食品中志贺氏菌快速检测免疫磁分离样品前处理技术研究

doi:10.3969/j.issn.1000-1158.2023.03.02

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摘要采用Protein A磁珠与志贺氏菌混合血清制备抗志贺氏菌多靶标免疫磁珠,建立了25mL志贺氏菌免疫磁分离体系,并通过测定代表性食品(牛奶、水果、面包)中志贺氏菌生长曲线,设计食品样品前处理可行性技术方案,与环介导等温扩增(LAMP)检测技术整合进行了食品中痕量志贺氏菌快速检测验证。结果显示:多靶标免疫磁珠能同时捕获福氏、宋内氏、鲍氏、痢疾氏等4种志贺氏菌,捕获效率为35%~94%,非特异性小于1%(金黄色葡萄球菌10%除外),1~2×10¹CFU志贺氏菌在25mL纯牛奶、25g梨/面包+25mL志贺氏菌增菌肉汤基础中,通过6h培养+免疫磁分离的策略可获得10²CFU/mL以上的志贺氏菌,将志贺氏菌检测样品前处理时间由48h缩短至8h内,并能够满足后续检测技术检出限的需求,实现了食品中志贺氏菌的快速检测。

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关键词 ：计量学;食品检测;志贺氏菌;免疫磁分离;捕获效率, Protein A磁珠;多价血清

Abstract：Anti-Shigella multi-target immunomagnetic beads were prepared by using Protein A magnetic beads and anti-Shigella mixed serum, and a 25mL immunomagnetic separation system for Shigella was established. Combined with determining the growth curve of Shigella bacteria in representative foods (milk, fruit, bread), a feasible strategy for the pretreatment of food samples was designed, which was integrated with the LAMP technology to conduct rapid detection of Shigella in food. The results showed that the multi-target immunomagnetic beads could simultaneously capture four Shigella species, including flexneri, sonnei, Bowman, and dysentery, with a capture efficiency of 35%~94% and a non-specificity of less than 1% (except for Staphylococcus aureus 10%). Above 10²CFU/mL of target Shigella could be obtained when 1~2×10¹CFU Shigella in 25mL milk/in 25g bear/bread +25mL Shigella enrichent broth base by culturing for 6h following the operation of immunomagnetic separation, the time of samples pretreatment for Shigella detection is shortened from 48h to within 8h, which can meet the requirements of the detection limit of technologies subsequently used to realize the rapid detection of Shigella in food.

Key words： metrology food detection Shigella immunomagnetic separation capture efficiency protein A magnetic beads multivalent serum

收稿日期: 2022-08-01 发布日期: 2023-03-08

PACS:

TB99

基金资助:国家自然科学基金(31972146);北京科学技术研究院高水平创新团队项目(HIT202201);北京科学技术研究院“北科萌芽”计划(BGS202116)

通讯作者: 杜美红(1974-),女,山西太谷人,北京市科学技术研究院研究员,从事食品安全微生物快速检测研究工作。Email: dumeihong@bcpca.ac.cn E-mail: dumeihong@bcpca.ac.cn

作者简介: 李静雯(1991-),女,河南新乡人,北京市科学技术研究院助理研究员,从事食品安全微生物快速检测研究工作。Email: li.j.w@hotmail.com

引用本文:

李静雯,陈尔凝,康福英,洪甜,富迎辉,杜美红. 食品中志贺氏菌快速检测免疫磁分离样品前处理技术研究[J]. 计量学报, 2023, 44(3): 326-333.
LI Jing-wen,CHEN Er-ning,KANG Fu-ying,HONG Tian,FU Ying-hui,DU Mei-hong. Study on Samples Pretreatment Technology Based on Immunomagnetic Separation for Rapid Detection of Shigella in Food. Acta Metrologica Sinica, 2023, 44(3): 326-333.

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http://jlxb.china-csm.org:81/Jwk_jlxb/CN/10.3969/j.issn.1000-1158.2023.03.02 或 http://jlxb.china-csm.org:81/Jwk_jlxb/CN/Y2023/V44/I3/326

