Use of DNA aptamer for sandwich type detection of Listeria monocytogenes

Abstract

A single stranded (ss) DNA aptamer, specific to members of Listeria genus, was used to develop a two-site binding sandwich assay for capture and detection of L. monocytogenes. Antibody-immobilized immunomagnetic beads were used to capture L. monocytogenes, followed by their exposure to the aptamer detector. Detection was achieved by amplification of cell-bound aptamers by qPCR. The lower limit of detection for the combined assay was 2.5 CFU L. monocytogenes in 500 μl buffer. This is juxtaposed to a detection limit of 2.4 log₁₀ CFU in 500 μl buffer for immunomagnetic separation coupled with qPCR detection of L. monocytogenes targeting the hly gene. When applied to turkey deli meat, subjected to 24 h of non-selective enrichment, the two-site binding sandwich assay showed positive results at initial inoculum levels of 1-2 log₁₀ CFU per 25 g sample. Because of its lower limit of detection, the assay reported here could be useful for detection of L. monocytogenes in foods and environmental samples.

Keywords: Listeria monocytogenes; Magnetic bead; Nucleic acid aptamer; Pathogen detection; Two-site binding sandwich assay.