Detection and characterization of Cryptosporidium cuniculus by real-time PCR

Cryptosporidium cuniculus was originally detected in rabbits and has been identified as an emerging human pathogen, but the occurrence, prevalence, and epidemiology in human and rabbit populations are poorly understood. As identification of C. cuniculus can be time-consuming and costly using existing molecular assays, a real-time polymerase chain reaction (PCR)-based method targeting specific markers for this species was developed. The assay is based on amplification of the C. cuniculus-specific 60-kDa glycoprotein (GP60) gene using two PCRs targeting subtype families Va and Vb. PCR product formation was monitored by SYBR Green I fluorescence measurement followed by post-amplification melt curve analysis; high resolution melt curve analysis was found to give increased sensitivity over standard melt curve analysis. The real-time PCR correctly identified all 41 C. cuniculus isolates (40 from humans, one from a rabbit) tested, with subtype family in agreement with GP60 gene sequencing. Specificity was demonstrated by lack of detection of nine other Cryptosporidium species and genotypes, including 88 isolates of the closely related species, Cryptosporidium hominis. The PCRs were performed in separate tubes to maximize the possibility of detecting mixed Va-Vb infections; however, none were detected. The potential for multiplexing the reactions was also demonstrated, furthering the utility of the assay for large-scale occurrence and prevalence studies.