Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay

• Published in: Applied and Environmental Microbiology Vol. 57; no. 1; pp. 289 - 294
• Main Authors: Kim, C. (Centers for Disease Control, Atlanta, GA), Graves, L.M, Swaminathan, B, Mayer, L.W, Weaver, R.E
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.01.1991
• Subjects: ADN; Biological and medical sciences; Digoxigenin; DNA Probes; Evaluation Studies as Topic; Food Contamination; Food industries; Food Microbiology; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; LISTERIA; Listeria - genetics; Listeria - isolation & purification; LISTERIA MONOCYTOGENES; Listeria monocytogenes - genetics; Listeria monocytogenes - isolation & purification; Nucleic Acid Hybridization; PODER PATOGENO; POUVOIR PATHOGENE; Species Specificity; Molecular hybridization; Specificity; Blotting assay; Listeria monocytogenes; Bacteria; Colony; Chromosome DNA; Molecular probe; Chemical labelling; Detection; Nucleic acid
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.57.1.289-294.1991

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species