DEVELOPMENT OF AN ASSAY FOR HEPATITIS B VIRUS MOLECULAR SCREENING IN BLOOD DONORS

PABLO E. CASAL; GERMÁN R. PÉREZ; ELISA M. BOLATTI; DIEGO CHOUHY; MIGUEL A. TABORDA; DANIELA GARDIOL; ADRIANA A. GIRI

Rosario

Congreso; XVI Congreso y XXXIX Reunion Anual de Sociedad de Biologia de Rosario; 2014

Transmission of hepatitis B virus (HBV) infection during the serological window period is an important concern in transfusional medicine. Here we show the development of a PCR assay for the molecular detection of the HBV DNA that includes a universal internal control (UIC) to verify every analytical step, and the following steps: a fixed amount of the UIC is added to the plasma sample before the DNA extraction; amplification by multiplex PCR using biotinylated reverse primers for each target (HBV and UIC); liquid hybridization of the biotinylated amplicons with specific fluorescein-containing probes; hybrid capture into streptavidin-coated microplate wells; colorimetric detection with a monospecific antifluorescein antibody conjugated with horseradish peroxidase, and color production measurement in a microplate reader. The assay had a detection limit of 15 copies/reaction. Preliminary results of the test specificity indicated that the assay was able to discriminate among the different infectious conditions (acute and chronic HBV patients) and healthy donors. However, the clinical specificity of the test need to be determined using a large number of samples with different HBV infectious status and statistical analysis. The definitive validation of our molecular HBV detection method will be carried out using the ?Second WHO International Standard for Hepatitis B Virus DNA?, available in our lab. In conclusion, this HBV test prototype provides a sensitive, low cost molecular assay that could be evaluated for its incorporation in the molecular screening of HBV infection of blood donors attending to public blood banks in Santa Fe province, Argentina.