Abstract
Although Legionnaires' disease is an issue of global importance, the isolation of Legionella species is a lengthy process requiring incubation on agar plates for more than one week. A method for rapid detection is therefore required to restrict the increasing range of Legionnaires' disease and to control Legionella species. While the nucleic acid amplification test (NAT) has been applied to bacterial screening, it has not yet been applied for the detection of the few Legionella species commonly found in large environmental water bodies like hot springs given the relative limits of the sample volumes that can be processed using NAT. We therefore designed a genetic detection system using a metal-coated flat sheet (MCFS) membrane for Legionella species that operates as follows: The MCFS-membrane is coated with silver on a flat filtration membrane. Legionella species become concentrated on the MCFS-membrane when water from a hot spring is aspirated through the membrane. The genes of the Legionella species that become concentrated in the membrane are then isolated by applying an electric field. Legionella species can then be identified by polymerase chain reaction (PCR) using specific primers. Here, we report the first attempt to detect Legionella species from various hot springs. A 10-μm-pore size MCFS-membrane captured approximately 30% of the Legionella species in the hot spring. Genes were isolated from most of the Legionella species in the MCFS-membrane when an electric field of 20 V/cm was applied to the membrane before PCR amplification. In addition to detecting Legionella species, this technique also facilitated bacterial concentration and genetic isolation. Amplification of DNA was not inhibited by hot spring components or proteins and lipids derived from humans, as these were removed by the MCFS-membrane. The time required for bacterial identification using this method was shortened to four hours.
