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Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods.

https://arctichealth.org/en/permalink/ahliterature11066
Source
Int J Food Microbiol. 1997 Apr 15;35(3):275-80
Publication Type
Article
Date
Apr-15-1997
Author
A. Agersborg
R. Dahl
I. Martinez
Author Affiliation
Norwegian Institute of Fisheries and Aquaculture N-9005 Tromso, Norway.
Source
Int J Food Microbiol. 1997 Apr 15;35(3):275-80
Date
Apr-15-1997
Language
English
Publication Type
Article
Keywords
Animals
Base Sequence
DNA, Bacterial - analysis - chemistry - genetics
Decapoda (Crustacea) - microbiology
Detergents - pharmacology
Electrophoresis, Agar Gel
Endopeptidase K - pharmacology
Fish Products - microbiology
Food Microbiology
Food Poisoning - diagnosis - epidemiology - etiology
Gene Amplification
Heat
Humans
Listeria monocytogenes - drug effects - genetics - isolation & purification
Muramidase - pharmacology
Norway - epidemiology
Octoxynol - pharmacology
Polymerase Chain Reaction - methods
Prevalence
Research Support, Non-U.S. Gov't
Sensitivity and specificity
Time Factors
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.
PubMed ID
9105938 View in PubMed
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