Nuclease free water

Species-specific primers [9 ] were used (Table 2) to amplify cell-free repeat DNA fragments from filtered urine samples to confirm the presence of either S. mansoni or S. haematobium or both. For every reaction, three different controls were used: 1) S. mansoni and S. haematobium genomic DNA (BEI Resources, Manassas, VA, USA) as positive control; 2) extracted DNA from urine collected in-house as negative control; and 3) nuclease-free-water as water control (Sigma-Aldrich, St. Louis, MO, USA). A commercially purchased Mastermix (New England Biolabs, Ipswich, MA, USA), 10μM forward and reverse primers, magnesium, and nuclease-free-water (Sigma-Aldrich, St. Louis, MO, USA) was used for a 20μl PCR reaction. PCR amplification took place in an automated thermocycler with the following settings. For amplification of S. mansoni, the denaturation at 95°C for 10 minutes was followed by annealing process of 57°C for 90 seconds, which was repeated 35 times and ended with an extension at 72°C for 10 minutes. For the amplification of S. haematobium, the steps were same for denaturation expect that the annealing process was set at 56°C for 90 seconds and final extension of 72°C for 1 minute.

Genomic DNA was extracted from a total of 721 pools of mosquitoes samples (adult and larvae) using the phenol/chloroform extraction method described by Sambrook and Russell [35 (link)]. Several types of controls were put in place to guide against false positive and negative results. To reduce cross-contaminations, extractions were conducted in batches of 10 pools and the 10 pools completely processed (extraction and PCRs) before moving back to a new set of extractions. Negative controls (nuclease-free water, Sigma-Aldrich) were added at a frequency of 10% (1 control per batch of extraction) to monitor potential cross-contaminations. Pooled mosquitoes samples were ground using an electric grinder in sterile 100 µl 1x PBS and the homogenates were suspended in 300 µl preheated lysis buffer made of 5 M NaCl, 0.5 M EDTA, 1 M Tris-HCl (pH 8.0), 10% SDS, and proteinase K (Qiagen, Hilden). The mixture was heated at 60°C for one hour and DNA extracted with phenol/chloroform/isoamyl acid in the ratio 25 : 24 : 1. This was briefly mixed by a pulse vortex and centrifuged for 2 min at 13,000g. The DNA was precipitated by adding 2 volumes of pure ethanol and the mixture was incubated for 2 hours and centrifuged 10 min at 13,000g. The DNA was washed by 70% cold ethanol, dried 20 min at room temperature, and eluted in 50 µl of nuclease-free water (Sigma-Aldrich).