Computational protocol: Culture Strategies for Isolation of Fastidious Leptospira Serovar Hardjo and Molecular Differentiation of Genotypes Hardjobovis and Hardjoprajitno

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Protocol publication

[…] To characterize the Leptospira strains, two molecular techniques were used. The MLVA identified isolates with five primer pairs for the VNTR loci 4, 7, 10, LB4, and LB5 as previously described (Salaün et al., ). For each of the five PCRs, the VNTR loci were used as positive controls for the reference strains of L. interrogans serovar Canicola serogroup canicola strain Canicola Hond Utrecht IV, L. interrogans serovar Hardjo serogroup sejroe genotype Hardjoprajitno strain Hardjoprajitno, and L. borgpetersenii serovar Hardjo serogroup sejroe genotype Hardjobovis strain Sponselee. After amplification, the sequencing of secY was used to identify and confirm genetic species, as previously described (Ahmed et al., ).The products of the secY gene amplification were purified with a Purelink Genomic DNA extraction kit (Invitrogen Life Technologies, Eugene, OR, USA), quantified by a Qubit™ Fluorometer (Invitrogen Life Technologies, Eugene, OR, USA), and sequenced on a ABI3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using forward and reverse primers. Sequence quality was analyzed by the Phred program (http://asparagin.cenargen.embrapa.br/phph/). The consensus sequences were obtained by CAP3 software (http://asparagin.cenargen.embrapa.br/cgi-bin/phph/cap3.pl), and the identities were compared with the sequences in GenBank using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The identity matrix was created in the BioEdit program with the alignment and phylogenetic tree developed by the MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets (Kumar et al., ). […]

Pipeline specifications

Software tools CAP3, BioEdit, MEGA
Application Phylogenetics
Chemicals Superoxides