Development of a novel molecular assay based on conventional PCR liquid hybridization and ELISA (NAT-ELISA) for the detection of HIV, HCV, and HBV in blood donors.

PURPOSE

Development of a simple, affordable, and sensitive method based on gene amplification by PCR followed by liquid hybridization for detection of HIV-1 & 2, HCV and HBV in plasma samples (NAT-ELISA) for detection of these viruses particularly in their window period.

METHODS

Viral nucleic acid extracted from WHO International Standards for HIV1, HIV2, HCV and HBV and 199 blood donor plasma samples were amplified by reverse transcription -PCR with forward and biotinylated reverse primers designed from the conserved regions of p-24 gene of HIV-1 and HIV-2, 5' UTR of HCV and middle part of S gene of HBV respectively. The biotinylated amplicons were immobilized on streptavidin coated ELISA plates, denatured chemically, and were hybridized with digoxigenin labelled specific oligonucleotide probes of HIV-1, HIV2, HCV and HBV. Hybridization signals were measured colorimetrically by indirect ELISA using anti-digoxigenin HRP conjugate and TMB substrate at 450 ​nm. Analytical Sensitivity or lower limit of detection (LOD) of this assay was determined with WHO standards.

RESULTS

PCR amplified products of WHO standards were 321bp of HIV-1, 291bp of HIV- 2, 229bp of HBV, and 105bp of HCV. The LOD and LOD endpoints of HIV1, HIV2, HCV and HBV were 13, 6, 15 and 11 IU/ml and 15, 7, 17 and 12 IU/ml respectively. Diagnostic sensitivity and specificity of NAT-ELISA assay were calculated on 199 samples and was 97.4% and 99.4% respectively.

CONCLUSIONS

The results suggest that this assay is highly suitable for the blood banks which do not have facilities for exorbitantly expensive NAT test for the detection of these viruses during their window period particularly in the blood donors who test negative by antibody/antigen screening tests.