗诺杆菌[D]. 广州: 南方医科大学, 2014. http://cdmd.cnki.com.cn/Article/CDMD-90023-1014338464.htm [16] 毛燕, 黄小林, 许恒毅, 等.免疫磁分离技术在食源性单增李斯特菌检测中应用的研究进展[J].食品工业科技, 2015, 36(8):351-355. http://mall.cnki.net/magazine/Article/ZGWY201103004.htm [17] 刘细霞, 涂俊铭.免疫磁珠分离技术及其在食源性致病菌检测中应用的进展[J].中国抗生素杂志, 2014, 39(12):956-960. doi: 10.3969/j.issn.1001-8689.2014.12.017 [18] 封莉, 黄继超, 刘欣, 等.食源性致病菌快速检测技术研究进展[J].食品科学, 2012, 33(21):332-339. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=spaqzljcjs201509023 [19] 王文彬. 乳及乳制品中主要食源性致病菌的免疫快速检测方法研究[D]. 无锡: 江南大学, 2017. http://cdmd.cnki.com.cn/Article/CDMD-10295-1017018540.htm [20] 牛凯莉. 基于胶体金免疫层析法的食源性致病菌检测技术的研究[D]. 上海: 上海师范大学, 2013. http://cdmd.cnki.com.cn/Article/CDMD-10270-1013297969.htm [21] 潘秀华. 单核细胞增生李斯特菌胶体金免疫层析检测试纸条的制备[D]. 武汉: 华中农业大学, 2013. http://cdmd.cnki.com.cn/Article/CDMD-10504-1013336519.htm [22] 章小雨, 戴晓爱.免疫胶体金技术在医学检验领域的应用与进展[J].实验与检验医学, 2014, 32(3):279-281. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=jiangxyxjy201403015 [23] FENG P. Food microbiology, fundamentals and frontiers, 3rd edn[M]. ASM Press:Washington D C, 2007, 911-934. [24] 谢雪钦.食品微生物快速检测方法优劣比较[J].质量技术监督研究, 2013(3):2-7. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zljsjdyj201303001 [25] KUMAR B K, RAGHUNATH P, DEVEGOWDA D, et al. Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood[J]. International Journal of Food Microbiology, 2011, 145(1):244-249. doi: 10.1016/j.ijfoodmicro.2010.12.030 [26] 伍燕华, 牛瑞江, 赖卫华, 等.双抗夹心酶联免疫吸附法检测沙门氏菌[J].食品工业科技, 2014, 35(10):62-65. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=spgykj201410015 [27] INSALATA N K, SCHULTE S J, HERMAN J H. Immunofluorescence technique for detection of salmoiiellae in various foods[J]. Applied Microbiology, 1967, 15(5):1145-1149. http://cn.bing.com/academic/profile?id=35e19ff9a466402186d9e39ce61d5710&encoded=0&v=paper_preview&mkt=zh-cn [28] RAJKHOWA S, HUSSAIN I, RAJKHOWA C. Detection of heat-stable and heat-labile enterotoxin genes of Escherichia coli in diarrhoeic faecal samples of mithun(Bos frontalis) calves by polymerase chain reaction[J]. Journal of Applied Microbiology, 2009, 106(2):455-458. doi: 10.1111/jam.2009.106.issue-2 [29] VERSTRAETE K, ROBYN J, DEL-FAVERO J, et al. Evaluation of a multiplex-PCR detection in combination with an isolation method for STEC O26, O103, O111, O145 and sorbitol fermenting O157 in food[J]. Food Microbiology, 2012, 29(1):49-55. doi: 10.1016/j.fm.2011.08.017 [30] GUAN Z P, JIANG Y, GAO F, et al. Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR[J]. European Food Research and Technology, 2013, 237(4):627-637. doi: 10.1007/s00217-013-2039-1 [31] 商颖, 许文涛, 元延芳, 等.通用引物多重PCR技术检测3种病原微生物[J].食品科学, 2011, 32(10):103-106. http://www.cnki.com.cn/Article/CJFDTotal-WXHX201404012.htm [32] YUAN Y, XU W, ZHAI Z, et al. Universal primer-multiplex PCR approach for simultaneous detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in food samples[J]. Journal of Food Science, 2009, 74(8):446-452. doi: 10.1111/jfds.2009.74.issue-8 [33] WANG H, ZHANG C, XING D. Simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes using oscillatory-flow multiplex PCR[J]. MicrochimicaActa, 2011, 173:503-512. [34] RODA A, MIRASOLI M, RODA B, et al. Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection[J].Microchimica Acta, 2012, 178:7-28. doi: 10.1007/s00604-012-0824-3 [35] DELBEKE S, CEUPPENS S, HOLVOET K, et al. Multiplex real-time PCR and culture methods for detection of Shiga toxin-producing Escherichia coli and SalmonellaThompson in strawberries, a lettuce mix and basil[J]. International Journal of Food Microbiology, 2015, 193:1-7. doi: 10.1016/j.ijfoodmicro.2014.10.009 [36] TORKY H A, AHMED H A, OMRAN E S. Duplex real time PCR for simultaneous detection of Salmonella species and Listeria monocytogenes in frozen meat[J]. Alexandria Journal for Veterinary Sciences, 2015, 46:100-109. doi: 10.5455/ajvs. [37] FREEMAN W M, WALKER S J, VRANA K E. Quantitative RT-PCR: pitfalls and potential[J]. Biotechniques, 1999, 26(1):112-125. http://cn.bing.com/academic/profile?id=18cf2afd14b9389296b6a5f357e819e9&encoded=0&v=paper_preview&mkt=zh-cn [38] 蒋原.