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Real-time PCR for the detection of Cryptosporidium parvum
J A Higgins1, R Fayer, J M Trout
1USDA-ARS, Rm. 202, Bldg. 173, 10300 Baltimore Blvd., Beltsville, MD 20705, USA. jhiggins@anri.barc.usda.gov
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.
Insights
Real-time PCR assays for Cryptosporidium parvum were developed, showing feasibility for quantifying parasite DNA in diarrheic feces. However, nested PCR is required for detecting Cryptosporidium DNA in challenging samples like manure and human feces.
Area of Science:
- Veterinary Parasitology
- Molecular Diagnostics
- Microbiology
Background:
- Cryptosporidium parvum is a significant protozoan parasite causing gastrointestinal illness in humans and animals.
- Accurate detection and quantification of C. parvum are crucial for disease management and epidemiological studies.
- Existing diagnostic methods may face challenges with sensitivity and specificity, particularly in complex sample matrices.
Purpose of the Study:
- To develop and evaluate real-time TaqMan PCR assays for Cryptosporidium parvum detection.
- To assess the efficacy of commercial DNA extraction kits for various sample types.
- To compare the performance of real-time PCR and nested PCR for C. parvum DNA quantification.
Main Methods:
- Development of real-time TaqMan PCR assays targeting Cp11 and 18S rRNA genes of C. parvum.
- Evaluation of Mo Bio UltraClean Soil DNA and Qiagen QIAamp DNA Stool kits for DNA extraction from calf diarrhea, manure, and preserved human feces.
- Application of real-time PCR and nested PCR for C. parvum DNA detection and quantification in different sample types and time points post-infection in calves.
Main Results:
- Real-time quantitation of C. parvum DNA was successful in calf diarrhea samples.
- Nested PCR was necessary for the detection of C. parvum DNA in manure and human fecal samples.
- C. parvum DNA was detected in ileal tissue from calves at 7 days post-infection using nested PCR.
Conclusions:
- Real-time PCR offers feasible quantification of C. parvum DNA in diarrheic samples with high oocyst load and low inhibitor concentrations.
- Nested PCR is essential for sensitive detection of C. parvum DNA in samples with low oocyst numbers and high PCR inhibitor levels, such as manure and human feces.
- The choice of diagnostic method should consider the sample type and expected parasite load for optimal detection of Cryptosporidium parvum.