Human Infectiology MDx | Catalogue 2011 - Diagenode Diagnostics

LegionellaRef: Dia-Lpn-050Legionella (Gram negative bacterium), including species thatcause legionellosis (most notably Legionella pneumophila) hasbeen shown to cause community-, travel-, and nosocomiallyacquired pneumoniae. Infection of humans is usually via inhalationof aerosols from a variety of natural or water systems (Templeton,Scheltinga et al. 2003).Templeton, K. E., S. A. Scheltinga, et al. (2003). “Development and clinicalevaluation of an internally controlled, single-tube multiplex real-time PCRassay for detection of Legionella pneumophila and other Legionella species.”J Clin Microbiol 41(9): 4016-21.VALIDATIONResearched pathogens:Target genes:• Legionella species: FAM dye, emission: 520 nm• Legionella pneumophila: yellow dye, emission: 549 nm• P1 (Legionella species)• 16S (Legionella pneumophila)Number of wells by test: 1 (Legionella species / Legionella pneumophila)Samples used:• Bronchoalveolar lavage• Nasopharyngeal swabs/washesValidated extraction methods: • Nuclisens easyMAG ® System (Biomerieux)• MagNa pure LC system (Roche)• QIAamp ® DNA Mini Kit (50) (Blood and Body Fluid Spin Protocol) (Qiagen)Real-time PCR systems: • ABI (7000/7300/7500/7900)• Roche LightCycler ® (480/2.0 on request)• Biorad (iCycler/IQ5/CFX96)• Qiagen Rotor-Gene (6000)• Stratagene (MX3000P/3005P)• Cepheid (SmartCycler II)PCR volumes: 50 µl, 25 µl (depending of the real-time PCR system used)PCR reactions/kit:• 50 reactions (50 µl PCR, final volume)• 100 reactions (25 µl PCR, final volume)In combination with:• Universal inhibition control (DIA-UIC-050):DIA-UIC(DR)-050: orange dye, emission: 576 nmDIA-UIC(TR)-050: texas red dye, emission: 603 nmDIA-UIC(Cy5)-050: Cy5 dye, emission: 662 nm• DNA extraction & PCR inhibition control (DIA-EIC/DNA)-050:DIA-EIC/DNA(DR)-050: orange dye, emission: 576 nmDIA-EIC/DNA(TR)-050: texas red dye, emission: 603 nmDIA-EIC/DNA(Cy5)-050: Cy5 dye, emission: 662 nm33