A PLASMODIUM-FALCIPARUM-SPECIFIC REVERSE TARGET CAPTURE ASSAY

Plasmodium falciparum DNA is detected with an assay modeled according to the reverse target capture assay described by Morrissey et al. [19] for the detection of Listeria cells. A poly (A)-tailed oligonucleotide (pWZ34), derived from the partial sequence of a 4-kb repetitive unit of P. falciparum, functions as a capture probe and the labelled 21-bp repetitive units specific for P. falciparum serve as a reporter probe. Both probes are complementary to non-overlapping regions of the target DNA and in the presence of high concentrations of chaotropic salts, hybridization efficiently takes place at relatively low temperatures (15 min, 37-degrees-C). The addition of poly(dT)-derivatized ferromagnetic beads allows the formation of A:T base pairing between the tailed beads and the tailed capture probe. Upon applying magnetic force, the target-capture-reporter-probe complex attached to the beads is removed from the reaction mixture, leaving the bulk of unreacted reporter molecules behind. Subsequent washings of the immobilized complex reduces the amount of non-specifically bound reporter probe. After elution of the complex from the beads a new cycle of capture, washing and release of the target-capture-reporter-probe complex is initiated by the additions of unused (dT)-tailed beads. After 3 cycles, the signal-to-noise ratio with 0.1 pg of P. falciparum DNA as a target was as high as 21-27, with a background of 8-10 cpm. The assay is unique in its speed, well suited for large sample numbers, and allows the manipulation of the background at will by simply increasing the number of capture rounds.