Mgcl2

The haspB PCR for K26 antigen detection was used as species screening for L. donovani by primers K26f (5′-ACGAAGGACTCCGCAAAG-3′) and K26r (5′-TTCCCATCGTTTTGCTG-3′) [18 (link)]. The PCR master mix was prepared in 50 µl containing 1X Qiagen PCR buffer, 1.5 mM MgCl₂, 200 µm of each dNTP (Eurogentec), 0.5 µM of each primer (Sigma-Aldrich), and 1 U of HotStar Taq polymerase. Amplification was done with (i) initial denaturation step of 95°C for 5 minutes; (ii) 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 60 seconds; and (iii) a final extension at 72°C for 10 minutes. Out of two haspB genotypes identified, genotype A has amplicon size of 640 bp and genotype B has amplicon size of 320 bp.

DNA was extracted using the UltraClean^TM Soil DNA isolation kit (MoBio Laboratories, Inc., Carlsbad, CA, United States) and amplified by PCR using the 16S rRNA gene universal eubacterial primers E338F (5′-ACTCCTACGGGAGGCAGCAGT-3′) and E797R (5′-GGGTATCTAATCCTG-3′) (amplicon length of 459-bp covering the variable V3 region of the 16S rRNA gene). The PCR mixtures were prepared with 2.5 μl 10x PCR buffer (Eurogentec, Seraing, Belgium), 1 μl MgCl₂ (50 mM; Eurogentec), 2.5 μl dNTPs (2 mM; Thermo Scientific, Waltham, MA, United States), 1.0 μl of each primer (20 pmol μl^-1 each; Eurogentec), 0.2 μl Silverstar Taq DNA polymerase (5.0 U μl^-1; Eurogentec) and 1 μl standardized template DNA (10.74 ng μl^-1) in a total reaction volume of 25 μL. The PCR was performed with the follow temperature/time conditions: initial denaturation at 94°C for 2 min, 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and a final extension for 5 min (72°C). PCR products were evaluated on gel electrophoresis, the bands then were excised and purification was performed using a Qiaquick Gel extraction kit (Qiagen, Venlo, Netherlands). For the quantification of the PCR products, we used the PicoGreen dsDNA Assay Kit (Life Technologies, United States). Sequencing was performed at the Genomics Core of KU Leuven (Belgium) using a Roche 454 GS FLX+ (Switzerland).