Listeria monocytogenes. Listeria monocytogenes. Invasive pathogen WEAR GLOVES!!! Listeriosis YOPI Miscarriage, stillbirth, premature birth Bacteremia Meningitis Meningoencephalitis. Location of L. monocytogenes. Ubiquitous Raw foods Processed RTE foods Food processing environment.
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A: Conventional Method
Culturing on various selective and differential media (enrichment, isolation, biochemical analysis)
B: Rapid Method
Genetic based: PCR method
Positive and negative controls(L. monocytogenes and E. faecalis)
Transfer into microcentrifuge tubes. Mix with lysis buffer.
Cell lysis: heat at 37°C-30 min, add proteinase K and Buffer AL, then 70°C-30 min
Continue with DNA isolation using DNeasy spin column.
PCR Reaction mixture (Water, Reaction Buffer, primers, dNTPs, Taq)
Run the thermocycler. Cool or refrigerate.
Gel with DNA bands
Identify DNA band that corresponds to Listeria spp.
Need: DNA polymerase (Taq), reaction buffer containing Mg, dNTPs, oligonucleotide primers, water, and DNA sample
100 bp DNA ladder
1.5 Kb bp band
1.5 kb band Listeria (+)
Gram reaction: purple (+)
Catalase: bubbles (+)
MOX: black colonies
PALCAM: black colonies, red agar
TSAYE: blue-gray tintOverall Results
Both conventional and rapid methods are specific to the genus level
Further characterization may be completed with API strips, CAMP test, DNA sequencing, etc.