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Int. J. Mol. Sci. 2017, 18(5), 883; doi:10.3390/ijms18050883

Selection, Characterization and Interaction Studies of a DNA Aptamer for the Detection of Bifidobacterium bifidum

1
,
1
,
1,2
,
1,2
,
1,2
and
1,2,3,*
1
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
2
International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi 214122, China
3
Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, China
*
Author to whom correspondence should be addressed.
Academic Editor: Julian Alexander Tanner
Received: 7 March 2017 / Revised: 14 April 2017 / Accepted: 18 April 2017 / Published: 25 April 2017
(This article belongs to the Special Issue Aptamers)
View Full-Text   |   Download PDF [4468 KB, uploaded 25 April 2017]   |  

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. View Full-Text
Keywords: Bifidobacterium bifidum; aptamer; SELEX; sequence truncation; colorimetric bioassay Bifidobacterium bifidum; aptamer; SELEX; sequence truncation; colorimetric bioassay
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Hu, L.; Wang, L.; Lu, W.; Zhao, J.; Zhang, H.; Chen, W. Selection, Characterization and Interaction Studies of a DNA Aptamer for the Detection of Bifidobacterium bifidum. Int. J. Mol. Sci. 2017, 18, 883.

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