All clinical samples from patients with cutaneous leishmaniasis were extracted using a modified protocol for the “Puregene tissue kit” from Qiagen (Protocol: “DNA Purification from Tissue Using the Gentra Puregene tissue kit”, Gentra Puregene Handbook 12/2014). Briefly, the tissue was placed into 300 µl CLS and 1.5 µl Puregene proteinase K (20 mg/ml) was added for incubation at 55 °C overnight. Subsequently, 100 µl Protein Precipitation Solution was added on ice and after a centrifugation step, the supernatant was poured to 300 µl isopropanol. After centrifugation the DNA pellet was washed in 70% ethanol, air dried, and 50 µl DNA hydration solution was added to dissolve the pellet.
All other samples (including culture supernatants in 700 µl CLS) were extracted employing the Gentra Puregene method (Qiagen) as previously described [16 (link)].
All extractions included extraction controls for the subsequent (q)PCR as routinely conducted in the accredited laboratories of DITM.
All other samples (including culture supernatants in 700 µl CLS) were extracted employing the Gentra Puregene method (Qiagen) as previously described [16 (link)].
All extractions included extraction controls for the subsequent (q)PCR as routinely conducted in the accredited laboratories of DITM.
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