［4］	Meng Q, Bai X N, Zhao A, et al. Characterization of Shiga toxin-producing Escherichia coli isolated from healthy pigs in China［J］. Bmc Microbiology, 2014,14:5-19.
［6］	GB 4789.5—2012 食品微生物检验-志贺氏菌检验［S］.
［1］	Farshad S, Sheikhi R, Japoni A, et al. Characterization of Shigella strains in Iran by plasmid profile analysis and PCR amplification of ipa genes［J］. Journal of Clinical Microbiology, 2006,44(8):2879-2883.
［10］	Du M H, Li J W, Liu Q J, et al. Rapid detection of trace Salmonella in milk using an effective pretreatment combined with droplet digital polymerase chain reaction［J］. Microbiol Res, 2021,251:126838.
［13］	Islam D, Lindberg A A. Detection of Shigella-dysenteriae type-1 and Shigella-flexneri in feces by immunomagnetic isolation and polymerase chain-reaction［J］. Journal of Clinical Microbiology, 1992,30(11):2801-2806.
［15］	Achi-Berglund R, Lindberg A A. Rapid and sensitive detection of Shigella sonnei in feces by the use of an O-antigen-specific monoclonal antibody in a combined immunomagnetic separation-polymerase chain reaction assay［J］. Clin Microbiol Infect, 1996,2(1): 55-58.
	Wu H J, Sun Y P, Fan T L, et al. Detection of Live Listeria monocytogenes by EMA-qPCR［J］. Journal of Food Science and Biotechnology,2020,39(11):65-70.
	Li D H, Lu J F, Zeng J L, et al. Evaluation of Uncertainty in Determination of Total Residues of Ribavirin and its Metabolites in Eggs by LC-MS/MS［J］. Acta Metrologica Sinica, 2021, 42 (9): 1250-1256.
［19］	张明明,肖剑,林秀敏,等. 多重微滴数字PCR同时定量检测三种食源性致病菌DNA拷贝数［J］.农业生物技术学报,2022,30(3):606-618.
［7］	GB 4789.31—2003 食品微生物学检验-沙门氏菌、志贺氏菌和致泻大肠埃希氏菌的肠杆菌科噬菌体诊断检验［S］.
［16］	Achi R, Mata L, Siles X, et al. Immunomagnetic separation and PCR detection show Shigellae to be common faecal agents in children from urban marginal communities of Costa Rica［J］. Journal of Infection, 1996,32(3): 211-218.
［22］	王斌, 隋志伟, 刘思渊,等. 基于流式分析技术的奶粉中金黄色葡萄球菌活菌快速定量检测方法研究［J］. 计量学报, 2021, 42 (2): 250-258.
［2］	Lee J Y, Kang D H. Development of an improved selective medium for the detection of Shigella spp［J］. Lwt-Food Science and Technology, 2016,65:311-317.
［3］	Iseki H, Alhassan A, Ohta N, et al. Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites［J］. Journal of Microbiological Methods, 2007,71(3):281-287.
［5］	Lauri A, Castiglioni B, Mariani P. Comprehensive analysis of Salmonella sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes［J］. Applied Microbiology and Biotechnology, 2011,91(1):189-210.
［9］	Kaluzewski S, Sienicka J. Quantitative evaluation of growth-promoting properties of selected culture media used for isolation of Salmonella and Shigella strains［J］. Med Dosw Mikrobiol, 1990,42(1-2):35-43.
［11］	Li J W, Liu Q J, Wan Y P, et al. Rapid detection of trace Salmonella in milk and chicken by immunomagnetic separation in combination with a chemiluminescence microparticle immunoassay［J］. Anal Bioanal Chem, 2019,411:6067-6080.
［23］	李德恒,陆晶芳,曾金林,等.液相色谱串联质谱法测定鸡蛋中利巴韦林及其代谢物总残留的不确定度评定［J］. 计量学报, 2021,42 (9): 1250-1256.
［8］	Mokhtari W, Nsaibia S, Majouriet D, et al. Detection and characterization of Shigella species isolated from food and human stool samples in Nabeul, Tunisia, by molecular methods and culture techniques［J］. Journal of Applied Microbiology, 2012,113(1):209-222.
［12］	Du M H, Li J W, Zhao R X, et al. Effective pre-treatment technique based on immune-magnetic separation for rapid detection of trace levels of Salmonella in milk［J］. Food Control, 2018, 91:92-99.
［14］	Islam D, Tzipori S, Islam M, et al. Rapid Detection of Shigella-dysenteriae and Shigella-Flexneri infeces by an immunomagnetic assay with monoclonal-antibodies［J］. European Journal of Clinical Microbiology & Infectious Diseases, 1993,12(1):25-32.
［17］	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.
［18］	Xia S Q, Yu Z B, Liu D F, et al. Developing a novel immunochromatographic test strip with gold magnetic bifunctional nanobeads (GMBN) for effificient detection of Salmonella choleraesuis in milk［J］. Food Control, 2016,59,507-512.
［21］	Dolka B, Cisek A A, Szeleszczuk P. The application of the loop-mediated isothermal amplification (LAMP) method for diagnosing Enterococcus hirae-associated endocarditis outbreaks in chickens［J］.BMC Microbiology,2019,19:48.
	Zhang M M, Xiao J, Lin X M, et al. Simultaneous Quantification Method of DNA Copy Number for Three Food-borne Pathogens by Multiplex Droplet Digital PCR［J］. Journal of Agricultural Biotechnology, 2022,30(3):606-618.
［20］	吴海江,孙玉萍,范田丽,等. EMA结合实时荧光PCR方法检测单核细胞增生李斯特氏菌［J］.食品与生物技术学报,2020,39(11):65-70.
	Wang B, Sui Z W, Liu S Y, et al. Rapid and Quantitative Detection of Viable Staphylococcus aureus in Milk Powder Based on Flow Cytometry Technology［J］. Acta Metrologica Sinica, 2021, 42 (2): 250-258.