DETECTION OF DENGUE-2 VIRAL-RNA BY REVERSIBLE TARGET CAPTURE HYBRIDIZATION

A reversible target capture (RTC) sandwich hybridization technique has been developed for the detection of dengue-2 viral RNA. The RTC is a form of sandwich hybridization that utilizes two probes: a poly(dA)-tailed capture probe and a labeled detector probe. Following hybridization of both probes to the analyte in solution, the poly(dA)-tailed capture probe is used to selectively remove the hybrids by capture on oligo(dT)-coated paramagnetic beads. After elution from the beads, the presence of specific hybrids is revealed by detection of the labeled probe. After optimization of all parameters by using P-32-labeled probes, digoxigenin was used as a label to preclude the use of radioisotopes. The sensitivity of the developed RTC procedure was determined. The lowest amount of virus detectable in cell culture lysates by using P-32-labeled probes was 20 PFU, while with digoxigenin-labeled probes, 200 PFU was detectable. The RTC procedure also detected dengue-2 virus in infected mosquitoes, both individually and in pools. The RTC has the advantage of being performed directly on crude samples, eliminating the need for phenol extraction and purification of target nucleic acids. These results indicate that the RTC procedure is sensitive, rapid, and easy to perform and that its use in surveillance programs will allow detection of dengue virus in pools of mosquitoes more rapidly than current procedures.