Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB).
The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.
Ko
Kwan Soo
KS
Department of Microbiology and Cancer Research Institute, SNUMRC, Seoul National University College of Medicine, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-799, South Korea.
Hong
Seong-Karp
SK
Lee
Keun-Hwa
KH
Lee
Hae Kyung
HK
Park
Mi-Yeoun
MY
Miyamoto
Hiroshi
H
Kook
Yoon-Hoh
YH
D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D016952:Legionella pneumophila / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D007877:Legionnaires' Disease / Q000175:diagnosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D014871:Water Microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit