Abstract
Listeria monocytogenes, a foodborne pathogen, is considered as one of the major problems in food safety. With strong safety regulations, a monitoring measure is essential for protecting the health and safety of consumers. Thus, a reliable monitoring method is required. In this study, a rapid assay based on a combination of helicase dependent amplification (HDA) and DNA signal detection via nucleic acid hybridization in blue silver nanoplates (AgNPls) was established. The assay started directly after short term enrichment in terrific broth using cotton ball swapping technique on seafood surface. A HDA amplification of hly gene of L. monocytogenes at 65 °C allowed DNA signals to be increased, whereas the rendered DNA products were detected via nucleic acid hybridization with an oligonucleotide probe in AgNPls solution. The positive specimens induced blue silver nanoplates’ aggregation resulting in pale gray change to colorless, while the negative specimens showed the blue color of non-aggregated nanoplates. The method had a detection limit at 100 copies of L. monocytogenes DNA per 50 g of sample. This method was rapid, simple, did not require laboratory facilities and was suitable for field food safety monitoring
Highlights
Listeria monocytogenes, a gram-positive, causes serious invasive disease or listeriosis in humans and animals
Infections by ingestion of food contaminated by L. monocytogenes cause severe complications such as non-bloody diarrhea, nausea and vomiting, high fever, and serious septicemia and meningitis (Bortolussi, 2008; Kalorey, 2008)
The results of primer specificity was in consistent with those studies using the Polymerase Chain Reaction (PCR) technique (Furrer, et al, 1991), indicating the reliability of primer for Listeria amplification. All these results confirmed the specificity of the helicase dependent amplification (HDA) primer set for L. monocytogenes DNA amplification
Summary
A gram-positive, causes serious invasive disease or listeriosis in humans and animals. For DNA amplification, Helicase Dependent Amplification (HDA) was selected because it has similar principles to that of PCR (Vincent, et al, 2004), except for the use of thermophillic helicase enzyme to unwind doublestranded DNA instead of heating It provided several advantages over PCR such as its rapidity, its equal sensitivity, specificity and its condition involved with the use of isothermal temperature conditions enable it to perform DNA amplification without the need of a thermocycler. The DNA amplification via helicase dependent amplification (HDA) platform and the DNA hybridization using oligonucleotide in non-functionalized blue silver nanoplates (AgNPls) was applied for Listeria monocytogenes detection in a simple colorimetric assay platform. The use of it for on-site frozen seafood products screening was demonstrated
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