Optimized Nucleic Acid Extraction for Parasite DNA

For the initial diagnostic assessment, nucleic acid from all samples had been extracted using the QiaAMP DNA Stool Mini Kit (Qiagen, Hilden, Germany) as described by the manufacturer without particular focus on specialized procedures for parasite DNA (later referred to as “without bead beating”). If any helminth real-time PCR signal had been detectable in the initial diagnostic process and residual stool material was still available stored at −80 °C, nucleic acid from these residual materials were extracted using polyvinylpyrrolidone pretreatment, bead-beating using garnet beads (0.7–1.2 mm, Biolabproducts, Bebensee, Germany) with a TissueLyser LT device (Qiagen) for 3 min at 30 s⁻¹, proteinase K digestion, and QiaAMP spin column extraction (Qiagen) as described previously [33 (link)]. This procedure is later referred to as “with bead beating”. Identical elution volumes as in the QiaAMP DNA Stool Mini Kit-based approach were achieved. To ensure sufficient sample volumes for the real-time PCR assays, photometric assessment of DNA concentrations within the samples using a Pico 100 Picodrop Microliter Spectrophotometer (Picodrop Ltd., Hinxton, UK) according to the manufacturer’s instructions was only performed from remaining residual DNA samples after the real-time PCRs had been completed.