6 9 Vol. 6 No. 9 2015 9 Journal of Food Safety and Quality Sep., 2015 * 章钢刚, 赖卫华 (, 330047) 摘要 :,,,,,,, 关键词 : ; ; Research progress of immunological detection methods of foodborne pathogen ZHANG Gang-Gang, LAI Wei-Hua * (National Key Laboratory of Food Science and Technology in Nanchang University, Nanchang 330047, China) ABSTRACT: Foodborne pathogen widely exists and has drawn more and more attention as it can cause human disease even to death. Traditional detection method of foodborne pathogens is very time-consuming and cannot meet the need of rapid detection. Immunological detection methods are convenient and rapid, including enzyme-linked immunosorbent assay, immunochromatography, immunomagnetic beads separation techniques, chemiluminescence immunoassay and latex agglutination assays. In this paper, the principle, application, advantages, and disadvantages of these methods were reviewed. The future development trend of the immunological detection method was prospected. KEY WORDS: foodborne pathogens; immunological method; rapid detection 1 引言,,,, 8~18 h, 18~24 h, 18~48 h, 36~48 h,,,, 基金项目 : (SKLF-ZZB-201307) (2012-CYH-DW-SP-001) Fund: Supported by the Free Explore Issue of State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-ZZB-201307), Science and Technology project of Nanchang (2012-CYH-DW-SP-001) * 通讯作者 :,,, E-mail: talktolaiwh@163.com *Corresponding author: LAI Wei-Hua, Ph.D, Professor, National Key Laboratory of Food Science and Technology in Nanchang University, No.235. Nanjing East Road, Nanchang 330047, China. E-mail: talktolaiwh@163.com
9, : 3415,,, 2 免疫学检验方法进展 2.1 食源性致病菌 2.1.1 沙门氏菌 (Salmonella), 2600, A B C D E [1], [2],,,, CDC 16, 145 [3] 2.1.2 致病性大肠杆菌,,,, 4 : (Enteropathogenic E. coli, EPEC) 6~54% [4], EPEC [5] ; (Enterotoxigenic E. coli, ETEC) [6] ; (Enteroinvasive E.coli, EIEC) [7] ; (Enterohemorrhagic E.coli, EHEC) 2011, EHEC O157, 0~4 [8] 2.1.3 志贺氏菌 (Shigella), (Shigella dysenteriae) (Shigella flexneri) (Shigella sonnei) (Shigella boydii) 4 [9],,,, [10] 2.1.4 副溶血性弧菌 (Vibrio parahemolyticus) 和霍乱弧菌 (Vibrio cholerae), [11] 1000 MPN/g [12], [13] [14] 2.1.5 蜡样芽孢杆菌 (Bacillus cereus), [15] 2003, 4.0%, 4,, [16] 2.1.6 肉毒梭状芽孢杆菌 (Clostridium botulinum),,,,,, 0.1 μg 2.2.7 金黄色葡萄球菌 (Staphylococcus aureus), 10-15% NaCl, [17], 2000, 14000 [18] 2.1.8 阪崎肠杆菌 (Cronobacter),, [19] 2007,,, [20] 1,, 40%~80% [21] 2.1.9 空肠弯曲杆菌 (Campylobacter jejuni) - [22] 2.1.10 单核增生李斯特菌 (Listeria monocytogenes),, ( ), [23], [24,25] 2.2 酶联免疫吸附法 (enzyme-linked immunosorbent assay, ELISA), ELISA,,, ELISA, ( ), [26],,, C1359, ELISA A B C D E,, 1 10 4 cfu/ml,,
3416 6 [27], InternalinA(InlA), ELISA 1.7 10 5 cfu/ml Bang [28], 10 6 cfu/ml, O157, 2.3 免疫层析法 (immunochromatography, IC) 2.3.1 胶体金免疫层析技术 (gold immunochromatography assay, GICA), GICA, ( ),,, [29] ( CMCC 25922 NCTC 12900 CMCC 45401 ATCC 43864 ; PBS ) (, 10 5 cfu/ml; 10 6 cfu/ml) ( 6 ) [30] 16.8 μg/ml ph 8.0, PEG 20000,, T C O157:H7 ( ), O157:H7, 10 4 cfu/ml, Chalinan [31] O139, 10 4 cfu/ml,, 1 cfu/ml 2.3.2 碳纳米颗粒免疫层析 (carbon nanoparticles immunochromatography assay), GICA,,, ( NC ), [32,33] Martina [34], 16S rrna,, ( ), (NC) (T ), (C ) - - -16S rdna- -, 8 ng(pcr ) 2.3.3 荧光微球免疫层析技术 (fluorescent microsphere immunochromatographic technique),,, [35] O157:H7 NC T C O157:H7, 1.5 mg/ml, 0.75 μl/cm; O157:H7, 100 μg/mg 1.2 10 4 cfu/ml, 6.1 10 3 cfu/ml, ( ) Il-Hoon [36] O157:H7, 5 cfu/ml Xu [37] PEB1 T, C,, 10 6 cfu/ml 2.3.4 量子点免疫层析技术 (quantum dots immunochromatographic technique) (quantum dots, QD) ( 2~6 nm),, Ⅱ-VI Ⅲ-V,, [38] Chen [39] DNA, PCR DNA, QDs, 3 10 0 cfu/ml 3 10 1 cfu/g Bruno [40] O157, 365 nm,,
