Mastercycler nexus pcr thermal cycler

Droplet digital PCR was performed as described.^{21 (link)} In brief, each reaction contained 11 μl 2x ddPCR Supermix for Probes (Bio-Rad), 2 μl target forward and reverse primer mix (20 μM), 1 μl target TaqMan Probe (FAM, 5 μM), 2 μl CCR5 internal control forward and reverse primer mix (20 μM), 1 μl CCR5 internal control TaqMan Probe (HEX, 5 μM), 100 ng genomic DNA, and water to a final volume of 22 μl. Droplets were generated on a QX200 Droplet Generator per the manufacturer’s instruction (Bio-Rad). PCR reactions were performed on a Mastercycler Nexus PCR Thermal Cycler (Eppendorf) using a thermal cycling program consisting of 40 cycles of denaturation at 95°C for 30 s and annealing/elongation at 57°C for 1 min. The droplets were then detected and analyzed by a QX200 Droplet Reader (Bio-Rad). Sequences of all primers and probes are listed in Supplementary Table S2. Primer and probe combinations for ddPCR reactions are listed in Supplementary Table S3.

The genomic DNA extraction process has been described in detail in our previous study [16 (link)]. Quawell Q5000 UV–Vis Spectrophotometer (Quawell Technology, Inc., San Jose, CA, USA) was used to determine DNA concentration and purity. Polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) analysis was used for the identification of LRP2 rs2228171 c.8614G > A and CUBN rs1801222 c.758 T > C polymorphisms, as described elsewhere [21 (link)], with identical PCR conditions except for an annealing temperature of 57 °C for both PCR reactions, using FspI (NIPPON Genetics EUROPE, Düren, Germany) and BbsI (New England BioLabs, Hitchin, UK) restriction enzymes, respectively. The opposite strand was genotyped for the detection of both polymorphisms, and thus variant alleles are assigned throughout the manuscript as LRP2 rs2228171C > T and CUBN rs1801222A > G. All PCR–RFLP procedures were carried out in Mastercycler® nexus PCR Thermal Cycler (Eppendorf SE, Hamburg, Germany).

The amplification of the ssu rRNA gene was performed using a nested PCR as previously described [23 (link),24 (link)]. Briefly, PCR reactions were prepared in total volumes of 25 µL, consisting of 10× PCR buffer 2.5 µL, 25 mM MgSO₄ 1.5 µL, 10 mM dNTPs 0.5 µL, 10 pmol of primers 1 µL, 0.5 unit of Taq DNA polymerase 0.5 µL (ABM Good, Richmond, Canada), 5% dimethyl sulfoxide (DMSO) 1 µL (DMSO, Carl Roth, Karlsruhe, Germany), and 2 µL of template genomic DNA. Sterile distilled water was used as a negative control and a positive control was extracted from a G. duodenalis trophozoite-positive fecal sample. A Mastercycler nexus PCR thermal cycler (Eppendorf, Hamburg, Germany) was used for PCR amplification. The first PCR amplification consisted of initial degeneration at 94 °C for 3 min, followed by 35 cycles of degeneration at 94 °C for 45 s, annealing at 58 °C for 45 s and extension at 72 °C for 1 min, with a final extension for 1 cycle at 72 °C for 3 min. The nested PCR conditions were same as the first conditions.