Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability
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.
KeywordsCryptosporidium 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.Yagita K, Izumiyama S, Tachibana H, Masuda G, Iseki M, Furuya K et al (2001) Parasitol Res 87:950–955Google Scholar
- 3.Wilkinson SL (1997) Chem Eng News 75:24–33Google Scholar
- 5.Chappell CL, Okhuysen PC, Sterling CR, DuPont HL (1996) J Infect Dis 173:232–236Google Scholar
- 6.Environmental Protection Agency. Method 1622: Cryptosporidium in water by filtration/IMS/FA. Vol., 2005:45–60Google Scholar
- 10.Hartley HA, Baeumner AJ (2003) Anal Bioanal Chem 376:319–327Google Scholar
- 15.Stinear T, Matusan A, Hines K, Sandery M (1996) Appl Environ Microbiol 62:3385–3390Google Scholar