title: Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods.
abstract: Five grams of seafood products were inoculated with one to 500 viable or 10(9) heat-killed cells of Listeria monocytogenes. The presence of the pathogen was detected by the polymerase chain reaction (PCR) with primers specific for fragments of the listeriolysin O (hly) gene (two sets) and for the invasion-associated protein (iap) gene (one set). For DNA preparation, boiling, either alone or in combination with lysozyme and proteinase K treatment, was not always sufficient to lyse L. monocytogenes, while treatment with Triton X-100 produced consistently good DNA suitable for amplification. To avoid false-negative and false-positive results, 48 h incubations were necessary and a subculturing step after an initial 24 h incubation greatly improved the results. The primers that amplified regions of the listeriolysin O gene gave clearer and stronger products than primers for the invasion-associated protein gene. Using this method we were able to detect one to five L. monocytogenes cells in 5 g of product in a total of 55 h.
authors.affiliation: Norwegian Institute of Fisheries and Aquaculture N-9005 Tromso, Norway.
authors.forename: A
authors.initials: A
authors.lastname: Agersborg
authors.forename: R
authors.initials: R
authors.lastname: Dahl
authors.forename: I
authors.initials: I
authors.lastname: Martinez
mesh_terms: D000818:Animals; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D033364:Decapoda / Q000382:microbiology; D003902:Detergents / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D019286:Endopeptidase K / Q000494:pharmacology; D005396:Fish Products / Q000382:microbiology*; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000175:diagnosis / Q000453:epidemiology / Q000209:etiology; D005784:Gene Amplification; D006358:Hot Temperature; D006801:Humans; D008089:Listeria monocytogenes / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D009113:Muramidase / Q000494:pharmacology; D009664:Norway / Q000453:epidemiology; D017830:Octoxynol / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; D003902:Detergents; D017830:Octoxynol; D009113:Muramidase; D019286:Endopeptidase K