Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability

  • John T. Connelly
  • Sam R. Nugen
  • Wlodek Borejsza-Wysocki
  • Richard A. Durst
  • Richard A. Montagna
  • Antje J. Baeumner
Original Paper


A bioanalytical detection method for specific detection of viable human pathogenic Cryptosporidium species, C. parvum, C. hominis, and C. meleagridis is described. Oocysts were isolated from water samples via immunomagnetic separation, and mRNA was extracted with oligo-dT magnetic beads, amplified using nucleic acid sequence-based amplification (NASBA), and then detected in a nucleic acid hybridization lateral flow assay. The amplified target sequence employed was hsp70 mRNA, production of which is stimulated via a brief heat shock. The described method was capable of detecting one oocyst in 10 μL using flow-cytometer-counted samples. Only viable oocysts were detected, as confirmed using 4′,6-diamidino-2-phenylindole and propidium iodide (DAPI/PI) staining. The detection system was challenged by detecting oocysts in the presence of large numbers of common waterborne microorganisms and packed pellet material filtered from environmental water samples. When the method was compared with EPA Method 1622 for C. parvum detection, highly comparable results were obtained. Since the described detection system yields unambiguous results within 4.5 h, it is an ideal method for monitoring the safety of drinking water.


Cryptosporidium mRNA Detection Liposome Lateral flow Human pathogen Oligo-dT 



This study was funded in part by EPA Contract Number EP-D-06–034, NYSTAR. This research was also supported in part by the Cornell University Agricultural Experiment Station federal formula funds, Project No. 123–314 received from Cooperative State Research, Education and Extension Service, US Department of Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the US Department of Agriculture. The authors would like to thank Jennifer Clancy and Randi McQuin of Clancy Environmental Consultants, Inc., Becky Hoffman and Martin Collins of the Wisconsin State Laboratory of Hygiene, and Giovanni Widmer and Sal Tzipori of Tufts University School of Veterinary Medicine for providing samples used in this study.


  1. 1.
    Yagita K, Izumiyama S, Tachibana H, Masuda G, Iseki M, Furuya K et al (2001) Parasitol Res 87:950–955Google Scholar
  2. 2.
    Leoni F, Amar C, Nichols G, Pedraza-Diaz S, McLauchlin J (2006) J Med Microbiol 55:703–707CrossRefGoogle Scholar
  3. 3.
    Wilkinson SL (1997) Chem Eng News 75:24–33Google Scholar
  4. 4.
    Mead P, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C et al (1999) Emerg Infect Dis 5:607–625CrossRefGoogle Scholar
  5. 5.
    Chappell CL, Okhuysen PC, Sterling CR, DuPont HL (1996) J Infect Dis 173:232–236Google Scholar
  6. 6.
    Environmental Protection Agency. Method 1622: Cryptosporidium in water by filtration/IMS/FA. Vol., 2005:45–60Google Scholar
  7. 7.
    Robertson LJ, Campbell AT, Smith HV (1993) Parasitology 106:13–19CrossRefGoogle Scholar
  8. 8.
    Hou L, Li X, Dunbar L, Moeller R, Palermo B, Atwill ER (2004) Appl Environ Microbiol 70:642–646CrossRefGoogle Scholar
  9. 9.
    Baeumner AJ, Humiston M, Montagna RA, Durst RA (2001) Anal Chem 73:1176–1180CrossRefGoogle Scholar
  10. 10.
    Hartley HA, Baeumner AJ (2003) Anal Bioanal Chem 376:319–327Google Scholar
  11. 11.
    Baeumner AJ, Schlesinger NA, Slutzki NS, Romano J, Lee EM, Montagna RA (2002) Anal Chem 74:1442–1448CrossRefGoogle Scholar
  12. 12.
    Baeumner AJ, Cohen RN, Miksic V, Min JH (2003) Biosens Bioelectron 8:405–419CrossRefGoogle Scholar
  13. 13.
    Esch MB, Baeumner AJ, Durst RA (2001) Anal Chem 73:3162–3167CrossRefGoogle Scholar
  14. 14.
    Aguilar ZP, Fritsch I (2003) Anal Chem 75:3890–3897CrossRefGoogle Scholar
  15. 15.
    Stinear T, Matusan A, Hines K, Sandery M (1996) Appl Environ Microbiol 62:3385–3390Google Scholar
  16. 16.
    Edwards KA, Baeumner AJ (2006) Anal Bioanal Chem 386:1335–1343CrossRefGoogle Scholar
  17. 17.
    Baeumner AJ, Pretz J, Fang S (2004) Anal Chem 76:888–894CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • John T. Connelly
    • 1
  • Sam R. Nugen
    • 1
  • Wlodek Borejsza-Wysocki
    • 2
  • Richard A. Durst
    • 2
  • Richard A. Montagna
    • 3
  • Antje J. Baeumner
    • 1
  1. 1.Department of Biological and Environmental EngineeringCornell UniversityIthacaUSA
  2. 2.Department of Food Science and TechnologyCornell UniversityGenevaUSA
  3. 3.Innovative Biotechnologies International, Inc.Grand IslandUSA

Personalised recommendations