Single and duplex fluorescence quantitative PCR for rapid detection of Legionella

Abstract

Objective To develop the single and duplex fluorescence quantitative PCR for detection of Legionella and Legionella pneumophila respectively.Methods Specific primers and probes were designed according to the 16 S rRNA gene of Legionella and mip gene of Legionella pneumophila.The probes were labeled with reporter genes, FAM (for 16 S rRNA) and HEX (for mip gene) respectively.Moreover, the reaction systems and conditions for PCR were optimized.Then the single gene probe (single fluorescence quantitative PCR) and duplex gene probe (duplex fluorescence quantitative PCR) were used to detect the pneumophila and non-Legionella pneumophila strains, as well as other non-Legionella, in order to validate the specificity and sensitivity of these two methods.In addition, duplex fluorescence quantitative PCR was employed to detect Legionella on filtering membrane and extracted Legionella DNA in water samples from air - conditioners, to compare the consistency between the both.Results The 16 S rRNA genes of Legionella and mip genes of Legionella pneumophila were well detected with fluorescence quantitative PCR,with the lowest detectable templates being 8 and 10 copies respectively.An ideal reaction system after optimization of conditions was established successfully.Single fluorescence quantitative PCR demonstrated positive expression of 16 S rRNA gene in all of 8 Legionella pneumophila and 4 nonLegionella pneumophila strains, and positive expression of mip gene in Legionella pneumophila but not in non- Legionella pneumophila strains.Duplex fluorescence quantitative PCR demonstrated 2 false negative strains among 23 Legionella pneumophila strains and 1 false positive strain among 9 nonLegionella pneumophila and non - Legionella strains.The results of DNA detection were consistent between 49 filtering membranes and extracted DNA in water samples from air-conditioners.There were 26 water samples tested positive for Legionella, including 20 for Legionella pneumophila and 6 for nonLegionella pneumophila strains.One Francisella strain was found to be HEX-positive (false positive) ,accounting for 1/26 of the cultures actually isolated.Conclusions The single and duplex fluorescence quantitative PCR are specific, rapid and sensitive methods that detect pneumophila and non- Legionella pneumophila strains at the same time.These tests may meet the need to detect Legionella in water samples from air-conditioners or natural environment. Key words: Polymerase chain reaction; Fluorescence; Legionella; 16 S rRNA gene; mip gene