DEVELOPMENT OF A PCR PROTOCOL FOR SENSITIVE DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN WATER SAMPLES

The development of a reliable method of using PCR for detection of Cry ptosporidium oocysts in environmental samples with oligonucleotide pri mers which amplify a portion of the sequence encoding the small (185) subunit of rRNA producing a 435-bp product was demonstrated. The PCR a ssay was found to provide highly genus-specific detection of Cryptospo ridium spp. after release of nucleic acids from oocysts by a simple fr eeze-thaw procedure, The assay routinely detected 1 to 10 oocysts in p urified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay, The sensitivity of the PCR assay in s ome seeded environmental water samples was up to 1,000-fold lower. How ever, this interference was eliminated by either how cytometry or magn etic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide pr obe detected by chemiluminescence, Confirmation of the presence of Cry ptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis,, was obtained with this technique, and PCR was found to be as sen sitive as immunofluorescence for detection of oocysts in wastewater co ncentrates.