9, : 3417 6000 cfu/ml 600 cfu/ml 2.4 免疫磁珠分离技术 (immunomagnetic beads separation techniques, IMS), Xiong [41] O157:H7, ( O157:H7) 98%( 0.05 mg, 10~10 6 ), 2%( 23.6%), ( 94.4% 99.8%) [42], : 0.4 mg, 50 μg/mg( ), 10 2 ~10 6 cfu/ml, 65%~82% Lin [43] 1.35T 90 T/m, 30 nm 180 nm O157, 10 2 ~10 5 cfu/ml 90%, IMS, Cui [44] O157:H7, O157:H7, 7.6 10 3 cfu/ml 2.5 化学发光免疫分析法 (chemiluminescence immuno assay, CLIA) ( ),,,, 3 [45] [46], (HRP) IgG,, H 2 O 2, (Salmonella choleraesuis) (Salmonella typhimurium) (Salmonella enteritidis), 1 10 4 cfu/ml, 97.33% Shiro [47], ( Q1, )- -,, 10 3 CFU/mL Park [48],, O157, -,, H 2 O 2, 1.8 10 3 ~1.8 10 8 cfu/ml 2.6 乳胶凝集法 (latex agglutination assays, LTA) ( ),, Medina [49] LTA (O26, O45, O103, O111, O121, O145) IgG- ( ), (O26 O103 O145 ) Fusun [50] meca, meca fema femb femx 3 结论, ;,,,, 参考文献 [1] Hoerner R, Feldpausch J, Gray RL, et al. Reveal Salmonella 2.0 Test for detection of Salmonella spp. in foods and environmental samples [J]. J AOAC Int, 2011, 94(5): 1467 1480. [2],, 冮,. inva PCR [J]., 2013, 34(12): 68 70. Du XW, Li Y, Gang J, et al. Establishment of real-time fluorescent PCR detection method of inva gene in Salmonella [J]. Sci Technol Food Ind, 2013, 34(12): 68 70. [3] Centers for Disease Control and Prevention (CDC). Multistate outbreaks of Salmonella infections associated with raw tomatoes eaten in restaurants-united States, 2005-2006 [J]. MMWR, 2007, 56(35): 11 909. [4],,. [N]., 2014, 11(12): 162 165. Liu WD, Li MY, Xu CB. Research progress on epidemiology molecular mechanisms of the intestinal pathogenic Escherichia coli [N]. China Medical Herald, 2014, 11(12):162 165. [5] Cedric NB, Robert KS, Fernando RP, et al. Interaction of enteroaggregative Escherichia coli with salad leaves [J]. Env Microbiol Rep, 2009, 1(4): 234 239. [6] Fernando RP, Rezwanul W, Christina SF, et al. Serine protease
3418 6 autotransporters from Shigella flexneri and pathogenic Escherichia coli target a broad range of leukocyte glycoproteins [J]. Proc Natl Acad Sci USA, 2011, 108(31): 12881 12886. [7] Justin M, Brooke W, Yoonjie KC, et al. The heat-resistant agglutinin family includes a novel adhesin from enteroaggregative Escherichia coli Strain 60A [J]. J Bacteriol, 2011, 193(18): 4813 4820. [8] Koushik R, Rita K, Scott RB, et al. Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli [J]. J Bio Chem, 2011, 286(34): 29771 29779. [9],,. [J]., 2011, 44(9): 1939 1944. Kang JJ, Yang YR, Liang HD. Progress in Research of Pathogenesis of Shigellosis [J]. Sci Agric Sin, 2011, 44(9): 1939 1944. [10] Jin Q, Yuan Z, Xu J, et al. Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157 [J]. Nucleic Acids Res, 2002, 30(20): 4432 4441. [11] Angelo DP, Jessica LNo, John CB, et al. Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters [J]. Appl Environ Microbiol, 2003, 69: 1521 1526. [12] GB29921-2013 [S]. GB29921-2013 Limitation of pathogenic bacteria in food [S]. [13],,. [J]., 2010, 31(S1): 124 126. Zhang PP, Li J, Yu Y. Prevalence of Vibrio cholerae in aquatic products and related enviroment in Zhoushan, Zhejiang province [J]. Progress Vet Med, 2010, 31(S1): 124 126. [14],. [J]., 2011, 6(2): 113 116. Shen XT, Zhao XT. Advances in detection methods for Vibrio choleraein fection [J]. J Microbes Infect, 2011, 