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1 6 12 Vol. 6 No Journal of Food Safety and Quality Dec., 2015 段 * 诺, 张田力, 吴世嘉, 王周平 (,, ) 摘要 : 目的, 方法 Whole bacteria-selex( ),, 15,,,, 2 (Seq.1 Seq.28) 结果 Seq.1 Seq.28 ( 15%), 45.06± ±6.002 nmol/l 结论 SELEX,, 关键词 : ; ; Whole bacteria-selex ; Selection of an aptamer targeted to Enteropathogenic Escherichia coli DUAN Nuo, ZHANG Tian-Li, WU Shi-Jia, WANG Zhou-Ping * (State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi , China) ABSTRACT: Objective Foodborne diseases caused by foodborne pathogenic bacteria have become a great threat to the global food security. The most common is Enteropathogenic Escherichia.coli (EPEC). Therefore, it will be of great significance to develop rapid and accurate detection method for ensuring food safety and national health. Methods In this work, a whole-bacteria Systemic Evolution of Ligands by Exponential Enrichment (SELEX) method was applied to identify aptamers specific binding to EPEC. After fifteen rounds of selection, a highly enriched oligonucleotide pool was sequenced. The binding affinity and specificity assay were analyzed by fluorescence analysis. Results Two sequences (Seq.1 and Seq.28), which presented high affinity and specificity with the target, were regarded as aptamers of EPEC. The binding affinity of Seq.1 and Seq.28 to the other bacteria was all below 15%. The dissociation constant (K d value) was 45.06±6.797 and 32.31±6.002 nmol/l, respectively. Conclusion Owing to their high selectivity, stability, and easy synthesis and label, aptamers have application in detecting EPEC in food. KEY WORDS: Enteropathogenic Escherichia coli; aptamer; whole bacteria-selex; fluorescence analysis 基金项目 : ( , ) Fund: Supported by the National Natural Science Foundation of China ( , ) * 通讯作者 :,,, wangzp@jiangnan.edu.cn *Corresponding author: WANG Zhou-Ping, Professor, School of Food Science and Technology, State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi , China. wangzp@jiangnan.edu.cn

002 nmol/l 结论 SELEX,, 关键词 : ; ; Whole bacteria-selex ; Selection of an aptamer targeted to Enteropathogenic Escherichia coli DUAN Nuo, ZHANG Tian-Li, WU Shi-Jia, WANG Zhou-Ping * (State Key

2 引言, 6, (EPEC) (ETEC)(EIEC) (EHEC)(EAEC) (ESIES) [1], EPEC, [2] 20 EPEC, 25%~50% [3,4] EPEC + +,,,,, ( 7 d ) EPEC ATP [5] PCR [6] [7-8] [9],,, EPEC 1990 [10], (systematicevolution of ligands by exponential enrichment, SELEX) (DNA RNA), [11] : (1) ; (2), ; (3),, ; (4),, Whole bacteria-selex,, whole bacteria-selex [12] [13] [14] [15], whole bacteria-selex EPEC, whole bacteria-selex,, 2 材料与方法 2.1 试验材料 EPEC O26: K60 CICC10372 EHEC O157: H7 CICC21530 ETEC O126: K71 CICC CICC10983 CICC10473 CICC10392 CICC21483, ATCC (OD 600 =0.3), r/min 4 min,, 1 BB(50 mmol/l Tris-HCl (ph 7.4), 5 mmol/l KCl, 100 mmol/l NaCl, 1 mmol/l MgCl 2 ) 2.2 试剂与仪器试剂 trna, 30% (Sigma-Aldrich, USA); Taq DNA 10 PCR dntp 10%AP TEMED - - ( ); (, ) DNA ( Integrated DNA Technologies ): 5 -CAGCTCAGAAGCTTG ATCCT-N 40 -GACTCGAAGTCGTGCATCTG-3,, 40 : 5 -CAGCTCAGAAGCTTGATCCT-3 ; 5 : 5 - -CAGATGCACGACTTCG AGTC 仪器 Bio-rad, Bio-rad, C1000 Thermal Cycler PCR ( (Bio-Rad) ); UV-1800 ( ); Direct-Q3 ( (Millipore) ); Thermo NanoDrop 2000 ( )

与方法 2.1 试验材料 EPEC O26: K60 CICC10372 EHEC O157: H7 CICC21530 ETEC O126: K71 CICC 10415 CICC10983 CICC10473 CICC10392 CICC21483, ATCC25922 37 (OD 600 =0.

3 12, : 试验方法适配体筛选 whole bacteria-selex Hamula [16], 1 PCR : 1 μl ssdna, 0.5 μl (20 μmol/l), 0.5 μl (10 μmol/l), 1 μl dntp (5 mmol/l), 0.5 μl Taq DNA (5 U/μL), 5 μl 1 PCR Buffer, 41 μl : 95 5 min, s, s, s, 18, min, 4 10 min PCR dsdna Lambda 5 DNA min, min, 8 % ssdna 350 μl, ssdna 100 pmol, 克隆与测序 15 ssdna PCR, pmd19-t, DH5α, 适配体亲和力考察,, (FAM), : ( cfu/ml) ( , nmol/l) 37 1 h, 1 BB,, 600 μl 1 BB GraphPad Prism 5, K d 适配体特异性考察 EHEC ETEC 37 1 h, 1 BB,, 600 μl 1 BB, Fig. 1 1 Schematic representation of the aptamer selection

ssdna 350 μl, ssdna 100 pmol, 2.3.2 克隆与测序 15 ssdna PCR, pmd19-t, DH5α, 40 2.3.3 适配体亲和力考察,, (FAM), : ( 1 10 8 cfu/ml) (30 50 70 100, 150 200 300 400 nmol/l) 37 1 h, 1 BB,, 600 μl 1 BB GraphPad Prism 5, K d 2.

4 表 1 肠致病性大肠杆菌适配体代表性序列随机区序列 Table 1 Representative random sequences of aptamers to EPEC Seq.1 Seq.2 Seq.4 Seq.9 Seq.21 Seq.26 Seq.28 Seq.30 Seq.35 Seq.36 Seq.37 Seq.38 ACCAGTAGACTTTCAACTTTACTGCCATCGTGTGCCCTAA ATGTCGCGGGCGTTTACTGGGGGGTAGTGGGCGGTGTGGT GTGTTGTGGGGTTTTTCGGTTTATCTAGCTTGTTACTCTG CCCACCGCCATCTCCCAGATCTCCTTCCACCGTCGCCATT ACTATCATTGGGGGTCGTTCGCACTTTCTTTGTTCCGTTT GACTAGATCATTATGGCGGGGGGTGATTGTGTGGTCTGGG ACAGTGCTCGGGATATATCAATATGTCACCTCGGCTAATG CTGTAATGGTTTAAGAGTTTGATACGTCGGGATGTGTGGT CATTGGGCGTTTCATTGTGTTTTCGGCGATCCCTGTCCCA GGGTGGGGTTGGAGGGTGGGTTGGATCAGCGGTCGGTGCG GGGTGAGAGGGCGGGGTGGGTGATCATGTAGTCTGGCTGT ATCACACCCACACGTACCATCCCGTCTTGCTCTGGCGTCT 3 结果与讨论 3.1 测序结果分析 PCR, 60, 60 12, 12, FAM, 适配体的亲和性分析 5 (FAM), EPEC,, Seq.2 Seq.4 Seq.9 Seq.21 Seq.30 Seq.35, EPEC, nmol 2 6 EPEC,,, EPEC, 200 nmol/l, K d, K d, K d, [17] 2 6 K d 32.31~47.49 nmol/l, 3.3 候选适配体的特异性分析, 6, 6 6 EHEC ETEC, ( 3), 6 EPEC 70%, Seq.26 EHEC 28.7%, 26.4%; Seq.36 20%; Seq.37 EHEC ETEC 25%, Seq.38 24%, Seq.1 Seq.28 15%,, Seq.1 Seq 适配体的二级结构分析 4 Seq.1 Seq.28,, Seq.1 1 4, Seq DNA,

ACTATCATTGGGGGTCGTTCGCACTTTCTTTGTTCCGTTT GACTAGATCATTATGGCGGGGGGTGATTGTGTGGTCTGGG ACAGTGCTCGGGATATATCAATATGTCACCTCGGCTAATG CTGTAATGGTTTAAGAGTTTGATACGTCGGGATGTGTGGT

5 12, : 4807, [18], Seq.1 Seq kcal/mol -7.3 kcal/mol, 4 结论,, whole bacteria-selex 15,, Seq.1 Seq ± ±6.002 nmol/l,, 2 Fig. 2 Binding saturation curves of aptamers to the target EPEC

