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. 1995 Nov;61(11):3849–3855. doi: 10.1128/aem.61.11.3849-3855.1995

Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples.

D W Johnson 1, N J Pieniazek 1, D W Griffin 1, L Misener 1, J B Rose 1
PMCID: PMC167689  PMID: 8526496

Abstract

The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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