Construction and Application of Duplex Quantification Fluorescence-PCR Assay for Legionella Detection

Objectives  To develop a specific, rapid and sensitive real-time fluorescence quantitative PCR assay for the detection of legionella pneumophila (LP) and for the differentiation of it from other legionella spp. in clinical and environmental samples.

Methods  The primers and TaqMan probes were designed in conservative sequence of LP mip gene and all legionella spp. 23S rRNA gene.Fragments were amplified from the standard strain of LP serotype I by PCR to construct 2 plasmid standards. The PCR reaction conditions and system were optimized.The specificity, sensitivity and reproducibility of the assay were evaluated.Some environmental samples and 80 clinical sputum samples were detected.

Results  The sensitivity of this real-time PCR assay was 10 copies/reaction.This assay proved to be specific and reproducible. The whole process could be done in about 2 hours. Of the 35 environmental samples for testing legionella, 3 were found to be positive for LP and 3 were found to be positive for other legionella spp. All environmental samples tested by both ordinary PCR and culture method were negative. Of 80 clinical sputum samples detected by this assay, 2 were found to be positive for LP, and all others were negative.

Conclusions  The study has developed a highly sensitive and specific TaqMan real-time PCR assay, which can detect both legionella at the genus level and LP. This assay could be used for detecting clinical and environmental samples in small hospitals.