Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction

• Published in: Applied and Environmental Microbiology Vol. 57; no. 9; pp. 2576 - 2580
• Main Authors: Thomas, E.J.G. (Agriculture Canada, Lethbridge, Alberta, Canada), King, R.K, Burchak, J, Gannon, V.P.J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.09.1991
• Subjects: Animals; Bacterial Proteins - genetics; Bacterial Toxins; BIOCHIMIE; Biological and medical sciences; BIOQUIMICA; CARNE DE RES; Cattle; Food Microbiology; Fundamental and applied biological sciences. Psychology; GENE; GENES; Heat-Shock Proteins - genetics; Hemolysin Proteins; LAIT; LECHE; LISTERIA MONOCYTOGENES; Listeria monocytogenes - genetics; Listeria monocytogenes - growth & development; Listeria monocytogenes - isolation & purification; Meat - microbiology; Milk - microbiology; Molecular and cellular biology; Molecular genetics; Polymerase Chain Reaction; Sensitivity and Specificity; TECHNIQUE ANALYTIQUE; TECNICAS ANALITICAS; VIANDE BOVINE; Bovine; Listeria monocytogenes; Amplification; Polymerase chain reaction; Vertebrata; Sensitivity; Specificity; Mammalia; Gene; Substrate specificity; Bacteria; Artiodactyla; Detection; Ungulata; Milk
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.57.9.2576-2580.1991

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeria murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB sample cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively