Enhancement ofLegionella pneumophilaCulture Isolation from Microenvironments by Macrophage Infectivity Potentiator (mip) Gene-Specific Nested Polymerase Chain Reaction
| Autor: | Kanokrat Siripanichgon, Fuangfa Utrarachkij, Rungrat Nintasen, Yasuhiko Suzuki, Orasa Suthienkul, Adisak Bhumiratana |
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| Rok vydání: | 2007 |
| Předmět: |
Legionella
Molecular Sequence Data Immunology EcoRI Polymerase Chain Reaction Sensitivity and Specificity Microbiology Legionella pneumophila Viable but nonculturable Bacterial Proteins Virology Environmental Microbiology Electrophoresis Agar Gel Infectivity Base Sequence biology Peptidylprolyl Isomerase Amplicon biology.organism_classification Molecular biology Culture Media respiratory tract diseases genomic DNA biology.protein Water Microbiology Sequence Alignment Nested polymerase chain reaction |
| Zdroj: | Microbiology and Immunology. 51:777-785 |
| ISSN: | 0385-5600 |
| DOI: | 10.1111/j.1348-0421.2007.tb03967.x |
| Popis: | The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3' proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments. |
| Databáze: | OpenAIRE |
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