6(2): 113 116. [15],,,. [J]., 2010(5): 1 4. Zhang WW, Lu F, Zhang JL, et al. Research progress of Bacillus cereus in food [J]. China Brewing, 2010(5): 1 4. [16] Schoeni JL, Wong AC. Bacillus cereus food poisoning and its toxins [J]. J Food Protect, 2005, 68(3): 636 648. [17],,,. [J],, 2010, (4): 517 520. Chen J, Zang HP, Zhu XG, et al. Morphological observation and pilot study on growth traits of Staphylococcus aureus under High salt Condition [J]. J Yunnan Agric Univ, 2010, (4): 517 520. [18]. [J]., 2004, 14(4): 392 395. Li Y. Advancement in researches of Staphylococcus aureus and its enterotoxin [J]. Chin J Health Lab Technol, 2004, 14(4): 392 395. [19]. [J]., 2011, 1(17): 37 38. Xiong ZL. Research progress of detection techniques of Enterobacter sakazaki [J]. China Med Pharmacy, 2011, 1(17): 37 38 [20],. [J]., 2009, 20(4): 418 420. Liang Y, Wang HJ. Research prograssing of Enterobacter sakazakii [J]. Chin J Microecol, 2009, 20(4): 418 420. [21],,,. 2010-2013 [J]., 2014, 24(15): 2248 2251. Zhou SJ, Deng XL, Zhu HM, et al. Survey of Enterobacter sakazakii pollution in infant food in Guangdong province [J]. Chin J Health Lab Technol, 2014, 24(15): 2248 2251. [22] McCarthy N, Giesecke J. Incidence of Guillain-Barré syndrome following infection with Campylobacter jejuni [J]. Am J Epidemiol, 2001, 153(6): 610 614. [23]. LAMP [D]. :, 2013. Xu YM. Establishment of loop-mediated isothermal amplification(lamp) methods for detection of Listeria monocytogenes and Yersinia enterocolitica [D]. Changchun: Jiling University, 2013. [24],,.. [C]., 2005. Li Y, Wei JZ, Wang GJ. Listeria monocytogenes and food safety. Theory and practice of food safety[c]. Anhui Food Safety Forum, 2005. [25],,,. [J]., 2009, 36(15): 2841 2843. Liu Y, Wang YL, Tao Y, et al. Analysis for Listeria monocytogenes contamination statu in foods in Maanshan [J]. Mod Pre Med, 2009, 36(15): 2841 2843. [26],,,. [J]., 2014(10):62 65. Wu YH, Niu RJ, Lai WH, et al. Detection of Salmonella by double antibody sandwich ELISA [J]. Sci Technol Food Ind, 2014(10):62 65. [27],,,. ELISA [J]., 2010, 31(24):272 276. Duan X, Huang X, Huang LF, et al. Detection of Listeria monocytogenes in food by sandwich ELISA [J]. Food Sci, 2010, 31(24): 272 276. [28] Bang J, Shukla S, Kim YH, et al. Development of indirect competitive ELISA for the detection of Salmonella typhimurium [J]. Rom Biotech Lett, 2012, 17(2): 7194 7204. [29],,,. [J],, 2014, 35(22): 154 158. Xia SQ, Xu CL, Liu DF, et al. Preparation and optimization of colloidal gold strip for simultaneously detecting five typical Salmonella in foods [J]. Food Sci, 2014, 35(22): 154 158. [30],,,. O157:H7 [J]., 2010, 31(24): 355 359. Huang LF, Duan X, Chen Y, et al. Preparation of colloidal gold strip for the detection of Escherichia coli O157:H7[J]. Food Sci, 2010, 31(24): 355 359. [31] Chalinan P, Parin C, Siwaporn L, et al. Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafoodsamples [J]. Biosens Bioelectron, 2013, 42(15): 229 235. [32] Maria L, Jan C. Quantitative detection in the attomole range for immunochromatographic tests by means of a flatbed scanner [J], Anal Biochem, 2001, 293(2): 224 231. [33] Noguera P, Posthuma-Trumpie GA, Van Tui M, et al. Carbon
