Direct detection of Listeria monocytogenes using paramagnetic bead DNA extraction and enzymatic DNA amplification

The potential of the polymerase chain reaction (PCR) as a tool for direct detection of Listeria monocytogenes in food and clinical samples was investigated. A specific oligonucleotide, directed against the listeriolysin 0 gene of L. monocytogenes, was coupled to paramagnetic beads and used to isolate listerial DNA from food homogenates and blood. The isolated DNA was amplified with a seminested PCR procedure, which recognized the hly gene. The detection limit for L. monocytogenes in 50 ml of a buffer solution was between 1 and 10 colony forming units (cfu). In food homogenates consisting of 10 g food and 40 ml 0.85% NaCl solution, artificially spiked with an overnight culture of L. monocytogenes, the detection limit was 1-10 cfu. The method was further evaluated by application to naturally contaminated food samples. In 250 microliters whole human blood artificially spiked with L. monocytogenes 10,000 cfu could be detected.