In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

Aptamers are specifi c nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high affi nity and specifi city. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment(SELEX) was used to select and PCR amplify DNA sequences(aptamers) capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simplifi ed affi nity separation approach was employed, in which L. monocytogenes in exponential(log) phase of growth was used as the separation target. A fl uorescently-labeled aptamer assay scheme was devised for detecting L. monocytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high affi nity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest affi nity was further tested in aptamer-peroxidase and aptamer-fl uorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and purifi cation of complex markers or targets.