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Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples.
Appl Environ Microbiol 1995; 61(11):3849-55AE

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.

Authors+Show Affiliations

Department of Marine Science, University of South Florida, St. Petersburg 33701-5016, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

8526496

Citation

Johnson, D W., et al. "Development of a PCR Protocol for Sensitive Detection of Cryptosporidium Oocysts in Water Samples." Applied and Environmental Microbiology, vol. 61, no. 11, 1995, pp. 3849-55.
Johnson DW, Pieniazek NJ, Griffin DW, et al. Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl Environ Microbiol. 1995;61(11):3849-55.
Johnson, D. W., Pieniazek, N. J., Griffin, D. W., Misener, L., & Rose, J. B. (1995). Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Applied and Environmental Microbiology, 61(11), pp. 3849-55.
Johnson DW, et al. Development of a PCR Protocol for Sensitive Detection of Cryptosporidium Oocysts in Water Samples. Appl Environ Microbiol. 1995;61(11):3849-55. PubMed PMID: 8526496.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. AU - Johnson,D W, AU - Pieniazek,N J, AU - Griffin,D W, AU - Misener,L, AU - Rose,J B, PY - 1995/11/1/pubmed PY - 1995/11/1/medline PY - 1995/11/1/entrez SP - 3849 EP - 55 JF - Applied and environmental microbiology JO - Appl. Environ. Microbiol. VL - 61 IS - 11 N2 - 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. SN - 0099-2240 UR - https://www.unboundmedicine.com/medline/citation/8526496/Development_of_a_PCR_protocol_for_sensitive_detection_of_Cryptosporidium_oocysts_in_water_samples_ L2 - http://aem.asm.org/cgi/pmidlookup?view=long&pmid=8526496 DB - PRIME DP - Unbound Medicine ER -