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J Med Microbiol 57 (2008), 50-57; DOI: 10.1099/jmm.0.47216-0
© 2008 Society for General Microbiology
ISSN 1473-5644

Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia

Natsu Uemura1,2, Koichi Makimura1, Masanobu Onozaki3, Yoshihito Otsuka4, Yasuhiro Shibuya5, Hirohisa Yazaki6, Yoshimi Kikuchi6, Shigeru Abe1 and Shoji Kudoh2

1 Teikyo University Institute of Medical Mycology, 539 Otsuka, Hachioji, Tokyo 192-0395, Japan

2 Department of Pulmonary Medicine/Infection and Oncology, Nippon Medical School, Tokyo, Japan

3 Kanto Chemical Co. Inc., Tokyo, Japan

4 Social Insurance Central General Hospital, Tokyo, Japan

5 Tokyo Metropolitan Hiroo General Hospital, Tokyo, Japan

6 AIDS Clinical Center, International Medical Center of Japan, Tokyo, Japan

Correspondence
Natsu Uemura
s3016{at}nms.ac.jp

Received 7 February 2007
Accepted 13 September 2007

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 °C, was capable of detecting 50 copies per tube (2x103 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.

Abbreviations: BALF, bronchoalveolar lavage fluid; BLF, bronchial lavage fluid; HIV, human immunodeficiency virus; LAMP, loop-mediated isothermal amplification; PCP, Pneumocystis pneumonia.

The GenBank/EMBL/DDBJ accession number for the 18S rRNA gene sequence of Pneumocystis jirovecii reported in this paper is AB266392.






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