Detection of Cryptosporidium parvum Using Oligonucleotide-Tagged Liposomes in a Competitive Assay Format Mandy B. Esch, ² Antje J. Baeumner, ‡ and Richard A. Durst* ,² BioAnalytical Research Laboratories, Department of Food Science & Technology, Cornell University, Geneva, New York 14456-0462, and Analytical Biotechnology Research Laboratory, Department of Agricultural and Biological Engineering, Cornell University, Ithaca, New York 14853-5701 To meet the technical challenge of accurately and rapidly detecting Cryptosporidium parvum oocysts in environ- mental water, the authors developed a single-use visual- strip assay. The first step in the overall assay procedure involves extracting C. parvum’s mRNA coding for heat- shock protein hsp7 0 , followed by amplification using nucleic acid sequence-based amplification (NASBA) meth- odology as described previously (Baeumner, A. J.; Hu- miston, M.; Montagna, R. A.; Durst, R. A. Anal. Chem., in press). Subsequently, generated amplicons are hybrid- ized with dye-entrapping liposomes bearing DNA oligo- nucleotides (reporter probes) and biotin on their surface. The liposome-amplicon complex is then allowed to migrate upward on a nitrocellulose membrane strip. On the nitrocellulose strip, antisense-reporter probes are immobilized in a capture zone and antibiotin antibodies are immobilized in a second zone above the capture zone. Depending on the presence or absence of amplicon in the sample, the liposomes will bind to the capture zone, or they will be caught via their biotin tag in the second zone. Visual detection or gray-scale densitometry allows the quantification of liposomes that are present in either zone. The detection limit of the assay was determined to be 80 fmol amplicon/ test. High accuracy and an internal assay control is established using this competitive format, because the presence or absence of liposomes can be quantified in the two capture zones. No other species of Cryptosporidium has a more significant impact on the health of human beings than C. parvum. 2,3 Infections by this coccidian parasite can lead to life-threatening conditions in individuals with impaired immune systems, such as patients with acquired immune deficiency syndrome (AIDS). 4 It also causes acute gastrointestinal symptoms in healthy people. Between 1984 and 1996, massive outbreaks in the U.S.A., U.K., and Japan were provoked by the waterborne route of transmission of C. parvum. 5 Because standard disinfection procedures such as chlorination cannot effectively inactivate C. parvum’s oocysts, 6,7 it is important to develop sensitive tests for detecting C. parvum in drinking water. A successful detection scheme requires collecting and con- centrating the oocysts from environmental water samples, separat- ing the oocysts from contaminating debris, and finally, detecting them. Our paper focuses on detecting viable C. parvum oocysts after having concentrated them. A procedure for rapidly and accurately detecting oocyst viability would enable researchers to assess (1) the risk posed by the detected oocysts and (2) the effectiveness of newly developed disinfection procedures. The standard procedure for detecting C. parvum uses fluores- cently labeled antibodies that stain the oocysts, which can thereupon be identified microscopically. 8 However, a study by Moore et al. demonstrated that some carbohydrate epitopes at the oocyst wall are labile after chlorine treatment and under oxidizing conditions similar to those used to eliminate bacteria found in drinking water. 9 Therefore, although the oocysts would still be infectious, they would not be detected by the use of antibodies toward these epitopes. A further drawback of detection using epifluorescence microscopy is that commercially available antibodies cross-react with organisms other than C. parvum. 10 Finally, the standard procedure does not permit researchers to determine the viability of oocysts. Various detection methods that overcome the difficulties encountered with epifluorescence microscopy have been reported. 11-17 Slifko et al. developed a detection scheme that focuses on determining oocyst viability by specifically identifying * To whom correspondence should be addressed. Fax: 1-315-787-2284. E-mail: rad2@ cornell.edu. † Department of Food Science & Technology. ‡ Department of Agricultural and Biological Engineering. (1) Baeumner, A. J.; Humiston, M.; Montagna, R. A.; Durst, R. A. Anal. Chem., in press. (2) Current, W. L. Am. Soc. Microbiol. News 1988 , 54, 605-611. (3) Current, W. L.; Garcia, L. S. Clin. Microbiol. Rev. 1991 , 4, 325-358. (4) Cook, G. C. Q. J. Med. 1987 , 65, 967-983. (5) Smith, H. V.; Rose, J. B. Parasitol. Today 1998 , 14, 14-22. (6) Campbell, I.; Tzipori, A. S.; Hutchinson, G.; Angus, K. W. Vet. Rec. 1982 , 111, 414-415. (7) Korich, D. G.; Mead, J. R.; Madore, M. S.; Sinclair, M. A.; Sterling, C. R. Appl. Environ. Microbiol. 1990 , 56, 1423-1428. (8) Fricker, C. R.; Crabb, J. H. Adv. Parasitol. 1998 , 40, 241-278. (9) Moore, A. G.; Vesey, G.; Champion, A.; Scandizzo, P.; Deere, D.; Veal, D.; Williams, K. L. Int. J. Parasitol. 1998 , 28 (8), 1205-1212. (10) Graczyk, T. K.; Cranfield, M. R.; Fayer, R. Am. J.Trop. Med. Hyg. 1996 , 54, 274-279. (11) Slifko, T. R.; Friedman, D.; Rose, J. B.; Jakubowski, W. Appl. Environ. Microbiol. 1997 , 63 (9), 3669-3675. (12) Slifko, T. R.; Friedman, D. E.; Rose, J. B.; Upton, S. J.; Jakubowski, W. Water Sci. Technol. 1997 , 35 (11-12) , 363-368. (13) Wagner-Wiening, Ch.; Kimmig, P. Appl. Environ. Microbiol. 1995 , 61 (12), 4514-4516. Anal. Chem. 2001, 73, 3162-3167 3162 Analytical Chemistry, Vol. 73, No. 13, July 1, 2001 10.1021/ac010012i CCC: $20.00 © 2001 American Chemical Society Published on Web 05/24/2001