RRC ID 5175
著者 Uemura N, Makimura K, Onozaki M, Otsuka Y, Shibuya Y, Yazaki H, Kikuchi Y, Abe S, Kudoh S.
タイトル Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia.
ジャーナル J Med Microbiol
Abstract Loop-mediated isothermal amplification (LAMP) is a novel, rapid nucleic acid amplification method with high specificity and sensitivity under isothermal conditions. In this study a LAMP assay for diagnosing Pneumocystis pneumonia (PCP) was developed. Oligonucleotide primers specific for Pneumocystis species were designed corresponding to 18S rRNA gene sequences. The assay, performed for 30 min at 61 degrees C, was capable of detecting 50 copies per tube (2 x 10(3) copies ml(-1)) in 30 min and did not show cross-reactivity to other species of fungi, including the genera Candida, Aspergillus and Cryptococcus. A total of 21 of 24 clinical specimens (sputum and bronchoalveolar lavage fluid) from patients with suspected PCP tested positive using the LAMP assay by real-time fluorescence detection. The results of the LAMP reaction were also observed by real-time turbidity detection and end-point visual turbidity or fluorescence detection. With real-time fluorescence detection, melting curves of the products were effective at distinguishing specific amplification from non-specific amplification or self-amplification. Visual detection was also possible as a rapid and easy assay using only a heat block and a black light.
巻・号 57(Pt 1)
ページ 50-57
公開日 2008-1-1
DOI 10.1099/jmm.0.47216-0
PMID 18065667
MeSH Bronchoalveolar Lavage Fluid / microbiology* DNA Primers DNA, Bacterial / analysis Fluorescence Hot Temperature Humans Nucleic Acid Amplification Techniques / methods* Pneumocystis / genetics Pneumocystis / isolation & purification* Pneumonia, Pneumocystis / diagnosis* Pneumonia, Pneumocystis / microbiology Polymerase Chain Reaction Sensitivity and Specificity Sputum / microbiology*
IF 2.156
引用数 58
WOS 分野 MICROBIOLOGY
リソース情報
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