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楼秀芹, 俞骅, 汪皓秋, 张蔚, 刘涛, 王旭初. 基于全菌SELEX技术副溶血性弧菌适配体筛选及鉴定[J]. 中国公共卫生, 2019, 35(12): 1718-1723. DOI: 10.11847/zgggws1120138
引用本文: 楼秀芹, 俞骅, 汪皓秋, 张蔚, 刘涛, 王旭初. 基于全菌SELEX技术副溶血性弧菌适配体筛选及鉴定[J]. 中国公共卫生, 2019, 35(12): 1718-1723. DOI: 10.11847/zgggws1120138
Xiu-qin LOU, Hua YU, Hao-qiu WANG, . Selection and identification of DNA aptamers targeted to Vibrio parahemolyticus based on whole-bacterium SELEX[J]. Chinese Journal of Public Health, 2019, 35(12): 1718-1723. DOI: 10.11847/zgggws1120138
Citation: Xiu-qin LOU, Hua YU, Hao-qiu WANG, . Selection and identification of DNA aptamers targeted to Vibrio parahemolyticus based on whole-bacterium SELEX[J]. Chinese Journal of Public Health, 2019, 35(12): 1718-1723. DOI: 10.11847/zgggws1120138

基于全菌SELEX技术副溶血性弧菌适配体筛选及鉴定

Selection and identification of DNA aptamers targeted to Vibrio parahemolyticus based on whole-bacterium SELEX

  • 摘要:
      目的  筛选并鉴定可用于实验室快速检测副溶血性弧菌的适配体序列。
      方法  利用全菌SELEX技术,通过人工合成全长86 bp、中间含40 bp随机核苷酸序列的单链DNA文库,筛选与副溶血性弧菌高效结合的适配体,对候选适配体序列进行特异性分析,并评估其二级结构和Kd值。
      结果  第4轮筛选即出现比较明显的产物富集,继续筛选至第12轮结束。经克隆和测序后得到5条候选适配体序列(F2、F5、F6、F19、F30),它们与副溶血性弧菌的结合率分别为68.8 %,68.8 %,73.1 %,65.2 %和72.7 %;特异性实验显示,5条候选适配体均能与副溶血性弧菌高效结合,与5种其它常见致病菌则结合不明显;用DNA folding form对5条候选核酸适配体序列进行二级结构模拟,结果显示,其二级结构以茎环(凸环和圆环)为主,茎环结构中的茎多由G-C配对组成;采用荧光分析法对5条候选适配体的Kd值进行分析测定,其对靶细菌的解离常数分别为15.88、15.24、9.13、4.12和25.39 nM。
      结论  全菌SELEX技术可用于副溶血性弧菌适配体筛选,5条候选适配体与靶细菌结合力强,特异性高。

     

    Abstract:
      Objective  To obtain the DNA aptamers specific binding to Vibrio parahemolyticus (VP) for rapid and sensitive detection of VP.
      Methods  The oligonucleotide library was synthesized as a single-stranded 86-mer, with 40-mer random oligonucleotides in center for whole-bacterium systematic evolution of ligands by exponential enrichment (SELEX) targeted to VP. Determination of specificity and affinity of the library were then carried out for the candidate aptamers. Prediction of secondary structures and estimation of binding dissociation constants (Kd) of the aptamers were also performed.
      Results  The aptamer pools from the twelfth round of SELEX displayed the highest affinity for the target cells. The aptamer pools were cloned and sequenced, and a total of 5 sequences were obtained as the candidate aptamers (F2, F5, F6, F19, and F30); the binding affinity of these sequences were 68.8%, 68.8%, 73.1%, 65.2%, and 72.7%. All of these aptamer sequences showed preferential binding to VP over the other 5 types of bacteria. Predictions of secondary structures with DNA folding form demonstrated that all the five candidate aptamers were with several stem rings based on G-C base pairing. Kd value of these aptamers' sequences were 15.88, 15.24, 9.13, 4.12, and 25.39, respectively.
      Conclusion  The use of whole-bacterium SELEX to identify DNA aptamers that are specific for Vibrio parahemolyticus is feasible. All the 5 selected candidate aptamers are of high affinity and specificity, and are suitable for further development of rapid detection.

     

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