We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.The commonly used multilocus methods for genotyping Toxoplasma gondii strains employ PCR-restriction fragment length polymorphism (PCR-RFLP) analysis at 10 markers (16), sequencing at 8 introns of 5 loci (14), and length polymorphism analysis of 5 microsatellite (MS) markers (4). Advantages and disadvantages of these different markers have been reviewed elsewhere (16). Based on our experience within the Toxoplasma Biological Resource Center (BRC ToxoBS) and the National Reference Center for Toxoplasmosis, it appears that screening of a large number of clinical isolates for T. gondii strain typing should rely on an easy-to-use method with two different levels of discrimination. The first step of discrimination is the performed at the typing level: that is, employing the ability of markers to distinguish the major clonal lineages from atypical strains. In areas with a marked clonal population structure such as Europe, the genetic screening of clinical isolates should rapidly identify basic type II or III strains, which represent the majority of isolates, and atypical strains, which are the exception to the rule (3, 11). In this context, the identification of atypical strains is of clinical and epidemiological importance because atypical strains are usually associated with severe disease outcomes and contamination of individuals by non-European strains either during residence abroad or after consumption of imported meat (5, 9, 10). The second level of discrimination is the fingerprinting level, representing a high degree of discriminatory power for differentiating closely related strains belonging to the same lineage. This high-resolution analysis is required for identifying laboratory contaminations for diagnosis issues and for establishing a common source of infection among different infected individuals in an outbreak (7,8).Microsatellite (MS) sequences are tandem repeats of short (1 to 6 bp) DNA motifs that are ubiquitous in eukaryotic genomes and undergo length changes due to insertion or deletion of one or multiple repeat units. The most commonly proposed mutation mechanism for MS sequences is strand slippage, occurring predominantly during replication (13). The numbers of repeat motifs differ in a population, thereby creating multiple alleles at an MS locus. MS loci are amplified by PCR using fluorescently labeled forward and unlabeled reverse primers. The dye-labeled products are separated by size using automated electrophoresis and identified by fluorescence detection. We previously designed a multiplex PCR assay with 5 MS markers that were able to reach the typing level but not the fingerprinting level of discrimination between strains (4). Here we developed an easy-to-use and rapid genotyping method which aimed to ensure both levels of genetic discrimina...

No abstract

No abstract

No abstract