|T. Annaka, T. Kojima, M. Yoshino, T. Momoda, J. Nemoto, A. Sunada, S. Sadamoto, M. Ikedo
(Eiken Chemical Co., Ltd.)
Y. Ishii, K. Yamaguchi
(Toho Univ. School of Medicine)
Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. Rapid and accurate detection of Legionella spp. is important for diagnosis and treatment of patients. In order to detect Legionella spp., a new DNA amplification method was designed and evaluated. Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity under isothermal conditions at 65oC (1). This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.
The primers targeting 16S rRNA gene were designed in order to detect a wide range of Legionella species. The LAMP was carried out in a reaction mixture containing Bst DNA polymerase large fragment and its appended buffer, primers, dNTPs and betaine. The LAMP reaction was monitored in real-time on the ABI PRISM 7700 system in the presence of fluorescent intercalating dye. The specificity of this LAMP method was tested by using extracted DNA of 13 Legionella strains including L. pneumophila serogroups 1-6, L. bozemanii, L. micdadei, L. dumoffii, L. gormanii and L. longbeachae, and 7 non-Legionella strains related community-acquired pneumonia, as A. xylosoxidans, E. coli, P. aeruginosa, H. influenzae, P. fluorescens, K. pneumoniae and S. epidermidis.
With regard to 13 strains of Legionella spp., the significant amplification was observed within 30 minutes and completed for 60 minutes incubation. On the other hand, 7 of non-Legionella strains were not amplified for 120 minutes incubation.
The LAMP method was able to detect a wide range of Legionella species with high specificity, rapidity and simple procedure. This method would be applied to detect Legionella spp. in specimens from patients with pneumonia.
(1) Notomi,T. et al.2000.Nucleic Acids Research.28:e63.
American Society for Microbiology (2002.5.22;Salt Lake city)