Vibrio parahaemolyticus is a foodborne pathogen and their human infection is regularly associated with the consumption of raw or undercooked seafood and contaminated water supplies. Many conventional biochemical identification and confirmation procedures are performed to detect the presence of this pathogen, both from seafood or environmental samples. However, these procedures not only require two or more days to complete, they do not have the capabilities to determine the number of V. parahaemolyticus cells in any given samples. Thus, in this study we describe the development of a rapid SYBR green based real-time PCR assay, targeting the thermo labile (tl) gene of V. parahaemolyticus for the detection and enumeration of this bacterium from seafood and environmental samples. We report that the real-time PCR assay and the primers designed are highly specific, and only generated the desired amplicons with V. parahaemolyticus DNA samples against other bacteria and fungi species. Our assay is also highly sensitive, and, is able to detect V. parahaemolyticus with high coefficient values in concentrations as low as 1.0 pg/μl DNA for pure genomic DNA solutions and 10 cells/ml in serially diluted cell suspension and spiked samples. This assay can be completed in less than 3 hours and may be used as a tool for rapid determination of V. parahaemolyticus densities in the food industries, environmental risk assessment and for clinical diagnostics purposes.
This study was conducted to detect the presence of Listeria monocytogenes (L. monocytogenes)
and screen for its antibiotic susceptibility characteristic from wildlife and water samples at
Kubah National Park, Sarawak, Malaysia. Samples collected were incubated and streaked on
selective medium PALCAM agar to confirm the presence of Listeria spp. before they were
further tested using molecular analysis. Specific Polymerase Chain Reaction (PCR) assay were
performed to target specific virulence gene, haemolysin gene, hlyA to further distinguish the
presence of this pathogenic bacteria in the samples. Overall, out of the 30 samples tested, 10
samples were confirmed as to contain L. monocytogenes strains and selected to subsequent
antibiotic susceptibility test. Susceptibility patterns to 10 antibiotics were investigated
among the L. monocytogenes strains. All strains were uniformly resistant to tetracycline and
erythromycin. On the other hand, all strains were sensitive to gentamycin and tobramycin. The
multiple antibiotic resistance shown by the strains in this study indicate the potential health
hazard associated with the possible transmission between wildlife and water to its surrounding
environment especially visitors and workers of Kubah National Park, Sarawak, Malaysia.