期刊
JOURNAL OF MICROBIOLOGICAL METHODS
卷 67, 期 3, 页码 408-415出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2006.04.012
关键词
Legionella; nucleic acid sequence-based amplification; respiratory samples
Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneuniophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10 000 CFU of L. pneumophila serotype I depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneuniophila serotype I could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All I I PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneuniophila real-time NASBA and Legionella spp. real-time NASBA, respectively. (c) 2006 Elsevier B.V. All rights reserved.
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