title: Real-time PCR for the detection of Cryptosporidium parvum.
abstract: Real time, TaqMan PCR assays were developed for the Cp11 and 18S rRNA genes of the protozoan parasite Cryptosporidium parvum. The TaqMan probes were specific 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 UltraClean Soil DNA kit, and the Qiagen QIAamp DNA Stool kit, were evaluated for DNA extraction from calf diarrhea and manure, and potassium dichromate and formalin preserved human feces. Real-time quantitation was achieved with the diarrhea samples, but nested PCR was necessary to detect C. parvum 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 PCR detected C. parvum DNA in the 7-day post-infection sample, but neither of the other time point samples were positive. These results indicate that real-time quantitation of C. parvum DNA, extracted using the commercial kits, is feasible on diarrheic feces, with large numbers of oocysts and small concentrations of PCR inhibitor(s). For samples with few oocysts and high concentrations of PCR inhibitor(s), such as manure, nested PCR is necessary for detection.
authors.affiliation: USDA-ARS, Rm. 202, Bldg. 173, 10300 Baltimore Blvd., Beltsville, MD 20705, USA. jhiggins@anri.barc.usda.gov
authors.forename: J A
authors.initials: JA
authors.lastname: Higgins
authors.forename: R
authors.initials: R
authors.lastname: Fayer
authors.forename: J M
authors.initials: JM
authors.lastname: Trout
authors.forename: L
authors.initials: L
authors.lastname: Xiao
authors.forename: A A
authors.initials: AA
authors.lastname: Lal
authors.forename: S
authors.initials: S
authors.lastname: Kerby
authors.forename: M C
authors.initials: MC
authors.lastname: Jenkins
mesh_terms: D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology; D005838:Genotype; D006801:Humans; D008297:Male; D010270:Parasite Egg Count; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid
chemical_list: D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S; D011933:Reagent Kits, Diagnostic