Rapid Identification of Listeria Species by Using Restriction Fragment Length Polymorphism of PCR-Amplified 23S rRNA Gene Fragments
| Autor: | Delphine Paillard, Claudine Quentin, Pierre Caumette, Fany Nathier, Catherine Guittet, Robert Duran, Véronique Dubois |
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| Přispěvatelé: | Institut des sciences analytiques et de physico-chimie pour l'environnement et les materiaux (IPREM), Université de Pau et des Pays de l'Adour (UPPA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2003 |
| Předmět: |
Time Factors
Listeria Molecular Sequence Data medicine.disease_cause Polymerase Chain Reaction Waste Disposal Fluid Applied Microbiology and Biotechnology Microbiology 03 medical and health sciences Listeria monocytogenes 23S ribosomal RNA medicine [CHIM]Chemical Sciences Ribosomal DNA 030304 developmental biology 0303 health sciences Sewage Ecology biology 030306 microbiology Genes rRNA Sequence Analysis DNA biology.organism_classification Molecular biology Bacterial Typing Techniques RNA Ribosomal 23S Restriction enzyme Listeria welshimeri Food Microbiology Listeria seeligeri Restriction fragment length polymorphism Water Microbiology Polymorphism Restriction Fragment Length Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology Applied and Environmental Microbiology, American Society for Microbiology, 2003, 69 (11), pp.6386-6392. ⟨10.1128/AEM.69.11.6386-6392.2003⟩ |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.69.11.6386-6392.2003 |
| Popis: | A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes Xmn I or Cfo I and, if needed, S1 was digested by either Alu I or Cla I. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua . Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua , 4 Listeria seeligeri , and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology. |
| Databáze: | OpenAIRE |
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