Detection of Cryptosporidium parvum Using Oligonucleotide-Tagged Liposomes in a Competitive Assay Format

• Published in: Analytical chemistry (Washington) Vol. 73; no. 13; pp. 3162 - 3167
• Main Authors: ESCH, Mandy B., BAEUMNER, Antje J., DURST, Richard A.
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-07-2001
• Subjects: Animals; Biological and medical sciences; Chemistry; Cryptosporidium parvum; Cryptosporidium parvum - genetics; Cryptosporidium parvum - isolation & purification; Environmental monitoring; Fluorescence; Fundamental and applied biological sciences. Psychology; General aspects and techniques; Lipids; Liposomes; Parasites; Protozoa; RNA, Messenger - chemistry; RNA, Protozoan - chemistry; Sensitivity and Specificity; Water; Protozoa; Molecular hybridization; Apicomplexa; Vitamin; Liposome; Artificial membrane; Biotin; Immunological method; Messenger RNA; Cryptosporidium parvum; Heat shock protein; Molecular probe; Detection; Nucleic acid sequence based amplification
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac010012i

To meet the technical challenge of accurately and rapidly detecting Cryptosporidium parvum oocysts in environmental 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 hsp70, followed by amplification using nucleic acid sequence-based amplification (NASBA) methodology as described previously (Baeumner, A. J.; Humiston, M.; Montagna, R. A.; Durst, R. A. Anal. Chem., in press). Subsequently, generated amplicons are hybridized with dye-entrapping liposomes bearing DNA oligonucleotides (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.