检测大肠杆菌(Escherichia coli)O157:H7免疫磁珠的制备、保存与应用
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摘要
【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增(Loop-mediatedisothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(VitaminC)和防腐剂ProClin300,利用正交试验L18(3~7)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为:牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,Pro Clin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。
[Background] Escherichia coli O157:H7 is the main serotype of Enterohemorrhagic Escherichia coli causing outburst of foodborne disease. [Objective] Our goal is to prepare immunomagnetic beads(IMBS) which are high efficient, stable and broad-spectrum, and also to improve the detection rate of target bacteria from food samples by combining immunomagnetic separation and molecular detection technology like loop-mediated isothermal amplification(LAMP), PCR, etc. [Methods] We used MIX&GO as activating agent of carboxyl magnetic beads, and successfully prepared immunomagnetic beads coupled with commercial polyclonal antibody. Subsequently, its universality and specificity were evaluated. The preservation solution of immumomagnetic beads consisting of bovine serum albumin, casein, trehalose, polyvinyl pyrrolidone(PVP), ascorbic acid and ProClin300, has been optimized by orthogonal experimental design L18(3~7). Six detecting methods including IMBS-LAMP, IMBS-PCR, IMBS-biochemistry, LAMP, PCR and biochemistry respectively without IMBS were adopted to detect E. coli O157:H7 in 20 ground raw pork meat samples. [Results] The capture efficiency of prepared immunomagnetic beads is 81.5%±1.3% when applied in the sample matrix of PBS solution, but decreasing in complex food matrix. The optimal formula for the preservation solution of the immunomagnetic beads is bovine serum albumin 15.0 g/L, casein 10.0 g/L, trehalose 10.0 g/L, PVP 2.0 g/L, ascorbic acid 5.0 g/L, and ProClin300 2.5 g/L. The results showed that the most sensitive method was the combination of IMBS and LAMP; 9 positive samples were respectively detected by self-prepared IMBS-LAMP and commercial IMBS-LAMP. But there was an inconsistency in two groups of positive samples because of different antibody sources of self-prepared and commercial IMBS. [Conclusion] Compared to commercial immunomagnetic beads, the prepared beads have good specificity and broader spectrum of E. coli O157 strains. Our study also showed that the IMBS-LAMP scheme could effectively enhanced detection rate of target bacteria. The IMBS-LAMP technique could be considered a high sensitive detection method of application prospect.
[Background] Escherichia coli O157:H7 is the main serotype of Enterohemorrhagic Escherichia coli causing outburst of foodborne disease. [Objective] Our goal is to prepare immunomagnetic beads(IMBS) which are high efficient, stable and broad-spectrum, and also to improve the detection rate of target bacteria from food samples by combining immunomagnetic separation and molecular detection technology like loop-mediated isothermal amplification(LAMP), PCR, etc. [Methods] We used MIX&GO as activating agent of carboxyl magnetic beads, and successfully prepared immunomagnetic beads coupled with commercial polyclonal antibody. Subsequently, its universality and specificity were evaluated. The preservation solution of immumomagnetic beads consisting of bovine serum albumin, casein, trehalose, polyvinyl pyrrolidone(PVP), ascorbic acid and ProClin300, has been optimized by orthogonal experimental design L18(3~7). Six detecting methods including IMBS-LAMP, IMBS-PCR, IMBS-biochemistry, LAMP, PCR and biochemistry respectively without IMBS were adopted to detect E. coli O157:H7 in 20 ground raw pork meat samples. [Results] The capture efficiency of prepared immunomagnetic beads is 81.5%±1.3% when applied in the sample matrix of PBS solution, but decreasing in complex food matrix. The optimal formula for the preservation solution of the immunomagnetic beads is bovine serum albumin 15.0 g/L, casein 10.0 g/L, trehalose 10.0 g/L, PVP 2.0 g/L, ascorbic acid 5.0 g/L, and ProClin300 2.5 g/L. The results showed that the most sensitive method was the combination of IMBS and LAMP; 9 positive samples were respectively detected by self-prepared IMBS-LAMP and commercial IMBS-LAMP. But there was an inconsistency in two groups of positive samples because of different antibody sources of self-prepared and commercial IMBS. [Conclusion] Compared to commercial immunomagnetic beads, the prepared beads have good specificity and broader spectrum of E. coli O157 strains. Our study also showed that the IMBS-LAMP scheme could effectively enhanced detection rate of target bacteria. The IMBS-LAMP technique could be considered a high sensitive detection method of application prospect.
