Research Article: Approach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments

Date Published: February 6, 2017

Publisher: Public Library of Science

Author(s): Wen-Chien Huang, Hsin-Chi Tsai, Chi-Wei Tao, Jung-Sheng Chen, Yi-Jia Shih, Po-Min Kao, Tung-Yi Huang, Bing-Mu Hsu, Ulrich Nübel.


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.

Partial Text

In developing countries, about 80% of diseases and one-thirds of fatal cases are associated with waterborne pathogens [1]. As a potential threat to public health, Legionella species are omnipresent in the natural aquatic environments, for example, river, lake, hot spring, and drinking water. Under specific environmental conditions, the density of these microorganisms can increase rapidly, as agreed to causing outbreaks of disease. Various systems (e.g., water supplies, cooling towers, hydrotherapic establishments, spa) provide ideal growth conditions and thus represent a worrying source of exposure for humans.

Nested amplification, a Legionella-like product was obtained from the river water sample. Semi-nested, nested and double PCR amplification protocols were used previously to improve the sensitivity of detection [10–15]. Furthermore, the nested PCR method, using degenerate and specific primers were found to increase the detection sensitivity of 100 copies of a gene per reaction [15]. The PCR products gained by the two and three step nested PCR process indicated that members of the Legionella were dominant in the river water sample. However, Legionella might be present in low concentrations, as these were detected only after by nested PCR method. The improved detection signal strength in the nested PCR may possibly be owing to the first round resulting in the PCR amplification of enough quantities of DNA level from the Legionella present in low concentrations and also owing to the dilution of inhibitory materials such as urea, calcium ions, humic acids, and melanin present in the water sample [16–18].

Each species of Legionella was found to have its own bands pattern after DGGE. The three step nested PCR-DGGE method made it a very useful to study the diversity of Legionella with high-resolution in low concentration in river water samples. Moreover, the specificity of the PCR primer pairs targeting various phylogenetic tree of Legionella is of primary significance for the achievement of this method. With advantages such as cloning-independence, economic, and the high-throughput procedure, DGGE may be potential for rough specie of unknown Legionella or even other pathogens in a great amount of environmental samples.