基于抗体-适配体夹心生物传感器检测大肠杆菌O157: H7

doi:10.13560/j.cnki.biotech.bull.1985.2023-0698

生物技术通报 ›› 2023, Vol. 39 ›› Issue (12): 81-89.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0698

基于抗体-适配体夹心生物传感器检测大肠杆菌O157: H7

侯炜辰¹(), 叶柯¹, 李洁², 张洋子³, 许文涛³, 朱龙佼³, 李相阳¹()

1.北京农学院食品科学与工程学院食品质量与安全北京实验室农产品有害微生物及农残安全检测与控制北京市重点实验室，北京 102206
2.中国农业大学食品科学与营养工程学院，北京 100083
3.中国农业大学营养与健康系食品精准营养与质量控制教育部重点实验室，北京 100083

收稿日期:2023-07-19 出版日期:2023-12-26 发布日期:2024-01-11
通讯作者: 李相阳，男，副教授，研究方向：食品安全；E-mail: lxy2002cn@163.com
作者简介:侯炜辰，女，硕士研究生，研究方向：食品加工与安全；E-mail: houweichen007@163.com
基金资助:
国家重点研发计划(2022YFF0607900);北京市景观休闲农业创新团队项目(BAIC09-2023);北京市科技计划(Z221100007122004)

Detection of Escherichia coli O157: H7 Based on Antibody Aptamer Sandwich Biosensor

HOU Wei-chen¹(), YE Ke¹, LI Jie², ZHANG Yang-zi³, XU Wen-tao³, ZHU Long-jiao³, LI Xiang-yang¹()

1. Beijing Laboratory of Food Quality and Safety, Beijing Key Laboratory of Detection and Control of Spoilage Organisms and Pesticide Residue in Agricultural Product, College of Food Science and Engineering, Beijing University of Agriculture, Beijing 102206
2. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083
3. Key Laboratory of Precision Nutrition and Food Quality, Department of Nutrition and Health, China Agricultural University, Beijing 100083

Received:2023-07-19 Published:2023-12-26 Online:2024-01-11

摘要/Abstract

摘要：

大肠杆菌O157: H7（Escherichia coli O157：H7）是一种重要的与公共卫生相关的食源性病原体。抗体分子是体液免疫应答中最重要的效应分子，具有多种生物学活性，最主要的生物学功能是与相应抗原特异性结合。适配体是体外合成的较短DNA序列，可通过识别特定区域和靶标特异性结合。利用抗体和适配体的特异性识别功能及免疫磁珠的捕获作用和磁分离，并通过多克隆抗体和裁剪得到的适配体搭建生物传感器，使用实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction，qPCR）可以实现对靶标在（8×10³）-（8×10⁶） CFU/mL范围内的定量检测，检测限为800 CFU/mL。

关键词: 多克隆抗体, 适配体, 裁剪, 免疫磁珠, 大肠杆菌O157: H7

Abstract:

Escherichia coli O157:H7 is an important food borne pathogen related to public health. Antibody molecules are the most important effector molecules in the humoral immune response. They have a variety of biological activities. The main biological function is to specifically bind to the corresponding antigen. Aptamers are shorter DNA sequences synthesized in vitro, which can bind specifically by recognizing specific regions and targets. Based on the specific recognition function of antibody and aptamer, the capture effect and magnetic separation of immunomagnetic beads, and the construction of biosensor by polyclonal antibody and tailoring aptamer, real-time fluorescence quantitative polymerase chain reaction（qPCR）may achieve the detection of target at (8×10³)-(8×10⁶) CFU/mL. The limit of detection is 800 CFU/mL for quantitative detection.

Key words: polyclonal antibody, aptamer, tailoring, immunomagnetic beads, Escherichia coli O157: H7

侯炜辰, 叶柯, 李洁, 张洋子, 许文涛, 朱龙佼, 李相阳. 基于抗体-适配体夹心生物传感器检测大肠杆菌O157: H7[J]. 生物技术通报, 2023, 39(12): 81-89.

HOU Wei-chen, YE Ke, LI Jie, ZHANG Yang-zi, XU Wen-tao, ZHU Long-jiao, LI Xiang-yang. Detection of Escherichia coli O157: H7 Based on Antibody Aptamer Sandwich Biosensor[J]. Biotechnology Bulletin, 2023, 39(12): 81-89.

