Abstract
BackgroundCyclospora 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.MethodsIn 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.ResultsAll 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.ConclusionsThe 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.
Highlights
Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks
We developed a quantitative PCR targeting the polymorphic link region of concatenated copies of mitochondrial genomes of C. cayetanensis, and assessed genetic heterogeneity among specimens from several countries by melt curve and electrophoresis analyses, and the ability for geographical source-tracking by DNA sequence analysis of the qPCR products
Development of a mitochondrial DNA-based qPCR Based on results of the previous comparison of the mitochondrial genomes of C. cayetanensis isolates from China (CHN_HEN01; KP796149) and the USA (Cyclo_CDC_2013; KP658101) [13], we designed a set of qPCR primers that amplifies a ~357 bp link region between different copies of the mitochondrial genome
Summary
Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. Cyclospora spp. are protozoan parasites that mainly cause diarrhea in humans and animals. Of the more than 20 known Cyclospora species, Cyclospora cayetanensis is the only one infecting humans. Cyclosporiasis caused by this species presents a serious challenge to food safety [1, 2]. The recent whole genome sequencing of C. cayetanensis has facilitated the development of genotyping tools for this pathogen [8,9,10]. To improve the investigation of cyclosporiasis outbreaks, a high-resolution multilocus sequence typing (MLST) tool has been developed recently for C. cayetanensis based on whole genome sequence data [11, 12]. Like most other eukaryotic parasites, C. cayetanensis has a mitochondrial genome
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