引文
[1] Zhu PX, Shelton DR, Li SH, et al. Detection of E. coli O157:H7by immunomagnetic separation coupled with fluorescence immunoassay[J]. Biosensors and Bioelectronics, 2011, 30:337-341
[2] Phillips CA. The epidemiology, detection and control of Escherichia coli O157[J]. Journal of the Science of Food and Agriculture, 1999, 79(11):1367-1381
[3] Perna NT, Plunkett III G, Burland V, et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7[J]. Nature, 2001,409(6819):529-533
[4] Zhang J, Xia SL, Ma H. The epidemiological investigation on infection cases of Shiga’s toxin-producing E. coli O157:H7 in eastern Henan, China[J]. Strait Journal of Preventive Medicine,2003, 9(5):26-28(in Chinese)张锦,夏胜利,马宏.河南豫东地区产志贺样毒素E. coli O157:H7感染病例的流行病学调查研究[J].海峡预防医学杂志, 2003, 9(5):26-28
[5] Zhou Y, Wan Q, Cai ZH, et al. Evaluation of loop-mediated isothermal amplification based kits for rapid detection of Escherichia coli O157:H7[J]. Microbiology China, 2017, 44(8):1996-2004(in Chinese)周杨,万强,蔡芷荷,等.基于环介导恒温扩增技术的大肠杆菌O157:H7快速检测试剂盒的评价[J].微生物学通报,2017, 44(8):1996-2004
[6] Schrader C, Schielke A, Ellerbroek L, et al. PCR inhibitors-occurrence, properties and removal[J]. Journal of Applied Microbiology, 2012, 113:1014-1026
[7] Pyle BH, Broadaway SC, McFeters GA. Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry[J].Applied and Environmental Microbiology, 1999, 65(5):1966-1972
[8] Ochoa ML, Harrington PB. Immunomagnetic isolation of enterohemorrhagic Escherichia coli O157:H7 from ground beef and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searches[J].Analytical Chemistry, 2005, 77(16):5258-5267
[9] Omisakin F, MacRae M, Ogden ID, et al. Concentration and prevalence of Escherichia coli O157 in cattle feces at slaughter[J]. Applied and Environmental Microbiology, 2003,69(5):2444-2447
[10] Sánchez-Tirado E, Martínez-García G, González-Cortés A, et al.Electrochemical immunosensor for sensitive determination of transforming growth factor(TGF)-β1 in urine[J]. Biosensors and Bioelectronics, 2017, 88:9-14
[11] Ojeda I, Barrejón M, Arellano LM, et al. Grafted-double walled carbon nanotubes as electrochemical platforms for immobilization of antibodies using a metallic-complex chelating polymer:application to the determination of adiponectin cytokine in serum[J]. Biosensors and Bioelectronics, 2015, 74:24-29
[12] Jadeja R, Janes ME, Simonson JG. Immunomagnetic separation of Vibrio vulnificus with antiflagellar monoclonal antibody[J].Journal of Food Protection, 2010, 73(7):1288-1293
[13] Shields MJ, Hahn KR, Janzen TW, et al. Immunomagnetic capture of Bacillus anthracis spores from food[J]. Journal of Food Protection, 2012, 75(7):1243-1248
[14] Shim WB, Choi JG, Kim JY, et al. Enhanced rapidity for qualitative detection of Listeria monocytogenes using an enzyme-linked immunosorbent assay and immunochromatography strip test combined with immunomagnetic bead separation[J].Journal of Food Protection, 2008, 71(4):781-789
[15] National Health and Family Planning Commission of PRC,China Food and Drug Administration. National food safety standard Food microbiological examination:Escherichia coli O157:H7/NM[S]. Beijing:China Standards Press, 2017(in Chinese)国家卫生和计划生育委员会,国家食品药品监督管理总局.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].北京:中国标准出版社, 2017
[16] Xiong QR, Cui X, Saini JK, et al. Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7[J]. Food Control, 2014, 37:41-45
[17] Zhang Y, Yan CH, Yang H, et al. Rapid and selective detection of E. coli O157:H7 combining phagomagnetic separation with enzymatic colorimetry[J]. Food Chemistry, 2017, 234:332-338
