Selection and Identification of a DNA Aptamer Targeted to Vibrio parahemolyticus

Abstract

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus. FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K d ) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Aptamers against pathogenic microorganisms

“…Single cell detection in bacterial suspension.Chang et al (2013)  Whole live cellsDNAFlow cytofluorometry in biological samples.Cao et al (2009)  Potentiometric aptasensor based on carbon single-walled nanotubes. LOD is 800 CFU/ml in bacterial suspension and skin samplesZelada-Guillén et al (2012)  Simultaneous dual-color fluorescent detection of S. aureus and S. typhimurium using aptamer-functionalized magnetic nanoparticles LOD is 8 CFU/ml in bacterial suspensionDuan et al (2012a)  α-ToxinDNAInhibition of α-toxin-mediated cell death in Jurkat T cellsVivekananda et al (2014)  Enterotoxin BDNASpecific enterotoxin B detection in toxin-reach culture medium after cultivation of four different strains of S. aureus DeGrasse et al (2012)  Escherichia coli      OMPs of Crooks strainDNAFRET detection in bacterial suspensionBruno et al (2010)  Fimbriae protein of   K88 strainDNASandwich-type fluorescent assay LOD is 1.1 × 10 3  CFU/ml in pure culture and 2.1 × 10 3  CFU/ml in biological samplesPeng et al (2014)  DH5a strain whole live cells2′-F-RNAField transistor based on single-walled carbon nanotubes LOD is 310 CFU/ml in bacterial suspensionSo et al (2008)  Sandwich-type detection system LOD is 10 CFU/ml in suspension of two bacterial speciesLee et al (2009)  Potentiometric aptasensor LOD is 6 CFU/ml in milk and 26 CFU/ml in apple juice samplesZelada-Guillén et al (2010)  Mycobacterium tuberculosis      Whole live cells…”

Simultaneous detection of pathogenic bacteria using an aptamer based biosensor and dual fluorescence resonance energy transfer from quantum dots to carbon nanoparticles

“…sigmaaldrich.com). The aptamers used in our research were prepared in our laboratory using previously reported methods [26,27]. Bacteria aptamers were synthesized by the Shanghai Sangon Biological Science & Technology Company (Shanghai, China) (http://www.sangon.com).…”

Selection, characterization and application of aptamers targeted to Aflatoxin B2

“…SELEX was carried out using a procedure based on previous work (Duan, Wu, Chen, Huang, & Wang, 2012). A random ssDNA library at a concentration of 2 nmol (second round begins with each addition of 200 pmol) was added to 500 mL of BB buffer (pH 7.0:100 mmol/L NaCl, 20 mmol/L TriseHCl pH 7.6, 2 mmol/L MgCl 2 , 5 mmol/L KCl, 1 mmol/L CaCl 2 and 0.02% Tween 20) and placed in a water bath at 95 C for 5 min and then in an ice bath for 5 min.…”