Detection of Cryptosporidium parvum using oligonucleotide-tagged liposomesin a competitive assay format

To meet the technical challenge of accurately and rapidly detecting Cryptos poridium parvum oocysts in environmental water, the authors developed a sin gle-use visual-strip assay, The first step in the overall assay procedure i nvolves extracting C. parvum's mRNA coding for heat-shock protein hsp70, fo llowed by amplification using nucleic acid sequence-based amplification (NA SBA) methodology as described previously (Baeumner, A. J.; Humiston, M.; Mo ntagna, R. A.; Durst, R. A. Anal. Chem., in press). Subsequently, generated amplicons are hybridized with dye-entrapping liposomes bearing DNA oligonu cleotides (reporter probes) and biotin on their surface, The liposome-ampli con complex is then allowed to migrate upward on a nitrocellulose membrane strip. On the nitrocellulose strip, antisense-reporter probes are immobiliz ed in a capture zone and antibiotin antibodies are immobilized in a second zone above the capture zone. Depending on the presence or absence of amplic on 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 gra y-scale densitometry allows the quantification of liposomes that are presen t 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 establis hed using this competitive format, because the presence or absence of lipos omes can be quantified in the two capture zones.