Open AccessApproach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments
Author(s) -
WenChien Huang,
Hsin-Chi Tsai,
ChiWei Tao,
JungSheng Chen,
Yi-Jia Shih,
Po-Min Kao,
Tzu-Yuan Huang,
Bing-Mu Hsu
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0170992
Subject(s) - legionella , amplicon , biology , nested polymerase chain reaction , primer (cosmetics) , 16s ribosomal rna , microbiology and biotechnology , polymerase chain reaction , phylogenetic tree , legionella pneumophila , phylotype , library , sequence analysis , gene , genetics , bacteria , chemistry , organic chemistry
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.