图/表 10

表1 实验所用的核酸序列

Table 1 Nucleic acid sequences used in the experiment

名称 Name	序列 Sequence（5'-3'）
S1	CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT ATG GGT GGT GGA TGA GTG TGT GGC TCG CAC TGC TCC TGA ACG TAC
S1-1	CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT A TCG CAC TGC TCC TGA ACG TAC
S1-2	CAG GTC CAT CGA GTG GTA GGA TGG GTG GTG GAT GAG TGT GTG GC TCG CAC TGC TCC TGA ACG TAC
S1-3	CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TGG GTG GT TCG CAC TGC TCC TGA ACG TAC
S1-3-1	CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TCG CAC TGC TCC TGA ACG TAC
FP	CAG GTC CAT CGA GTG GTA GGA
RP	GTA CGT TCA GGA GCA GTG CGA

图1 抗体-适配体夹心法检测大肠杆菌原理图

Fig. 1 Schematic diagram of antibody aptamer sandwich method for detecting Escherichia coli

图2 适配体裁剪可行性分析 A：原始菌液-适配体和梯度稀释后的扩增曲线；B：原始菌液-适配体和梯度稀释后的变化趋势

Fig. 2 Feasibility analysis of adaptor clipping A：Amplification curve of original bacterial solution adaptor and gradient dilution.B：Change trend of original bacterial solution adaptor and gradient dilution

图3 裁剪后适配体和原始适配体比较 A：S1和S1-1扩增曲线；B：S1和S1-2扩增曲线；C：S1和S1-3扩增曲线；D：S1和S1-3-1扩增曲线

Fig. 3 Comparison of tailored adaptor and original adaptor A: S1 and S1-1 amplification curves; B: S1 and S1-2 amplification curves; C: S1 and S1-3 amplification curves; D: S1 and S1-3-1 amplification curves

图4 IMB的表征 A：粒径变化；B：电位变化

Fig. 4 Characterization of IMB A: Particle size change; B: potential change

图5 生物传感器可行性分析 A：IMB-靶标-适配体、IMB-适配体和IMB-靶标3种复合物扩增曲线；B：菌液4次10倍梯度稀释后扩增曲线

Fig. 5 Feasibility analysis for biosensor A: Amplification curves of three complexes of IMB target adaptor, IMB adaptor, and IMB target. B: Amplification curves after four times ten fold gradient dilution of bacterial solution

图6 条件优化结果 A：适配体裁剪结合缓冲液类型优化；B：适配体裁剪缓冲液pH优化；C：适配体裁剪孵育温度优化；D：生物传感器IMB-靶标孵育时间优化

Fig. 6 Condition optimization results A: Optimization of the type of adapted body scissor binding buffer; B: optimization of the pH of adapted body scissor buffer; C: optimization of adapted body scissor incubation temperature; D: optimization of sensor IMB target incubation time

图7 灵敏度测定

Fig. 7 Sensitivity measurement

图8 大肠埃希菌 O157∶H7 检测方法的特异性 A：裁剪适配体特异性；B：传感器属间特异性；C：传感器种间特异性

Fig. 8 Specificity of detection method for E. coli O157∶H7 A: Tailor adaptor specificity; B&C: interspecific specificity of sensors

表2 实际样品检测

Table 2 Actual sample testing

样品 Sample	传统培养法Traditional cultivation method /（CFU·mL^-1）	检测量Detection quantity/ （CFU·mL^-1）	回收率 Rate of recovery/%	RSD/%
1	3×10³	2.89×10³	96.3	2.15
2	3×10⁴	3.11×10⁴	103	2.07
3	3×10⁵	3.26×10⁵	108	1.35

