2 0 1 3 3 Journal of Chinese Institute of Food Science and Technology Vol. 13 No. 3 Mar. 2 0 1 3 1 1 1 2 1 1 2 1 1* ( 1 上海师范大学生命与环境科学学院上海 200234 2 上海市出入境检疫检疫局上海 200135) 研究了一种采用低场核磁共振技术 (NMR) 快速筛选检测沙门氏菌的方法以 Fe 3 O 4 为核心的纳米磁珠包裹上二氧化硅, 使其具有很好的分散性和生物相容性通过氨基硅烷修饰磁珠, 使磁珠能固定抗体在富集过程中, 免疫磁珠特异性结合在沙门氏菌表面, 由核磁共振仪测得其 T 2 值, 并以此判别采用同样方法对 5 株食源性致病菌大肠杆菌 O157 单核细胞增生李斯特菌 志贺氏菌 金黄色葡萄球菌 副溶血性弧菌进行分析, 可很好地区分这 5 种食源性致病菌与沙门氏菌, 表现出很好的特异性同时, 优化了试验参数, 结果显示确定磁珠质量浓度为 0.14 mg/ml 检出限为 10 3 cfu/ml 纳米磁珠 ; 免疫反应 ; 沙门氏菌 ; 核磁共振 ; 快速检测 1009-7848(2013)03-0171-05 1.1 ATCC 50013 (Salmonella [1] typhimurium ATCC 50013) ATCC 7644 (Listeria monocytogenes ATCC [2-3] PCR, 7644) O157 ATCC 25922 (Escherichia 4~7 d,,, coli O157 ATCC 25922) ATCC 51572, (Shigella flexneri ATCC 51572),PCR ATCC 12600(Staphylococcus aureus ATCC 12600), ATCC 17802 (Vibrio parahaemolyticus ATCC 17802), (, ), [4], A-F, (T 2 ) ;EDC HCL(1-(3- (LF-NMR) )-3- )NHS(N-, ), ; (NB) LB, [5], [6] [7] 1.2 1 : 2012-03-18 :,,1988, : 1.2.1 [8] Fe 3 O 4, Fe 3 O 4 [9] TEM(Hitachi-600) 0.1 ml EDC HCL NHS (ph 7.5 0.15 mol/ml)
172 2013 3,4, (GB/T 4789.4-4, 2010),25 g 225 ml 4 (BPW), (MagneticNanoparticles, O157 MNPs) 1.2.2, -NMR, (150 r/min) 37 18~24 h ( 3% NaCl), 2 (CFU/mL) (2.1 0.13 mol/l) Fe 3 O 4, 1.2.3 0.8 ml, Fe 3 O 4 0.8 ml, 37 [10], 30 min 4.5 ml 2% TEM ( ), 37 30 min, (Hitachi-600), NM120-Analyst, T 2 1, MNPs- 10 2 cfu/ml 10 3 cfu/ml 37 1, 70 LF-NMR nm, 1.2.4 2, A, 1~10 4 cfu/ml, B 0.08~0.16 mg/ml, A B 280 nm, OD, Origin7.0, 1.2.5, 2.2 3 2.3 ( ) ( + 2.3.1 )NMR T 2, O157
食源性致病菌低场磁共振快速筛选检验方法 173 - T 2 2 NMR ms 8 ms, 4 T 2 =16 ms,, 5 T 2,, T 2 T 2 =8 ms,, 8, 2.3.2 ml, 1 cfu/ml, 5, 2.4 -NMR 0.14 mg/ml,2% 5,,, 0.08 mg/ml 0.16 mg/ml,δt 2 1, O157, 0.14 mg/ml ΔT 2 ( 1 cfu/ ml,δt 2 =35 ms; 10 cfu/ml,δt 2 =40 ms),,0.14 mg/ ΔT 2,, 0~4 h,t 2 (ΔT 2 =21 ms), (20~24 h),, ml,, 2.3.3 (20 24 h),6 (10 0 ~10 4 cfu/ml) 3, 0.14 mg/, ml,6, ΔT 2, (R 2 =0.97837), 10 0 cfu/ml 10 4 cfu/ml, ΔT 2 5, 43 1 cfu/ml,, ΔT 2 (ΔT 2 =34 ms),t 2,,,,,, 10 3 cfu/ml,δt 2 (11 ms),, 10 0 ~10 3 cfu/,
174 2013 3 样品中菌浓度 / cfu ml -1 菌株 Table 1 1 Results of bacteria detection in food samples 孵育时间 (h)/δt 2 (ms) 0 4 20 24 10 2 沙门氏菌 24.20±0.53 21.15±1.17 ND ND 大肠杆菌 O157 ND ND ND ND 10 3 沙门氏菌 20.88±1.18 21.48±0.98 ND 11.24±1.03 大肠杆菌 O157 ND ND ND ND :ND (ΔT 2<8 ms),error bars=±sd(n=3),,, [1] Wang S, Duan H, Zhang W, et al. Analysis of bacterial foodborne disease outbreaks in China between 1994 and 2005[J]. FEMS Immunology & Medical Microbiology, 2007, 51(1): 8-13. [2],,,. [J]., 2009, 34(4): 247-251. [3],,,. PCR, [J]., 2010, 10(6): 173-179. [4] Josephson L, Perez J. M,Weissleder R. Magnetic nanosensors for the detection of oligonucleotide sequences [J]. Angewandte Chemie, 2001, 113(17): 3304-3306. [5] Ma W, Chen W, Qiao R., et al. Rapid and sensitive detection of microcystin by immunosensor based on nuclear magnetic resonance[j]. Biosensors and Bioelectronics, 2009, 25(1): 240-243. [6] Perez J. M, Simeone F. J, Saeki Y, et al. Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media[j]. Journal of the American Chemical Society, 2003, 125(34): 10192-10193. [7] Kaittanis C, Naser S. A, Perez J. M. One-step, nanoparticle-mediated bacterial detection with magnetic relaxation
食源性致病菌低场磁共振快速筛选检验方法 175 [J]. Nano Letters, 2007, 7(2): 380-383. [8] Yang H, Zhuang Y, Hu H, et al. Silica c\coated manganese oxide nanoparticles as a platform for targeted magnetic resonance and fluorescence imaging of cancer cells[j]. Advanced Functional Materials, 2010, 20 (11): 1733-1741. [9] Cheng F, Chen L, Wang W, et al. Immobilization and stabilization of papain on SiO 2 particles containing amine groups[j]. Chinese journal of biotechnology, 2004, 20(2): 287. [10] Vollath D, Szabo D. Coated nanoparticles: a new way to improved nanocomposites[j]. Journal of Nanoparticle Research, 1999, 1(2): 235-242. A Method for Rapid Detection of Pathogenic Bacteria Using Low Field NMR Li Xianfu 1 Chen Yan 1 Zhang Zhiyun 1 Han Wei 2 Xiao Ming 1 Yang Shiping 1 Gu Ming 2 Chen Jun 1 Zhao Yu 1* ( 1 Department of Life and Environment Science College, Shanghai Normal University, Shanghai 200234 2 Shanghai Entry-Exit Inspection and Quarantine Bureau of the People s Republic of China, Shanghai 200135) Abstract This paper reported a rapid detection method of Salmonella using low field nuclear magnetic resonance (NMR). The Fe 3 O 4 magnetic nanoparticles were coated with silica to empower the nanoparticles with high dispersion and broad compatibility to biomacromolecules. The magnetic beads were modified with amino silane, which could immobilize antibody. The immune magnetic beads specifically attached to the surface of Salmonella in the enrichment. Nuclear magnetic resonance was used to obtain the value of T 2 to determine microorganism polluting condition. In this study, five different bacteria strains (E. coil O157, S. typhimurium, S. flexneri, S. aureus, and V. parahemolyticus) were used as control to test the specificity of this method, which performanced high specificity. Moreover, the reaction conditions were optimized in the study. The results indicated that the optimal concentration of magnetic beads was 0.14 mg/ml and the minimum level of this method was 10 3 cfu/ml. Key words magnetic nanoparticles; immunoreaction; Salmonella; nuclear magnetic resonance; rapid detection,,, 8 45 74 13,,, 14%, 20%, 32%,,, ( 消息来源 : 南方都市报 )