Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Aug;63(8):3134–3138. doi: 10.1128/aem.63.8.3134-3138.1997

Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples.

M Q Deng 1, D O Cliver 1, T W Mariam 1
PMCID: PMC168611  PMID: 9251200

Abstract

A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.

Full Text

The Full Text of this article is available as a PDF (794.2 KB).

Selected References

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

  1. Awad-el-Kariem F. M., Warhurst D. C., McDonald V. Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction. Parasitology. 1994 Jul;109(Pt 1):19–22. doi: 10.1017/s0031182000077714. [DOI] [PubMed] [Google Scholar]
  2. Balatbat A. B., Jordan G. W., Tang Y. J., Silva J., Jr Detection of Cryptosporidium parvum DNA in human feces by nested PCR. J Clin Microbiol. 1996 Jul;34(7):1769–1772. doi: 10.1128/jcm.34.7.1769-1772.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Campbell A. T., Robertson L. J., Smith H. V. Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes. Appl Environ Microbiol. 1992 Nov;58(11):3488–3493. doi: 10.1128/aem.58.11.3488-3493.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casemore D. P. Epidemiological aspects of human cryptosporidiosis. Epidemiol Infect. 1990 Feb;104(1):1–28. doi: 10.1017/s0950268800054480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Current W. L., Garcia L. S. Cryptosporidiosis. Clin Microbiol Rev. 1991 Jul;4(3):325–358. doi: 10.1128/cmr.4.3.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DuPont H. L., Chappell C. L., Sterling C. R., Okhuysen P. C., Rose J. B., Jakubowski W. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med. 1995 Mar 30;332(13):855–859. doi: 10.1056/NEJM199503303321304. [DOI] [PubMed] [Google Scholar]
  7. Fayer R., Leek R. G. The effects of reducing conditions, medium, pH, temperature, and time on in vitro excystation of Cryptosporidium. J Protozool. 1984 Nov;31(4):567–569. doi: 10.1111/j.1550-7408.1984.tb05504.x. [DOI] [PubMed] [Google Scholar]
  8. Fayer R., Nerad T. Effects of low temperatures on viability of Cryptosporidium parvum oocysts. Appl Environ Microbiol. 1996 Apr;62(4):1431–1433. doi: 10.1128/aem.62.4.1431-1433.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Filkorn R., Wiedenmann A., Botzenhart K. Selective detection of viable Cryptosporidium oocysts by PCR. Zentralbl Hyg Umweltmed. 1994 Jun;195(5-6):489–494. [PubMed] [Google Scholar]
  10. Johnson D. W., Pieniazek N. J., Griffin D. W., Misener L., Rose J. B. Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl Environ Microbiol. 1995 Nov;61(11):3849–3855. doi: 10.1128/aem.61.11.3849-3855.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kehl K. S., Cicirello H., Havens P. L. Comparison of four different methods for detection of Cryptosporidium species. J Clin Microbiol. 1995 Feb;33(2):416–418. doi: 10.1128/jcm.33.2.416-418.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laxer M. A., Timblin B. K., Patel R. J. DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction. Am J Trop Med Hyg. 1991 Dec;45(6):688–694. doi: 10.4269/ajtmh.1991.45.688. [DOI] [PubMed] [Google Scholar]
  13. Mac Kenzie W. R., Hoxie N. J., Proctor M. E., Gradus M. S., Blair K. A., Peterson D. E., Kazmierczak J. J., Addiss D. G., Fox K. R., Rose J. B. A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply. N Engl J Med. 1994 Jul 21;331(3):161–167. doi: 10.1056/NEJM199407213310304. [DOI] [PubMed] [Google Scholar]
  14. Robertson L. J., Campbell A. T., Smith H. V. In vitro excystation of Cryptosporidium parvum. Parasitology. 1993 Jan;106(Pt 1):13–19. doi: 10.1017/s003118200007476x. [DOI] [PubMed] [Google Scholar]
  15. Robertson L. J., Campbell A. T., Smith H. V. Survival of Cryptosporidium parvum oocysts under various environmental pressures. Appl Environ Microbiol. 1992 Nov;58(11):3494–3500. doi: 10.1128/aem.58.11.3494-3500.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wagner-Wiening C., Kimmig P. Detection of viable Cryptosporidium parvum oocysts by PCR. Appl Environ Microbiol. 1995 Dec;61(12):4514–4516. doi: 10.1128/aem.61.12.4514-4516.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Weber R., Bryan R. T., Bishop H. S., Wahlquist S. P., Sullivan J. J., Juranek D. D. Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J Clin Microbiol. 1991 Jul;29(7):1323–1327. doi: 10.1128/jcm.29.7.1323-1327.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Webster K. A., Pow J. D., Giles M., Catchpole J., Woodward M. J. Detection of Cryptosporidium parvum using a specific polymerase chain reaction. Vet Parasitol. 1993 Oct;50(1-2):35–44. doi: 10.1016/0304-4017(93)90005-8. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES