Sterile 5 mm steel bead

DNA was extracted from placenta, fetal lung, liver, and kidney. Kidney was included in order to screen for the presence of Leptospira spp. DNA by sequencing and PCR. Tissue samples were thawed, and 20 mg of each tissue was transferred to a sterile plastic tube using a sterile scalpel and sterile forceps. Lung and liver samples were pooled by transferring 10 mg of each tissue into one tube. If only lung or only liver was available, 20 mg of the respective tissue was used. Samples were incubated at 37°C for 30 min in 300 mL lysozyme buffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X and 5 mg lysozyme per 100 mL) and 350 mL of lysis buffer (Promega, Madison, WI, USA), and a sterile 5-mm steel bead (Qiagen, Hilden, Germany) was added, and the tissue was homogenized using a TissueLyser II (Qiagen) at 20 Hz for 2 min. Protease K was added, and the sample was incubated at 56°C for 1 h. DNA was extracted on a Maxwell 16 Research Instrument System (Promega), using a Maxwell LEV Blood DNA Purification Kit (Promega). In order to control for potential contamination with 16S rDNA possibly originating from reagents or instruments, an extraction control was added for every 15 samples; i.e., a sterile plastic tube without tissue was processed in the same way and with the same reagents as the 15 samples.