For samples with a Ct value > 36 or with incomplete MLST results, a conventional PCR targeting the secY gene (657 bp) was performed to determine Leptospira species as previously described [54 (link)]. As positive control, DNA of a laboratory strain of L. interrogans serovar Icterohaemorrhagiae was used [55 (link)].
PCR products were prepared with DNA Gel Loading Dye (6x) (Thermo Fisher Scientific, Darmstadt, Germany) for gel electrophoresis in 2% agarose, and gels were stained with HDGreen Plus DNA Stain (Intas Science Imaging Instruments GmbH, Göttingen, Germany). Amplification products were visualised by UV light using the UVP GelSolo streamlined gel documentation (Analytik Jena AG, Jena, Germany). The samples were purified for sequencing using a NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) as recommended by the manufacturer. The sequences were trimmed using Bionumerics v.7.6.1. (Applied Maths Inc., Austin, TX, USA) and compared to available data in GenBank with the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi , accessed on 7 August 2022). The obtained sequences were uploaded to GenBank (accession numbers: OQ865429–OQ865435).
PCR products were prepared with DNA Gel Loading Dye (6x) (Thermo Fisher Scientific, Darmstadt, Germany) for gel electrophoresis in 2% agarose, and gels were stained with HDGreen Plus DNA Stain (Intas Science Imaging Instruments GmbH, Göttingen, Germany). Amplification products were visualised by UV light using the UVP GelSolo streamlined gel documentation (Analytik Jena AG, Jena, Germany). The samples were purified for sequencing using a NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) as recommended by the manufacturer. The sequences were trimmed using Bionumerics v.7.6.1. (Applied Maths Inc., Austin, TX, USA) and compared to available data in GenBank with the Basic Local Alignment Search Tool (BLAST) (
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