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Chonnam Med J. 2000 Jun;36(2):139-147. Korean. Original Article.
Jung DW , Lee HC , Chay KO , Chung JK , Ha DR , Ryu PY .
Department of Microbiology, Chonnam National University Medical School, Kwangju, Korea.
Department of Biochemistry, Chonnam National University Medical School, Kwangju, Korea.
Division of Microbiology, Kwangju City Health and Environment Institute, Kwangju, Korea.
Abstract

BACKGROUND: Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia, and the nonpneumonic syndrome Pontiac fever. Isolation of L. pneumophila from the patient specimens and environment is fastidious and time-consuming. By using Taq polymerase, DNA amplification of L. pneumophila-specific sequences may thus represent an interesting tool for detectection of L. pneumophila from various types of samples. The aim of this study was to determine the proper condition of polymerase chain reaction (PCR) of L. pneumophila. METHODS: Two sets of primers were used in this study, The first set (Lmip-1 and Lmip-2) was specific for macrophage infectivity potentiator (mip) gene of L. pneumophila and the second set (L5SR9 and L5SR93) was specific 5S rRNA of Legionella spp. After an initial predenaturation step at 95 degrees C for 1 min, each amplification cycle was performed as follows: 1 min for annealing at 62 degrees C, 1 min for elongation at 72 degrees C, 0.5 min for denaturation at 94 degrees C and the last cycle was followed by a post-elongation step at 72 degrees C for 5 min. RESULT: When the reaction mixture(50microliterl) contained 1.5 mM MgCl2, 1.0 unit DynaZyme DNA polymerase, 40microM dNTP and 0.2microM each primer (Lmip-1 and Lmip-2), this system detected as few as 5 X 10(2) CFU of L. pneumophila in pure culture and as little as 0.4 ng of purified chromosomal DNA. Another pair of primers, L5SR9 and L5SR93 were adopted into the system with the same reaction conditions except annealing temperature at 55 degrees C instead of 62 degrees C to amplify 104 bps DNA fragment specific to 5S rRNA gene from Legionella spp. For the primers L5SR9 and L5SR93, the amplified DNA bands were detected from the most of Legionella spp. examined. But the band was unique to L. pneumophila for the primers Lmip-1 and Lmip-2. The amplified DNA bands were detected from environmental isolates cultured and identified serologically. CONCLUSION: These results may be used in rapid and direct detections of Legionella spp. from clinical and environmental specimens.

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