title: Development of an aptamer-based concentration method for the detection of Trypanosoma cruzi in blood.
abstract: Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8-25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Bethesda, Maryland, United States of America.
authors.forename: Rana
authors.initials: R
authors.lastname: Nagarkatti
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Bist
authors.forename: Sirena
authors.initials: S
authors.lastname: Sun
authors.forename: Fernanda
authors.initials: F
authors.lastname: Fortes de Araujo
authors.forename: Hira L
authors.initials: HL
authors.lastname: Nakhasi
authors.forename: Alain
authors.initials: A
authors.lastname: Debrabant
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001769:Blood / Q000469:parasitology*; D006801:Humans; D059346:Magnets / Q000737:chemistry; D008863:Microspheres; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D052156:SELEX Aptamer Technique / Q000379:methods*; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide