肠致病性大肠杆菌适配体筛选研究.pdf

第6卷第12期
食品安全质量检测学报
Vol 6 No 12
2015年12月
Journal of Food Salcly and Quality
IDcc..2015
肠致病性大肠杄菌适配体筛选硏究
段诺,张田力,吴世嘉,王周平
(江南大学食品学院,食品科学与技术国家重点实验室,无锡214122)
摘要:目的食源性致病菌引起的食源性疾病已成为全球食品安全面临的巨大威胁,其中由大肠杆菌引起
的食源性疾病最为常见。因此发展快速、准确检测大肠杆菌的方法对于保障食品安全、保护国民健康具有重
要意义。方法应用 Wholc bactcria-SHI.HX(指数富集的配体系统进化技术,以肠致病性大肠杆菌为靶标,经过
15轮的筛选富集、将所得产物进行克隆测序,并结合荧光分析考察所得序列的亲和力和特异性,根据解离常数
值分析比较序列的亲和力和特异性,最终得到2条(Seqg.1和Seq.28)与肠致病性大肠杅菌高特异性结合的适配
体。结果适配体Seq,1和Seq28均对肠致病性大肠杄菌表现出了相对良好的亲和力和特异性(对其他菌的相
对亲和力均低于15%,解离常数分别为45.0616.797和32.3116.002 nmol/L。结论本文利用 SELEX技术筛选
特异性识别肠致病性大肠杄菌适配体,具有稳定性高、合成方便、易标记等特点,可进一步应用于食品中肠致
病性大肠杄菌的快速检测。
关键词:肠致病性大肠杆菌:适配体: Whole bacteria- SELEX技术;荧光分析
Selection of an aptamer targeted to Enteropathogenic Escherichia coli
DUAN Nuo. ZHANG Tian-li WU Shi-jia WANG Zhou-ping
(State Key aboratory of ood Science and Technolog, cho of ood cience and ecolog, iangnan niver ity
ABSTRACT: Objective Foodborne discascs causcd by foodborne pathogenic bacteria have bccomc a grcat
threat to the global lood security. The most common is Enteropathogenic Escherichia coli(EPEC).Therefore
it will be of grcat significance to develop rapid and accurate detection mcthod for ensuring food safcty and
national health. Methods In this work, a wholc-bactcria Systemic Evolution of Ligands by E
Enrichment(SELEX)method was applied to identify aptamers specific binding Lo EPEC. Alter fifteen rounds
of sclcction, a highly enriched oligonuclcotide pool was scqucnccd. The binding affinity and specificity assa
were analyzcd by fluoresccncc analysis. Results Two sequences(Scq.1 and Scq. 28), which prescnted high
finity and specificity with the target, were regarded as aptamers of EPEC. The binding allinity of Seq. 1 and
Scq. 28 to the other bacteria was all bclow 15%o. The dissociation constant(Kd valuc)was 45.06#6.797 and
2.31=6.002 nmol/T, respectively. Conclusion Owing to their high sclcctivity, stability, and casy synthesis
and labcl, aptamers have application in detecting EPEC in food
KEY WORDS: Tntcropatho cnic cherichia coli; aptamer; whole bacteria-stix, fluorescence analysis
基金项目:国家自然科学基金项目(21375049,31401575
Iund: Supported by the National Natural Science Ioundation of China (21375049, 31401575
通讯作者:王周平,教授,博士生导师,主要研究方向为营养与食品安全。-mail:wang/p(@jiangnan.edu.cn
Ichnology, annan University, Wuxi 214122, China. E-mail: wangzp(c jiangnan. hnology, State Kcy Laboraory of Food Scicnce and
Corresponding author: WANG Zhou-ping, Professor, School of Food Scicncc and To