First, serogroups of the isolates were determined through the MAT using a panel of eight polyclonal anti-sera against the eight serovars reported in Section 4.2 . The agglutination with specific antiserum was used to identify the presumptive strain’s serogroup [84 ].
Isolated Leptospira were genotyped using a multilocus sequence typing (MLST) scheme based on housekeeping genes [87 (link),88 (link),89 (link)].
Moreover, the Leptospira species were identified from positive pathogenic and intermediate Leptospira PCR reactions, using primer for rrs2 gene and 16S rRNA gene, respectively [86 (link),88 (link)].
The amplification of each target gene was realized with HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany), and further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [90 ]. Phylogenetic analysis was performed by the maximum likelihood method based on the Tamura–Nei model using MEGA 10 software [91 (link)].
Isolated Leptospira were genotyped using a multilocus sequence typing (MLST) scheme based on housekeeping genes [87 (link),88 (link),89 (link)].
Moreover, the Leptospira species were identified from positive pathogenic and intermediate Leptospira PCR reactions, using primer for rrs2 gene and 16S rRNA gene, respectively [86 (link),88 (link)].
The amplification of each target gene was realized with HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany), and further sequenced (BMR Genomics, Padova, Italy) using the same amplification primer sets and analyzed using BioEdit Software [90 ]. Phylogenetic analysis was performed by the maximum likelihood method based on the Tamura–Nei model using MEGA 10 software [91 (link)].
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