1 Seq.28 45.06±6.797 32.31±6.002 nmol/l,, 2 Fig.

6 Fig. 3 3 The evaluation of specificity of aptamers of enterocyte effacement conserved among diverse enterobacterial pathogens [J]. Proc Natl Acad Sci USA, 1995, 92: [5] Bushon RN, Brady AM, Likirdopulos CA, et al. Rapid detection of Escherichia coli and enterococci in recreational water using animmunomagnetic separation adenosine triphosphate technique [J]. Appl Microbiol, 2009, 106(2): [6],. PCR [J]., 2006, 28(2): Fig. 4 The secondary structures of aptamer sequences 参考文献 [1],,,. [J]., 2011, (1): Jin YJ, Liu Q, Chen YJ, et al. Advance in laboratory detection of four diagrrheagenic Escherichia coli [J]. China Tropical Med, 2011, (1): [2] Fagundes-Neto U, Scaletsky IC. The gut at war: the consequences of enteropathogenic Escherichia coli infection as a factor of diarrhea and malnutrition [J]. Sao Paulo Med J, 2000, 118(1): [3] PelayoJS, Scaletsky IC, Pedroso MZ, et al. Virulence properties of atypical EPEC strains [J]. J Med Microbiol, 1999, 48: [4] McDaniel TK, Jarvis KG, Donnenberg MS, et al. A genetic locus Yang SH, Chai TJ. Multiplex PCR for detection of rabbit EPEC and Clostridium perfringen [J]. Chin J Pre Vet Med, 2006, 28(2): [7] Martín MC, Martínez N, Rio BD, et al. A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries [J]. J Dairy Sci, 2010, (3): [8] İsmail HB, Zoraida PA, Mahmud HH, et al. Amperometric determination of live Escherichia coli using antibody-coated paramagnetic beads [J]. Anal Bioanal Chem, 2005, (5): [9] YouYH, Fu CX, ZengX, et al. A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool specimens of patients with diarrhea [J]. J Microbiol Meth,2008, 75(3): [10] Tuerk C, Gold L. Systematic evolution of ligands by exponential

Appl Microbiol, 2009, 106(2): 432 441. [6],. PCR [J]., 2006, 28(2): 228 231. 4 Fig. 4 The secondary structures of aptamer sequences 参考文献 [1],,,. [J]., 2011, (1): 116 118.

7 12, : 4809 enrichment: RNA ligands to bacteriophage T4 DNA polymerase [J]. Science, 1990, 249(4968): [11],. [J]., 2014, 8: Yao Y, Zhang B. The research progress of aptamer [J]. Int J Lab Med, 2014, 8: [12] Moon J, Kim G, Lee S, et al. Identification of Salmonella Typhimurium-specific DNA aptamersdeveloped using whole-cell SELEX and FACS analysis [J]. J Microbiol Meth, 2013, 95: [13] Cao X, Li S, Chen L, et al. Combining use of a panel of ssdna aptamers in the detection of Staphylococcus aureus [J]. Nucleic Acids Res, 2009, 37: [14] Chen F, Zhou J, Luo F, et al. Aptamer from whole-bacterium SELEX as new therapeutic reagent againstvirulent Mycobacterium tuberculosis [J]. Biochem Biophy Res Commun, 2007, 357(3): [15] DuanN, Wu SJ, ChenXJ. Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus [J]. J Agric Food Chem, 2012, 60: [16] Hamula CLA, Zhang H, Guan LL, et al. Selection of aptamers against live bacterial cells [J]. Anal Chem, 2008, 80(20): [17] Dezhi K, Jiangjie W, Weiyun Z. Selection of DNA aptamers against glioblastoma cells with high affinity andspecificity [J]. PloS One, 2012, 7(10): [18] Cho SS, Pincus DL, Thirumalai D. Assembly mechanisms of RNA pseudoknots are determined by the stabilities of constituent secondary structures [J]. Proc Natl Acad Sci USA, 2009, 106(41): 作者简介 ( 责任编辑 : 白洪健 ) 段诺, 副教授, 主要研究方向为食品安全检测与控制 duannuo@jiangnan.edu.cn 王周平, 教授, 主要研究方向为营养与食品安全 wangzp@jiangnan.edu.cn

J Microbiol Meth, 2013, 95: 162 166. [13] Cao X, Li S, Chen L, et al. Combining use of a panel of ssdna aptamers in the detection of Staphylococcus aureus [J]. Nucleic Acids Res, 2009, 37: 4621 4628.

Microsoft Word - 4839-4843 20150924002-李建煌-二校全出

Microsoft Word - 4839-4843 20150924002-李建煌-二校全出 6 12 Vol. 6 No. 12 215 12 Journal of Food Safety and Quality Dec., 215 ONPG 李建煌 1, 姚蕾 1, 李玉优 1, 李焕仪 1, 何亮辉 1, 姜春宝 2, 曹维强 (1., 51852; 2., 5166) 1* 摘要 : 目的 βd(onitrophenylβdgalactopyranoside,