Real-time PCR for the detection of Cryptosporidium parvum

Real time. TaqMan PCR assays were developed for the Cp11 and 18S rRNA genes of the protozoan parasite Cryptosporidium parvum. The TaqMan probes were s pecific for the genus Cryptosporidium, but could not hybridize exclusively with human-infectious C. parvum species and genotypes. In conjunction with development of the TaqMan assays, two commercial kits, the Mo Bio UltraClea n (TM) Soil DNA kit, and the Qiagen QIAamp (TM) DNA Stool kit, were evaluat ed for DNA extraction from calf diarrhea and manure, and potassium dichroma te and formalin preserved human feces. Real-time quantitation was achieved with the diarrhea samples., but nested PCR was necessary to detect C. parvu m DNA in manure and human feces. Ileal tissues were obtained from calves at 3, 7, and 14 days post-infection, and DNA extracted and assayed, Nested PC R detected C. part,um DNA in the 7-day post-infection sample, but neither o f the other time point samples were positive. These results indicate that r eal-time quantitation of C. parvum DNA, extracted using the commercial kits , is feasible on diarrheic feces, with large numbers of oocysts and small c oncentrations of PCR inhibitor(s). For samples with few oocysts and high co ncentrations of PCR inhibitor(s), such as manure, nested PCR is necessary f or detection. Published by Elsevier Science B.V.