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Title: REAL TIME PCT FOR THE DETECTION OF CRYPTOSPORIDIUM PARVUM

Author
item Higgins, James
item Fayer, Ronald
item Trout, James
item XIAO, LIHUA - CDC, ATLANTA, GA
item LAL, ALTAF - CDC, ATLANTA, GA
item KERBY, STEVE - USAMRIID, FT. DETRICK, MD
item Jenkins, Mark

Submitted to: American Society for Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2001
Publication Date: 11/1/2001
Citation: N/A

Interpretive Summary: Cryptosporidium parvum is an important protozoan parasite of humans and cattle. The oocyst stage of the parasite is responsible for causing infections, and it is present in the feces of infected individuals. The authors developed a molecular biology-based assay to detect Cryptosporidium oocysts in feces from infected people, and in manure from infected calves. The assay also detected Cryptosporidium in gut tissues from infected calves. An internal positive control was used to determine if the DNA obtained from the manure and tissues was of satisfactory quality for use in molecular assays. The assay shows promise for use in monitoring a variety of environmental samples for the presence of Cryptosporidium parvum.

Technical Abstract: Real time, TaqMan PCR assays were developed for the Cp11 and 18S rRNA genes of the protozoan parasite Cryptosporidium parvum. The assays were specific for Cryptosporidium, but did not hybridize with C. parvum to the exclusion of other species or genotypes. The assays were evaluated on DNA extracted from calf diarrhea and manure, and human feces, using two commercial kits, the Mo Bio UltraClean# Soil DNA kit, and the Qiagen QIAamp# DNA Stool kit. 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. PCR results were related to the quantity of oocysts present in the feces. We conclude that real time quantitation is dependant on the number of parasites in fecal samples, as well as the continued presence of PCR inhibitors in DNA preparations derived from these samples.