This assay consistently detected 10 copies of the cloned target fragment and can be considered more efficient and sensitive than microscopy flotation methods for detecting multiple species of oocysts in human feces. Cystoisospora belli (×3), Cryptosporidium parvum (×3), Cryptosporidium hominis (×5), Cryptosporidium meleagridis (×1), Cryptosporidium canis (×1), and Cyclospora cayetanensis (×9) were detected by qPCR-MCA and confirmed by sequencing. Putative positive samples were also tested by conventional PCR and microscopy. Samples were subjected to qPCR using universal coccidia primers targeting 18S rDNA to detect oocysts followed by MCA to identify oocyst species based on amplicon melting temperature. A quantitative polymerase chain reaction assay with melt curve analysis (qPCR-MCA) was applied for the detection of protozoan oocysts in 501 human fecal samples collected in Dominican Republic.
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