食源性病原微生物检测指南[M].北京:中国标准出版社, 2010. [39] MARTIN B, RAURICH S, GARRIGA M, et al. Effect of amplicon length in propidiummonoazidequantitative PCR for the enumeration of viable cells of Salmonella in cooked ham[J]. Food Analytical Methods, 2013, 6(2):683-690. doi: 10.1007/s12161-012-9460-0 [40] WANG L, SHI L, ALAM M J, et al. Specific and rapid detection of foodborne Salmonella by loop-mediated isothermal amplification method[J]. Food Research International, 2008, 41(1):69-74. doi: 10.1016/j.foodres.2007.09.005 [41] MORI Y, NAGAMINE K, TOMITA N, et al. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J]. Biochemical & Biophysical Research Communications, 2001, 289(1):150-154. http://cn.bing.com/academic/profile?id=15d852a14eb835a4624717c66c804082&encoded=0&v=paper_preview&mkt=zh-cn [42] MARUYAMA F, KENZAKA T, YAMAGUCHI N, et al. Detection of bacteria carrying the stx2gene by in situ loop-mediated isothermal amplification[J]. Applied & Environmental Microbiology, 2003, 69(8): 5023-5028. http://cn.bing.com/academic/profile?id=910a8f1124c3349713949fd9b8ebe419&encoded=0&v=paper_preview&mkt=zh-cn [43] MORI Y, NOTOMI T.Loop-mediated isothermal amplification(LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases[J]. Journal of Infection and Chemotherapy, 2009, 15(2):62-69. doi: 10.1007/s10156-009-0669-9 [44] NAGAMINE K, WATANABE K, OHTSUKA K, et al. Loop-mediated isothermal amplification reaction using a nondenaturedtemplate[J]. Clinical Chemistry, 2001, 47(9):1742-1743. http://www.freepatentsonline.com/article/Clinical-Chemistry/209697539.html [45] LEONE G, VAN SCHIJNDEL H, VAN GEMEN B, et al. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA[J]. Nucleic Acids Research, 1998, 26(9):2150-2155. doi: 10.1093/nar/26.9.2150 [46] 高闪电, 常惠芸, 丛国正, 等. NASBA(依赖核酸序列的扩增)技术及其在病毒检测中的应用[J].中国生物工程杂志, 2009, 29(1):80-85. http://kns.cnki.net/KCMS/detail/detail.aspx?filename=swgj200901015&dbname=CJFD&dbcode=CJFQ [47] 雷质文, 姜英辉, 王妍婷, 等.沙门氏菌的依赖于核酸序列恒温扩增检测方法的建立[J].食品安全质量检测学报, 2011, 2(5):22-26. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=spaqzljcjs201105004 [48] 倪鑫, 王志聪, 雷质文, 等.依赖于核酸序列恒温扩增技术快速检测副溶血性弧菌方法的建立[J].中国预防兽医学报, 2011, 33(11):882-886. doi: 10.3969/j.issn.1008-0589.2011.11.13 [49] LAW J W, AB MUTALIB N S, CHAN K G, et al. An insight into the isolation, enumeration, and molecular detection ofListeria monocytogenes in food[J]. Frontiers in Microbiology, 2015, 6:1-16. http://jjmicrobiol.com/en/articles.rss [50] SHOMAKER T S, WARD K. Microarray technology in biomedical research[J]. Hawaii Medical Journal, 2006, 65(9):253-256. http://cn.bing.com/academic/profile?id=6d909e050f756b64af01f007161e53ae&encoded=0&v=paper_preview&mkt=zh-cn [51] KUPRADIT C, RODTONG S, KETUDATCAIRNS M. Development of a DNA macroarray for simultaneous detection of multiple foodborne pathogenic bacteria in fresh chicken meat[J]. World Journal of Microbiology & Biotechnology, 2013, 29(12):2281-2291. doi: 10.1007/s11274-013-1394-1 [52] SUO B, HE Y, PAOLI G, et al. Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens[J]. Molecular & Cellular Probes, 2010, 24(2):77-86. http://cn.bing.com/academic/profile?id=4bd7f18d5bb3b24fbd5cb4669fd6f6c7&encoded=0&v=paper_preview&mkt=zh-cn [53] SUN H, MO Q H, LIN J C, et al. Rapid simultaneous screening of seven clinically important enteric pathogens using a magnetic bead based DNA microarray[J]. World Journal of Microbiology & Biotechnology, 2011, 27(1):163-169. http://cn.bing.com/academic/profile?id=1aedb78e7825a79b75b8f461c4ecc21b&encoded=0&v=paper_preview&mkt=zh-cn [54] 杨春光, 王宏伟, 彭心婷, 等.