Real-Time PCR amplification of Leishmania DNA

Parasite load was determined by qPCR in lymph nodes. DNA was extracted using the High Pure PCR Template Preparation Kit (Roche). L. infantum DNA was specifically detected and quantified with a TaqMan qPCR Assay (Applied Biosystems) following a previously reported protocol [40] with some modifications. The qPCR assay was designed to target conserved DNA regions of the kinetoplast from L. infantum genome. Primer sequences were LEISH-1 5′-AAC TTT TCT GGT CCT CCG GGT AG-3′, LEISH-2 5′-ACC CCC AGT TTC CCG CC-3′, and the TaqMan-MGB probe FAM-5′-AAA AAT GGG TGC AGA AAT-3′- MGB. The thermal cycling profile was 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. Analyses were performed in a StepOnePlus Real Time PCR System device (Applied Biosystems). Each sample plus a negative control was analysed in triplicate. Parasite quantification was performed by comparison with a standard curve generated with L. infantum DNA extracted from 1 × 10⁷ parasites by using serial dilutions from 10³ to 10⁻³ parasites. This technique was sensitive enough to detect 0.001 parasites per reaction with a dynamic range of 10⁷. The median slope of three different standard curves was −3.44, and the qPCR efficiency was 98%. Quantification was linear between 10³ and 10⁻² parasites per reaction tube (correlation = 0.99).