参考文献 21

[1]	Odonkor ST, Ampofo JK. Escherichia coli as an indicator of bacteriological quality of water: an overview[J]. Microbiol Res(Pavia), 2013, 4(1): e2.
[2]	Jang J, Hur HG, Sadowsky MJ, et al. Environmental Escherichia coli: ecology and public health implications-a review[J]. J Appl Microbiol, 2017, 123(3): 570-581. doi: 10.1111/jam.13468 pmid: 28383815
[3]	Torres-Chavolla E, Alocilja EC. Aptasensors for detection of microbial and viral pathogens[J]. Biosens Bioelectron, 2009, 24(11): 3175-3182. doi: 10.1016/j.bios.2008.11.010 pmid: 19117748
[4]	Riley LW, Remis RS, Helgerson SD, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype[J]. N Engl J Med, 1983, 308(12): 681-685. doi: 10.1056/NEJM198303243081203 URL
[5]	樊晓洁. 食源性致病菌大肠杆菌O157: H7检测方法的研究进展[J]. 食品安全质量检测学报, 2020, 11(7): 2144-2149.
	Fan XJ. Research progress on detection methods of foodborne pathogenic Escherichia coli O157: H7[J]. J Food Saf Qual, 2020, 11(7): 2144-2149.
[6]	Rani A, Ravindran VB, Surapaneni A, et al. Review: trends in point-of-care diagnosis for Escherichia coli O157: H7 in food and water[J]. Int J Food Microbiol, 2021, 349: 109233. doi: 10.1016/j.ijfoodmicro.2021.109233 URL
[7]	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: GB 4789.36—2016[S]. 北京: 中国标准出版社, 2017.
	National Health and Family Planning Commission, China Food and Drug Administration. National Food Safety Standard - - Food Microbiological Examination - - Examination of Escherichia coli O157: H7/MN: GB 4789.36—2016[S]. Beijing: Standards Press of China, 2017.
[8]	Zhao YN, Zeng DX, Yan C, et al. Rapid and accurate detection of Escherichia coli O157: H7 in beef using microfluidic wax-printed paper-based ELISA[J]. Analyst, 2020, 145(8): 3106-3115. doi: 10.1039/D0AN00224K URL
[9]	Fan W, Gao XY, Li HN, et al. Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157: H7, and Listeria monocytogenes in meat using multiplex immunomagnetic separation and multiplex real-time PCR[J]. Eur Food Res Technol, 2022, 248(3): 869-879. doi: 10.1007/s00217-021-03933-5
[10]	Zhou SS, Lu C, Li YZ, et al. Gold nanobones enhanced ultrasensitive surface-enhanced Raman scattering aptasensor for detecting Escherichia coli O157: H7[J]. ACS Sens, 2020, 5(2): 588-596. doi: 10.1021/acssensors.9b02600 URL
[11]	Li Y, Liu HM, Huang H, et al. A sensitive electrochemical strategy via multiple amplification reactions for the detection of E. coli O157: H7[J]. Biosens Bioelectron, 2020, 147: 111752. doi: 10.1016/j.bios.2019.111752 URL
[12]	Darmostuk M, Rimpelova S, Gbelcova H, et al. Current approaches in SELEX: an update to aptamer selection technology[J]. Biotechnol Adv, 2015, 33(<W>6 Pt 2):1141-1161. doi: 10.1016/j.biotechadv.2015.02.008 pmid: 25708387
[13]	Kim YS, Raston NH, Gu MB. Aptamer-based nanobiosensors[J]. Biosens Bioelectron, 2016, 76: 2-19. doi: 10.1016/j.bios.2015.06.040 pmid: 26139320
[14]	Qin CF, Li GC. Mammalian cell display technology coupling with AID induced SHM in vitro: an ideal approach to the production of therapeutic antibodies[J]. Int Immunopharmacol, 2014, 23(2): 380-386. doi: 10.1016/j.intimp.2014.09.017 URL
[15]	Yu XF, Chen F, Wang RH, et al. Whole-bacterium SELEX of DNA aptamers for rapid detection of E. coli O157: H7 using a QCM sensor[J]. J Biotechnol, 2018, 266: 39-49. doi: 10.1016/j.jbiotec.2017.12.011 URL
[16]	辛思培, 蒋蔚, 龙梦瑶, 等. 检测牛奶中肠出血性大肠杆菌O157: H7的双抗夹心化学酶联免疫方法的建立[J]. 畜牧与兽医, 2020, 52(4): 116-121.
	Xin SP, Jiang W, Long MY, et al. Development of a double-antibody sandwich chemiluminescence enzyme immunoassay for detection of Escherichia coli O157: H7 in milk[J]. Anim Husb Vet Med, 2020, 52(4): 116-121.
[17]	王淑娟, 范一灵, 冯震, 等. 多酶恒温核酸快速扩增法检测大肠杆菌O157∶H7[J]. 上海预防医学, 2022, 34(6): 511-518.
	Wang SJ, Fan YL, Feng Z, et al. Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157∶H7[J]. Shanghai J Prev Med, 2022, 34(6): 511-518.
[18]	Ma ZL, Meliana C, Munawaroh HSH, et al. Recent advances in the analytical strategies of microbial biosensor for detection of pollutants[J]. Chemosphere, 2022, 306: 135515. doi: 10.1016/j.chemosphere.2022.135515 URL
[19]	樊凯, 陈移平, 陈晶晶, 等. 电化学免疫生物传感器在食品中大肠杆菌O157∶H7检测中的应用[J]. 食品安全导刊, 2021, 30: 52-53.
	Fan K, Chen YP, Chen JJ, et al. Application of electrochemical immunosensor in detection of Escherichia coli O157∶H7 in food[J]. China Food Saf Mag, 2021, 30: 52-53.
[20]	郝良玉, 曲晗, 李志萍, 等. 适配体捕获磁珠富集大肠埃希菌O157: H7方法的建立[J]. 中国病原生物学杂志, 2020, 15(4): 387-391.
	Hao LY, Qu H, Li ZP, et al. Enrichment of E. coli O157: H7 using aptamer capture magnetic beads[J]. J Pathog Biol, 2020, 15(4): 387-391.
[21]	Jayasena SD. Aptamers: an emerging class of molecules that rival antibodies in diagnostics[J]. Clin Chem, 1999, 45(9): 1628-1650. pmid: 10471678

基于抗体-适配体夹心生物传感器检测大肠杆菌O157: H7

Detection of Escherichia coli O157: H7 Based on Antibody Aptamer Sandwich Biosensor

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