RRC ID 50843
著者 Hu L, Wang L, Lu W, Zhao J, Zhang H, Chen W.
タイトル Selection, Characterization and Interaction Studies of a DNA Aptamer for the Detection of Bifidobacterium bifidum.
ジャーナル Int J Mol Sci
Abstract A whole-bacterium-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure was adopted in this study for the selection of an ssDNA aptamer that binds to Bifidobacterium bifidum. After 12 rounds of selection targeted against B. bifidum, 30 sequences were obtained and divided into seven families according to primary sequence homology and similarity of secondary structure. Four FAM (fluorescein amidite) labeled aptamer sequences from different families were selected for further characterization by flow cytometric analysis. The results reveal that the aptamer sequence CCFM641-5 demonstrated high-affinity and specificity for B. bifidum compared with the other sequences tested, and the estimated Kd value was 10.69 ± 0.89 nM. Additionally, sequence truncation experiments of the aptamer CCFM641-5 led to the conclusion that the 5'-primer and 3'-primer binding sites were essential for aptamer-target binding. In addition, the possible component of the target B. bifidum, bound by the aptamer CCFM641-5, was identified as a membrane protein by treatment with proteinase. Furthermore, to prove the potential application of the aptamer CCFM641-5, a colorimetric bioassay of the sandwich-type structure was used to detect B. bifidum. The assay had a linear range of 10⁴ to 10⁷ cfu/mL (R² = 0.9834). Therefore, the colorimetric bioassay appears to be a promising method for the detection of B. bifidum based on the aptamer CCFM641-5.
巻・号 18(5)
公開日 2017-4-25
DOI 10.3390/ijms18050883
PII ijms18050883
PMID 28441340
PMC PMC5454810
MeSH Aptamers, Nucleotide / chemistry* Aptamers, Nucleotide / metabolism Bacterial Proteins / chemistry Bacterial Proteins / metabolism Bifidobacterium bifidum / chemistry Bifidobacterium bifidum / isolation & purification* Bifidobacterium bifidum / metabolism Binding Sites Biological Assay / methods* Colorimetry / methods Fluorescein / chemistry Kinetics Membrane Proteins / chemistry Membrane Proteins / metabolism SELEX Aptamer Technique
IF 4.556
引用数 5
リソース情報
一般微生物 JCM 11658