Table 4. Overview of the different DNA extraction and NAAT protocols used in the HEMQAS pilot study.
The number between brackets corresponds to the number of laboratories. ITS-1, intergenic transcribed spacer-1; ITS-2, intergenic transcribed spacer-2; 18S rRNA, 18S ribosomal ribonucleic acid; 28S rRNA, 28S ribosomal ribonucleic acid.
| DNA extraction |
|---|
| Bead beating: Yes (12), No (3) |
| Type of beads: MagNA Lyser Green Beads (Roche) (1); Garnet beads 0.7 mm (SanBio) (1); Zirconia/silica beads 0.5 mm (Daintree Scientific) (1); Silica beads (1); Included in FastDNA SPIN kit for Soil (MP Biomedicals) (4); Mixture of ceramic and silica particles (1); Included in ISOLATE II Faecal DNA extraction kit (Bioline) (1); Zirconium beads 1.5 mm (Benchmark Scientific) (1); Included in Precellys Lysing Kit (Bertin technologies) (1) |
| Bead beater: TissueLyser LT (Qiagen) (1); FastPrep (MP Biomedicals) (6); Mini-Beadbeater (Biospec Products) (1); Beadbug (Benchmark Scientific) (1); Precellys 24 Tissue Homogenizer (Bertin technologies) (1); Vortex (2) |
| Proteinase K pretreatment: Yes (4), No (11) |
| Freeze-thaw cycle: Yes (5), No (10) |
| DNA extraction kit: MagnaPure (Roche) (1); Isolate II Faecal DNA Kit (Bioline) (1); Maxwell (Promega) (1); Maxwell RSC PureFood GMO and Authentication kit (Promega) (1); FastDNA SPIN Kit for Soil (MP-Biomedicals) (5); QIAamp DNA Mini Kit (Qiagen) (1); DNeasy Blood and Tissue Kit (Qiagen) (2); QIAamp Stool Kit (Qiagen) (1); DNeasy PowerSoil Kit (formerly Powersoil kit sold by MO BIO) (3); QIA Symphony (Qiagen) (1) |
| NAAT |
| NAAT technology: qPCR (15) |
| qPCR format: Singleplex (9), Multiplex (6) |
| Detection: Hydrolysis probes (15) |
| Master mix: Qiagen (1); Quantitect (Qiagen) (2); HotStarTaq MasterMix (Qiagen) (2); Applied Biosystems (4); VWR (1); Roche (1); Promega (1); Platinum qPCR Supermix-UDG with ROX (Thermofisher) (1); TaqPath ProAmp Master (Thermofisher) (1); Technologies Brilliant II QPCR Master Mix (Agilent) (1) |
| Stool volume used for DNA extraction (mean, range): 223 μl, 50–500 μl |
| Elution volume (mean, range): 109 μl, 50–200 μl |
| DNA used for NAAT (mean, range): 3.2 μl, 1–10 μl |
| Stool volume used per NAAT reaction (mean, range): 8.6 μl, 1.5–36.4 μl |
| Ascaris primer and probe target: ITS-1 (10), non-coding repetitive DNA sequence (2) |
| Trichuris primer and probe target: 18S rRNA (4), 28S rRNA (1), ITS-1 (1), non-coding repetitive DNA sequence (6) |
| Necator primer and probe target: ITS-1 (1), ITS-2 (6), non-coding repetitive sequence (5) |
| Schistosoma primer and probe target: 28S rRNA (1), cytochrome c oxidase (1), non-coding repetitive DNA sequence (1), ITS-2 (4) |
| Strongyloides primer and probe target: 18S rRNA (8), non-coding repetitive sequence (5) |