Multilocus Microsatellite Genotyping of N. caninum

DNA samples obtained from N. caninum PCR-positive brains were used for genotyping by multilocus microsatellite analysis. Specifically, MS4, MS5, MS6A, MS6B, MS7, MS8, MS10, MS12 and MS21 markers were amplified using specific primers and nested-PCR conditions, as previously described [14 (link)]. For all microsatellites, the size of the PCR products was determined in a 48-capillary 3730 DNA Analyser (Applied Biosystems, Foster City, CA, USA) with GeneScan-500 (LIZ) size standards (Applied Biosystems) at the Unidad Genómica del Parque Científico de Madrid. The results were analysed with GeneMapper1 software, v3.5. For confirmation of allele identification, microsatellite alleles MS4, MS5, MS6A, MS7, MS10 and MS21 from representative samples were also sequenced using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3730 DNA Analyser (Applied Biosystems). The sequencing was conducted at the Unidad Genómica del Parque Cientifico de Madrid. Sequences were analysed using BioEdit Sequence Alignment Editor v.7.0.1 software (Copyright 1997–2004 Tom Hall, Ibis Therapeutics, Carlsbad, CA, USA). Allele assignment was performed as previously described [13 (link)].