[18] Lim MC, Lee GH, Huynh DTN, et al. Biological preparation of highly effective immunomagnetic beads for the separation,concentration, and detection of pathogenic bacteria in milk[J].Colloids and Surfaces B:Biointerfaces, 2016, 145:854-861
[19] Luciani M, di Febo T, Zilli K, et al. Rapid detection and isolation of Escherichia coli O104:H4 from milk using monoclonal antibody-coated magnetic beads[J]. Frontiers in Microbiology, 2016, 7:942
[20] Fu Z, Rogelj S, Kieft TL. Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR[J].International Journal of Food Microbiology, 2005, 99(1):47-57
[21] Varshney M, Yang LJ, Su XL, et al. Magnetic nanoparticle-antibody conjugates for the separation of Escherichia coli O157:H7 in ground beef[J]. Journal of Food Protection, 2005, 68(9):1804-1811
[22] Hosotani Y, Noviyanti F, Koseki S, et al. Growth delay analysis of high-salt injured Escherichia coli O157:H7 in fermented soybean paste by real-time PCR and comparison of this method with other estimation methods[J]. LWT, 2018, 96:426-431
[23] Kawasaki S, Hosotani Y, Noviyanti F, et al. Growth delay analysis of heat-injured Salmonella Enteritidis in ground beef by real-time PCR[J]. LWT, 2018, 90:499-504
[24] Qin Y, Puthiyakunnon S, Zhang YD, et al. Rapid and specific detection of Escherichia coli O157:H7 in ground beef using immunomagnetic separation combined with loop-mediated isothermal amplification[J]. Polish Journal of Food and Nutrition Sciences, 2018, 68(2):115-123
[25] Karch H, Janetzki-Mittmann C, Aleksic CS, et al. Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture[J]. Journal of Clinical Microbiology, 1996, 34(3):516-519
[26] Hepburn NF, MacRae M, Johnston M, et al. Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation[J]. Letters in Applied Microbiology, 2002, 34(5):365-369
[2] Phillips CA. The epidemiology, detection and control of Escherichia coli O157[J]. Journal of the Science of Food and Agriculture, 1999, 79(11):1367-1381
[3] Perna NT, Plunkett III G, Burland V, et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7[J]. Nature, 2001,409(6819):529-533
[4] Zhang J, Xia SL, Ma H. The epidemiological investigation on infection cases of Shiga’s toxin-producing E. coli O157:H7 in eastern Henan, China[J]. Strait Journal of Preventive Medicine,2003, 9(5):26-28(in Chinese)张锦,夏胜利,马宏.河南豫东地区产志贺样毒素E. coli O157:H7感染病例的流行病学调查研究[J].海峡预防医学杂志, 2003, 9(5):26-28
[5] Zhou Y, Wan Q, Cai ZH, et al. Evaluation of loop-mediated isothermal amplification based kits for rapid detection of Escherichia coli O157:H7[J]. Microbiology China, 2017, 44(8):1996-2004(in Chinese)周杨,万强,蔡芷荷,等.基于环介导恒温扩增技术的大肠杆菌O157:H7快速检测试剂盒的评价[J].微生物学通报,2017, 44(8):1996-2004
[6] Schrader C, Schielke A, Ellerbroek L, et al. PCR inhibitors-occurrence, properties and removal[J]. Journal of Applied Microbiology, 2012, 113:1014-1026
[7] Pyle BH, Broadaway SC, McFeters GA. Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry[J].Applied and Environmental Microbiology, 1999, 65(5):1966-1972
[8] Ochoa ML, Harrington PB. Immunomagnetic isolation of enterohemorrhagic Escherichia coli O157:H7 from ground beef and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searches[J].Analytical Chemistry, 2005, 77(16):5258-5267
[9] Omisakin F, MacRae M, Ogden ID, et al. Concentration and prevalence of Escherichia coli O157 in cattle feces at slaughter[J]. Applied and Environmental Microbiology, 2003,69(5):2444-2447
[10] Sánchez-Tirado E, Martínez-García G, González-Cortés A, et al.Electrochemical immunosensor for sensitive determination of transforming growth factor(TGF)-β1 in urine[J]. Biosensors and Bioelectronics, 2017, 88:9-14
