Rapid On-chip flow Cytometric Detection of Listeria monocytogenes in Milk

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, intracellular bacterium that can cause severe infections in humans. Contamination by L. monocytogenes in foods represents a potential public health problem. Here we describe a rapid method for the detection of L. monocytogenes in milk by flow cytometry based on microfluidics (on-chip flow cytometry). This rapid identification of L. monocytogenes is based on fluorescence in situ hybridization (FISH) with a Cy5-labeled rRNA-targeting oligonucleotide probe. FISH experiments were successfully analyzed using a commercially available on-chip flow cytometry. FISH results with bacterial cultures indicated that the L. monocytogenes probe RL-2 was hybridized with 4 major strains of L. monocytogenes (serotype 1/2a, 1/2b, 1/2c and 4b) but not with other Listeria strains or milk spoilage bacteria. Specific FISH detection of L. monocytogenes was accomplished using the probe RL-2 when the milk contained a mixture of other bacterial species. The positive identification of L. monocytogenes in milk was completed within 5 hr (milk clearing: 40 min, hybridization: 3.5 hr, on-chip analysis: 30 min). The method presented in this study allows the specific and rapid identification of L. monocytogenes in milk.