DNA extraction was performed at the Modena RRL using the ELITe STAR 200 Extraction kit (ELITechGroup S.p.A, Torino, Italy). DNA extracts were split in two aliquots to be analysed by real-time PCR at the Modena RRL and at the National Reference Laboratory at the Istituto Superiore di Sanità in Rome.
The Modena RRL analysed 5μL of DNA with the CE IVD marked real-time PCR commercial kit Legionella pn. Q-PCR Alert (ELITechGroup, CE IVD marked) detecting for Lp mip gene, according to the manufacturer’s instructions on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, California (CA), US). The NRL also analysed 5μL of DNA using an in-house real-time PCR assay in a final volume of 20μL, containing 10μL of EXPRESS qPCR SuperMix, (Invitrogen, Carlsbad, CA, US), with Chromo 4 BioRad instrument (Bio-Rad, Hercules, CA, US), updated to CFX-96, and the following thermal protocol: 5 minutes at 95°C followed by 45 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 60 °C for 15 seconds. Primers and probes were as described by Mentasti et al. [14 (link)], targeting mip and wzm genes for detection of Lp (sg1–15) and sg1 marker, respectively. Primers and probes for internal control DNA were also as already described [14 (link)].
The Modena RRL analysed 5μL of DNA with the CE IVD marked real-time PCR commercial kit Legionella pn. Q-PCR Alert (ELITechGroup, CE IVD marked) detecting for Lp mip gene, according to the manufacturer’s instructions on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, California (CA), US). The NRL also analysed 5μL of DNA using an in-house real-time PCR assay in a final volume of 20μL, containing 10μL of EXPRESS qPCR SuperMix, (Invitrogen, Carlsbad, CA, US), with Chromo 4 BioRad instrument (Bio-Rad, Hercules, CA, US), updated to CFX-96, and the following thermal protocol: 5 minutes at 95°C followed by 45 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 60 °C for 15 seconds. Primers and probes were as described by Mentasti et al. [14 (link)], targeting mip and wzm genes for detection of Lp (sg1–15) and sg1 marker, respectively. Primers and probes for internal control DNA were also as already described [14 (link)].