[11] Ojeda I, Barrejón M, Arellano LM, et al. Grafted-double walled carbon nanotubes as electrochemical platforms for immobilization of antibodies using a metallic-complex chelating polymer:application to the determination of adiponectin cytokine in serum[J]. Biosensors and Bioelectronics, 2015, 74:24-29
[12] Jadeja R, Janes ME, Simonson JG. Immunomagnetic separation of Vibrio vulnificus with antiflagellar monoclonal antibody[J].Journal of Food Protection, 2010, 73(7):1288-1293
[13] Shields MJ, Hahn KR, Janzen TW, et al. Immunomagnetic capture of Bacillus anthracis spores from food[J]. Journal of Food Protection, 2012, 75(7):1243-1248
[14] Shim WB, Choi JG, Kim JY, et al. Enhanced rapidity for qualitative detection of Listeria monocytogenes using an enzyme-linked immunosorbent assay and immunochromatography strip test combined with immunomagnetic bead separation[J].Journal of Food Protection, 2008, 71(4):781-789
[15] National Health and Family Planning Commission of PRC,China Food and Drug Administration. National food safety standard Food microbiological examination:Escherichia coli O157:H7/NM[S]. Beijing:China Standards Press, 2017(in Chinese)国家卫生和计划生育委员会,国家食品药品监督管理总局.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].北京:中国标准出版社, 2017
[16] Xiong QR, Cui X, Saini JK, et al. Development of an immunomagnetic separation method for efficient enrichment of Escherichia coli O157:H7[J]. Food Control, 2014, 37:41-45
[17] Zhang Y, Yan CH, Yang H, et al. Rapid and selective detection of E. coli O157:H7 combining phagomagnetic separation with enzymatic colorimetry[J]. Food Chemistry, 2017, 234:332-338
[18] Lim MC, Lee GH, Huynh DTN, et al. Biological preparation of highly effective immunomagnetic beads for the separation,concentration, and detection of pathogenic bacteria in milk[J].Colloids and Surfaces B:Biointerfaces, 2016, 145:854-861
[19] Luciani M, di Febo T, Zilli K, et al. Rapid detection and isolation of Escherichia coli O104:H4 from milk using monoclonal antibody-coated magnetic beads[J]. Frontiers in Microbiology, 2016, 7:942
[20] Fu Z, Rogelj S, Kieft TL. Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR[J].International Journal of Food Microbiology, 2005, 99(1):47-57
[21] Varshney M, Yang LJ, Su XL, et al. Magnetic nanoparticle-antibody conjugates for the separation of Escherichia coli O157:H7 in ground beef[J]. Journal of Food Protection, 2005, 68(9):1804-1811
[22] Hosotani Y, Noviyanti F, Koseki S, et al. Growth delay analysis of high-salt injured Escherichia coli O157:H7 in fermented soybean paste by real-time PCR and comparison of this method with other estimation methods[J]. LWT, 2018, 96:426-431
[23] Kawasaki S, Hosotani Y, Noviyanti F, et al. Growth delay analysis of heat-injured Salmonella Enteritidis in ground beef by real-time PCR[J]. LWT, 2018, 90:499-504
[24] Qin Y, Puthiyakunnon S, Zhang YD, et al. Rapid and specific detection of Escherichia coli O157:H7 in ground beef using immunomagnetic separation combined with loop-mediated isothermal amplification[J]. Polish Journal of Food and Nutrition Sciences, 2018, 68(2):115-123
[25] Karch H, Janetzki-Mittmann C, Aleksic CS, et al. Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture[J]. Journal of Clinical Microbiology, 1996, 34(3):516-519
[26] Hepburn NF, MacRae M, Johnston M, et al. Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation[J]. Letters in Applied Microbiology, 2002, 34(5):365-369
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