食品病原微生物快速检测技术研究进展[J].食品安全质量检测学报, 2015, 29(1):41-47. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=spaqzljcjs201501008 [55] 宋东晓. 多重PCR检测牛肉中沙门氏菌、单增李斯特菌和大肠杆菌O157: H7的研究[D]. 泰安: 山东农业大学, 2014. http://cdmd.cnki.com.cn/Article/CDMD-10434-1014347142.htm [56] Ranjbar R, Erfanmanesh M, Afshar D, et al. Visual detection of enterohemorrhagicEscherichia coli O157:H7 using loop-mediated isothermal amplification[J]. Electronic Physician, 2016, 8(6):2576-2585. doi: 10.19082/2576 [57] 孔超, 董娜, 高伟.应用环介导等温扩增法快速检测食品中沙门氏菌的研究[J].中国病原生物学杂志, 2015, 10(9):816-818. http://www.cnki.com.cn/Article/CJFDTOTAL-ZISC201509012.htm [58] HUANG A, QIU Z, JIN M, et al. High-throughput detection of food-borne pathogenic bacteria using oligonucleotide microarray with quantum dots as fluorescent labels[J]. International Journal of Food Microbiology, 2014, 185:27-32. doi: 10.1016/j.ijfoodmicro.2014.05.012 [59] VAISOCHEROVÁ-LÍSALOVÁ H, VÍŠOVÁ I, ERMINI M L, et al. Low-fouling surface plasmon resonance biosensor for multi-step detection of foodborne bacterial pathogens in complex food samples[J]. Biosensors & Bioelectronics, 2016, 80(3):84-90. http://cn.bing.com/academic/profile?id=dd72fbb45aaf7e49ef6cb4a06c06665b&encoded=0&v=paper_preview&mkt=zh-cn [60] FARKA Z, JUŘÍK T, PASTUCHA M, et al. Enzymatic precipitation enhanced surface plasmonresonance immunosensor for the detection of Salmonella in powdered milk[J]. Analytical Chemistry, 2016, 88(23):11830-11836. doi: 10.1021/acs.analchem.6b03511 [61] TAHERI R A, REZAYAN A H, RAHIMI F, et al. Development of an immunosensor using oriented immobilized anti-OmpW for sensitive detection of Vibrio cholerae by surface plasmon resonance[J]. Biosensors & Bioelectronics, 2016, 86:484-488. http://cn.bing.com/academic/profile?id=7393a92fa2f3f2e4dd11ce6ef7234665&encoded=0&v=paper_preview&mkt=zh-cn [62] HU C, DOU W, ZHAO G. Enzyme immunosensor based on gold nanoparticles electroposition and Streptavidin-biotin system for detection of S. pullorum & S. gallinarum[J]. Electrochimica Acta, 2014, 117:239-245. doi: 10.1016/j.electacta.2013.11.132 [63] VISWANATHAN S, RANI C, HO J A. Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode[J]. Talanta, 2012, 94(94):315-319. http://cn.bing.com/academic/profile?id=42eaeea8b91d6dca3ed0889c45af77ec&encoded=0&v=paper_preview&mkt=zh-cn [64] ZHANG W, LUO C, ZHONG L, et al. Sensitive detection of entero-pathogenic E.coli using a bfpA gene-based electrochemical sensor[J]. Microchimica Acta, 2013, 180(13-14):1233-1240. doi: 10.1007/s00604-013-1061-0 [65] 黄欣迪, 涂晓波, 亓双, 等.食源性致病菌的检测方法及其发展趋势[J].食品安全质量检测学报, 2016, 7(12):4794-4800. http://www.cqvip.com/QK/90648X/201419/663089706.html [66] LÓPEZ-CAMPOS G, MARTÍNEZ-SUÁREZ J V, AGUADO-URDA M, et al. Microarray detection and characterization of bacterial foodborne pathogens[M]. Boston:Springer, 2012, 13-32. [67] 唐倩倩, 叶尊忠, 王剑平, 等. ATP生物发光法在微生物检验中的应用[J].食品科学, 2008, 29(6):460-465. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=spkx200806103 [68] JASSON V, JACXSENS L, LUNING P, et al. Alternative microbial methods: An overview and selection criteria[J]. Food Microbiology, 2010, 27(6):710-730. doi: 10.1016/j.fm.2010.04.008 [69] HUNTER D M, LESKINEN S D, MAGAÑA S, et al. Dead-end ultrafiltration concentration and IMS/ATP-bioluminescence detection of Escherichia coliO157:H7 in recreational water and produce wash[J]. Journal of Microbiological Methods, 2011, 87(3):338-342. doi: 10.1016/j.mimet.2011.09.010 -
