Method Description

This method employs a target-specific detection system using fluorescent resonance energy transfer (FRET) hybridization probes designed for a specific sequence found within the Legionella 5S ribosomal RNA gene. The LightCycler (LC) instrument amplifies and monitors target nucleic acid sequences by fluorescence during polymerase chain rection (PCR) cycling. This is an automated PCR system that can rapidly detect amplified product development through stringent air-controlled temperature cycling and capillary cuvettes. The detection of amplified products is based on the FRET principle. For FRET product detection, a hybridization probe with a donor fluorophore, fluorescein, on the 3' end is excited by an external light source, which emits light that is absorbed by a second hybridization probed with an acceptor fluorophore, LC-Ted 640, on the 5' end. The acceptor fluorophore then emits light of a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product. The detection process is completed in under an hour using a closed tube system.(Cunningham SA, Sloan LM, Uhl JA, et al: Validation of a real-time PCR assay for the detection of Legionella species in respiratory samples. Abstracts of the Annual Meeting of the Association for Molecular Pathology, 2009 General Meeting, Nov. 19-22, 2009; Rucinski SL, Murphy MP, Kies KD, Cunningham SA, Schuetz AN, Patel R. Eight years of clinical Legionella PCR testing illustrates a seasonal pattern. J Infect Dis. 2018 Jul 13;218(4):669-670. doi: 10.1093/infdis/jiy201)