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The Sensitivity of PCR Detection of Cryptosporidium Oocysts in Fecal Samples Using Two DNA Extraction Methods

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Abstract

Background: The implementation of cost-effective intervention strategies for zoonotic protozoa relies on the development of sensitive and accurate diagnostic methods. We carried out a study to evaluate the accuracy of a PCR method for the detection of Cryptosporidium spp. oocysts in fecal samples from cattle.

Methods: Fecal samples were spiked with different numbers of oocysts and the limit of detection of the method was determined. Two methods of DNA extraction were assessed: glass beads and freeze-thawing using liquid nitrogen. A nested PCR approach was developed targeting the Cryptosporidium SSU rRNA and TRAP-C2 genes. Agreement between the diagnosis of Cryptosporidium spp. at the SSU rRNA and TRAP-C2 loci was quantified using the κ-coefficient.

Results: Compared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA from Cryptosporidium oocysts (sensitivities were 83 and 100%, respectively). The limits of detection for glass beads and freeze-thaw were low, 1 and 10 oocyst/g fecal samples, respectively. Forty-six percent of the field samples previously classified as negative for Cryptosporidium parvum by the floatation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol.

Conclusion: Primers for SSU rRNA are more successful in producing an amplification than primers for the TRAP-C2 gene which makes the former PCR protocol the approach of choice for detecting Cryptosporidium parvum oocysts in field samples.

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Acknowledgements

This study was funded by a grant from the USGS through the Water Resources Institute, Cornell University (NY01-B-1841), and from the USDA (grant no. 98-38420-5803).

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Authors and Affiliations

  1. Section of Epidemiology, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, New York, 14853, USA

    Gabriella Lindergard, Daryl V. Nydam &  Hussni O. Mohammed

  2. Section of Parasitology, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA

    Susan E. Wade & Stephanie L. Schaaf

Authors
  1. Gabriella Lindergard
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  2. Daryl V. Nydam
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  3. Susan E. Wade
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  4. Stephanie L. Schaaf
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  5. Hussni O. Mohammed
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Correspondence to Hussni O. Mohammed.

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Lindergard, G., Nydam, D.V., Wade, S.E. et al. The Sensitivity of PCR Detection of Cryptosporidium Oocysts in Fecal Samples Using Two DNA Extraction Methods. CNS Drugs 7, 147–153 (2003). https://doi.org/10.1007/BF03260031

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