Single Tube Multiplex PCR, Liquid Hybridization and ELISA (Multiplex NAT-ELISA) for rapid detection of HIV,HBV and HCV in window period

Abstract Introduction: A molecular assay based on single tube multiplex PCR, liquid hybridization and indirect ELISA has been designed for rapid detection of HIV-1 & 2, HCV and HBV in plasma samples (multiplex NAT-ELISA) during the window period of these infections. Methods Nucleic acid extracted from the virus standards and blood donor plasma samples were amplified by conventional reverse transcription-PCR with a mixture of optimized concentrations of forward and biotinylated reverse primers followed by liquid hybridization of immobilized denatured biotinylated amplicons with digoxigenin labelled specific probes and indirect ELISA to measure hybridization signals colorimetrically. Performance of Multiplex NAT-ELISA was validated by Analytical Sensitivity, Analytical Specificity, and by the “Accuracy” (Trueness) of the assay. Results Performance characteristics of the assay were established. Analytical sensitivity (LOD95) was 17 IU/ml (10 geq/ml) for HIV-1) 13 IU/ml (73 geq /ml) for HBV, and 15 IU/ml (66 geq/ml) for HCV. Comparison of the assay with the serological screening tests for HIV/HCV antibodies and HBsAg shows a concordance of 98%. Five serologically negative samples yielded HBV DNA in 4 samples and HCV RNA in one sample by NAT-ELISA. Conclusions Multiplex NAT-ELISA assay described here is performed on individual donations. The main advantage of the assay is interdiction of HIV/HCV/HBV virus infections offering a significantly higher sensitivity for detecting blood-borne infections at affordable cost (less than US$ 5 per sample). Moreover, it has analytical sensitivity that matches with that of Procleix Ultrio System and Cobas TaqScreen MPX.