Absolute quantification program

The leptospiral burden in blood and urine was determined by quantitative real-time PCR (qPCR), as described [40 (link)]. The Maxwell 16 automat was used to extract total DNA from 50 μl of blood and from a drop of urine, using the Maxwell blood DNA and cell LEV DNA purification kits (Promega), respectively. Primers and probe designed in the lpxA gene of L. interrogans strain Fiocruz L1-130 [4 (link)] were used to specifically detect pathogenic Leptospira spp. [40 (link)]. qPCR reactions were run on a Step one Plus real-time PCR apparatus using the absolute quantification program (Applied Biosystems), with the following conditions for FAM TAMRA probes: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 15 s and annealing temperature 60°C for 1 min, according to the manufacturer’s instructions.

The leptospiral load in blood, urine and organs was determined by quantitative real-time PCR (qPCR), as described (22 (link)). Total DNA from blood and urine (around 50 μL) was extracted using a Maxwell 16 automat with the Maxwell blood DNA and cell LEV DNA purification kits (Promega), respectively. DNA was extracted with the QIAamp DNA kit (Qiagen) from organs mechanically disrupted for 3 min at 4°C with metal beads using an automat (Labomodern). Primers and probe designed in the lpxA gene of L. interrogans strain Fiocruz L1-130 (4 (link)) were used to specifically detect pathogenic Leptospira sp. (22 (link)), using the nidogen gene for normalization in kidneys. qPCR reactions were run on a Step one Plus real-time PCR apparatus using the absolute quantification program (Applied Biosystems), with the following conditions for FAM-TAMRA probes: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 15 s and annealing temperature 60°C for 1 min.