Development of genosensor based on helicase dependent dna amplification and fluorescence assay for detection of listeria monocytogenes in seafood

Abstract

Listeriosis, a foodborne disease caused by ingesting food contaminated with Listeria monocytogenes. This serious illness posed hazard especially to pregnant women, newborn, elderly as well as people with weakened immune systems (immunocompromised). The introduced legislation to control the incidence of listeriosis in several countries including a zero-tolerance in ready-to-eat (RTE) foods in the United Stated and a low acceptable level setting in the EU made a requirement for a rapid monitoring method. In this study, a rapid assay based on a combination of both helicase dependent amplification (HDA) and DNA signal detection via fluorescence DNA-DNA hybridization and analog probe as clamp lock was established to detect hly gene of Listeria monocytogens in seafood. Assay processes did include a short period of enrichment in terrific broth using cotton ball swabbing technique on seafood surface. HDA amplification of hly gene at 65oC allowed DNA signals to be increased, whereas the rendered DNA products were detected via fluorescence visualization based on FRET and analog DNA probe via DNA hybridization. The positive specimens induced fluorescence signals from a reporter at 5’ (FAM) while the negative specimens did not due to a resonance transfer of energy from 5’ (FAM) to 3’ (BHQ-1). The method had a detection limit at 100 copies or equivalence to 100 CFU of L. monocytogenes per 50 g of sample. This method was the first report of the application of molecular beacon and analog probe hybridization with Helicase Dependent Amplification to detection of hly gene. Furthermore these techniques could be used to discriminate the single nucleotide polymorphic mutant of hly determinant in food. This developed method is rapid, simple, and relied less on laboratory facilities which is suitable for monitoring of safety in frozen seafood in the field.