Detection and differentiation of Cryptosporidium parasites that are pathogenic for humans by real-time PCR.

Cryptosporidiosis is a significant cause of food-borne and waterborne outbreaks of diarrheal diseases. To better understand the route of transmission of Cryptosporidium parasites, a number of genotyping techniques have been developed, based on PCR-restriction fragment length polymorphism or sequencing analysis of antigen, structural, and housekeeping genes. In this study, a real-time assay for the detection of Cryptosporidium oocysts is described. This technique had a detection limit of five oocysts. By melting curve analysis of PCR products with fluorescence-labeled hybridization probes, this technique was able to differentiate five common Cryptosporidium parasites that are pathogenic for humans in a single PCR. We evaluated and validated the test using samples from presently known Cryptosporidium parasites that are pathogenic for humans. This technique provides an alternative molecular tool in epidemiologic studies of human cryptosporidiosis.