DNA was extracted from each tissue sample using the Quick-DNA Plus Kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions. All samples were tested by multiplex Real-Time PCR, identifying the Leptospira genus-specific target located on 16S rRNA gene and the specific target for pathogenic species (lipL32 gene) [32 (link),33 (link)]. The Real-Time PCR assay was performed on a Rotorgene Corbett 6000 (Corbett Research, Sidney, Australia) with the following thermal conditions: a holding stage of 95°C for 5 min, 45 cycles of 95°C for 15 sec, and 60°C for 30 sec. Samples were considered positive with a Ct < 40.
Finally, from PCR-positive tissue samples, Leptospira species were identified using a primer for the rrs2 gene [34 (link)]. Amplification of each target gene was carried out using a HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany). Amplicons were further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [35 ]. Phylogenetic analysis based on the rrs2 gene was performed using the Maximum Likelihood method based on the Tamura-Nei model using MEGA 10 software [36 (link)].
Finally, from PCR-positive tissue samples, Leptospira species were identified using a primer for the rrs2 gene [34 (link)]. Amplification of each target gene was carried out using a HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany). Amplicons were further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [35 ]. Phylogenetic analysis based on the rrs2 gene was performed using the Maximum Likelihood method based on the Tamura-Nei model using MEGA 10 software [36 (link)].
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