9, : 3419 nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin producing Escherichia coli [J]. Anal Bioanal Chem, 2011, 399(2): 831 838. [34] Martina B, Barbora J, Ladislav F, et al. Immunochromatographic strip test for detection of genus Cronobacter [J]. Biosens Bioelectron, 2011, 26(6): 2828 2834. [35],,,. O157:H7 [J]., 2013, 34(16): 353 357. Xie QY, Lai WH, Liu CM, et al. Development of fluorescent microsphere immunochromatographic strip for detection of Escherichia coli O157:H7 [J]. Food Sci, 2013, 34(16): 353 357. [36] Il-Hoon C, Lisa M, Joseph I. In-situ fluorescent immunomagnetic multiplex detection of foodborne pathogens in very low numbers [J]. Biosens Bioelectron, 2014, 57: 143 148. [37] Xu D, Wu XL, Li B, et al. Rapid detection of Campylobacter jejuni using fluorescent microspheres as label for immunochromatographic strip test [J]. Food Sci Biotechnol, 2013, 22(2): 585 591. [38] Li ZH, Wang Y, Wang J, et al. Rapid and sensitive detection of protein biomarker using a portable fluorescence biosensor based on quantum dots and a lateral flow test strip [J]. Anal Chem, 2010, 82(16): 7008 7014. [39] Chen XX, Gan M, Xu H, et al. Development of a rapid and sensitive quantum dot-based immunochromatographic strip by double labeling PCR products for detection of Staphylococcus aureus in food [J]. Food Control, 2014, 46: 225 232. [40] Bruno GJ. Application of DNA aptamers and quantum dots to lateral flow test strips for detection of foodborne pathogens with improved sensitivity versus colloidal gold [J]. Pathogens, 2014, 3(2), 341 355. [41] Xiong QR, Cui X, Jasdeep KS, et al. Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7 [J]. Food Control, 2014, 37: 41 45. [42],,,. [J]., 2013, 34(13):153 156. Shan S, Niu RJ, Lai WH, et al. Optimization and application of immunomagnetic beads for enriching Salmonella spp [J]. Sci Technol Food Ind, 2013, 34(13): 153 156. [43] Lin JH, Li M, Li YB, et al. A high gradient and strength bioseparator with nano-sized immunomagnetic particles for specific separation and efficient concentration of E. coli O157:H7 [J]. J Magn Magn Mater, 2015, 378(15): 206 213. [44] Cui X, Xiong QR, Xiong YH, et al. Establishing of a method combined immunomagnetic separation with colloidal gold lateral flow assay and its application in rapid detection of Escherichia coli O157:H7 [J]. Chin J Anal Chem, 2013, 41(12): 1812 1816. [45],,,. [J]., 2014, 5(3):840 845. Jing MJ, Wang J, Yang LH, et al. Latest progress of chemiluminescent immunoassay in food safety detection [J]. J Food Saf Qual, 2014, 5(3): 840 845. [46],,,. [J]., 2014, 39(8): 281 286. Fang F, Jing SQ, Li JF, et al. Establishment of chemiluminescent enzyme immunoassay for Salmonella [J]. Food Sci Technol, 2014, 39(8): 281 286. [47] Shiro Y, Atsushi A, Susumu K, et al. Chemiluminescent assay for detection of viable microorganisms [J]. Anal Biochem, 2004, 333(2): 303 308. [48] Park S, Kim H, Paek SH, et al. Enzyme-linked immuno-strip biosensor to detect Escherichia coli O157:H7 [J]. Ultramicroscopy, 2008, 108(10): 1348 1351. [49] Medina MB, Shelver WL, Fratamico PM, et al. Latex agglutination assays for detection of non-o157 shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 [J]. J Food Prot, 2012, 75(5): 819 826. [50] Fusun ZA, Gulgun BT, Onur K, et al. Evaluation of methicillin resistance by cefoxitin disk diffusion and PBP2a latex agglutination test in meca-positive Staphylococcus aureus, and comparison of meca with fema, femb, femx positivities [J]. Microbiol Res, 2009, 164(4): 400 403. 作者简介 ( 责任编辑 : 白洪健 ) 章钢刚, 硕士研究生, 主要研究方向为食品质量安全 E-mail: joezgg@sina.com 赖卫华, 博士, 教授, 主要研究方向为食品质量安全 E-mail: talktolaiwh@163.com