Mitochondrial genome sequence variation as a useful marker for assessing genetic heterogeneity among Cyclospora cayetanensis isolates and source-tracking

Background: Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks. Methods: In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products. Results: All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking. Conclusions: The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.