Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia.
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.
Uemura
Natsu
N
Department of Pulmonary Medicine/Infection and Oncology, Nippon Medical School, Tokyo, Japan.|Teikyo University Institute of Medical Mycology, 539 Otsuka, Hachioji, Tokyo 192-0395, Japan.
Makimura
Koichi
K
Teikyo University Institute of Medical Mycology, 539 Otsuka, Hachioji, Tokyo 192-0395, Japan.
Onozaki
Masanobu
M
Kanto Chemical Co. Inc., Tokyo, Japan.
Otsuka
Yoshihito
Y
Social Insurance Central General Hospital, Tokyo, Japan.
Shibuya
Yasuhiro
Y
Tokyo Metropolitan Hiroo General Hospital, Tokyo, Japan.
Yazaki
Hirohisa
H
AIDS Clinical Center, International Medical Center of Japan, Tokyo, Japan.
Kikuchi
Yoshimi
Y
AIDS Clinical Center, International Medical Center of Japan, Tokyo, Japan.
Abe
Shigeru
S
Teikyo University Institute of Medical Mycology, 539 Otsuka, Hachioji, Tokyo 192-0395, Japan.
Kudoh
Shoji
S
Department of Pulmonary Medicine/Infection and Oncology, Nippon Medical School, Tokyo, Japan.
D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005453:Fluorescence; D006358:Hot Temperature; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011010:Pneumocystis / Q000235:genetics / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*
D017931:DNA Primers; D004269:DNA, Bacterial