Genotyping of human isolates from human toxoplasmosis outbreak: Restriction Fragment Length Polymorphism and Sanger Sequencing.

Abstract

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii, capable of infecting a wide range of hosts. The parasite exhibits a broad genetic diversity, necessitating genotypic characterization for genotype identification and associations with epidemiological information. Therefore, the Restriction Fragment Length Polymorphism (RFLP) technique is used for characterization. This study aimed to perform genotypic characterization of isolates from pregnant women infected during a human toxoplasmosis outbreak, comparing the RFLP and Sanger Sequencing methodologies. For this purpose, six human isolates were subjected to conventional PCR, Multiplex PCR, Nested PCR, Enzymatic Digestion, and Sanger Sequencing. Additionally, the standard strains GTI (Type I), PTG (Type II), and CTG (Type III) were also subjected to the same techniques described above. Subsequently, the amplified DNA products were compared with the standard strains. As a result, it was observed that Sanger Sequencing provides the same information as RFLP PCR, as well as the possibility of cost reduction for genotypic characterization, and providing greater agility in issuing results. Additionally, Sanger Sequencing of T. gondii isolates allows for detailed evaluation of nucleotide sequences, including the assessment of SNPs and enzymatic restriction sites, which the RFLP technique does not.