Approach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments.
In this study, we describe a nested PCR-DGGE strategy to detect Legionella communities from river water samples. The nearly full-length 16S rRNA gene was amplified using bacterial primer in the first step. After, the amplicons were employed as DNA templates in the second PCR using Legionella specific primer. The third round of gene amplification was conducted to gain PCR fragments apposite for DGGE analysis. Then the total numbers of amplified genes were observed in DGGE bands of products gained with primers specific for the diversity of Legionella species. The DGGE patterns are thus potential for a high-throughput preliminary determination of aquatic environmental Legionella species before sequencing. Comparative DNA sequence analysis of excised DGGE unique band patterns showed the identity of the Legionella community members, including a reference profile with two pathogenic species of Legionella strains. In addition, only members of Legionella pneumophila and uncultured Legionella sp. were detected. Development of three step nested PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella community. The method is rapid and provided sequence information for phylogenetic analysis.
Department of Medicine, Mackay Medicine College, Taipei, Taiwan, ROC.|Department of Thoracic Surgery, Mackay Memorial Hospital, Taipei, Taiwan, ROC.|Mackay Junior College of Medicine, Nursing, and Management, Taipei, Taiwan, ROC.
Wen-Chien
WC
Huang
School of Medicine Tzu-Chi University, Hualien, Taiwan, ROC.|Department of Psychiatry, Tzu-Chi General Hospital, Hualien, Taiwan, ROC.
Hsin-Chi
HC
Tsai
Section of Respiratory Therapy, Cheng Hsin General Hospital, Taipei, Taiwan, ROC.
Chi-Wei
CW
Tao
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
Jung-Sheng
JS
Chen
Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC.
Yi-Jia
YJ
Shih
Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC.
Po-Min
PM
Kao
Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC.
Tung-Yi
TY
Huang
Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC.
Bing-Mu
BM
Hsu
D058645:Denaturing Gradient Gel Electrophoresis; D007875:Legionella / Q000145:classification* / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D045483:Rivers / Q000382:microbiology; D017422:Sequence Analysis, DNA; D014871:Water Microbiology
D012336:RNA, Ribosomal, 16S