Multi-Locus Sequencing for Strain Typing of Toxoplasma gondii.

Toxoplasma gondii is a globally distributed apicomplexan parasite characterized by substantial genetic diversity, necessitating accurate molecular approaches for strain discrimination. The goal of this protocol is to establish a reproducible workflow for targeted multi-locus genotyping of cultured T. gondii isolates. The procedure involves in vitro propagation of representative Type I (RH) and Type II (ME49) strains, extraction of genomic DNA, locus-specific amplification of the SAG2 and SAG3 genes, and sequencing-based analyses to detect single-nucleotide polymorphisms (SNPs) and insertion/deletion (INDEL) events. Sequencing data are processed to generate high-confidence consensus sequences and to perform comparative alignment for the identification of strain-specific polymorphisms. Application of this workflow to archetypal strains demonstrated high mapping efficiency, consistent read depth across target loci, and accurate recovery of expected amplicon sizes, confirming reliable detection of locus-level genetic variation. Although comprehensive genotype assignment requires inclusion of additional standardized markers, this protocol provides a scalable and reproducible framework for targeted genetic characterization of laboratory-maintained T. gondii isolates and supports consistent multi-locus sequence analysis.