IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Desarrollo de un ensayo para el tamizaje molecular del virus de hepatitis B en donantes de sangre.
Autor/es:
PABLO E. CASAL; GERMÁN R. PÉREZ; ELISA M. BOLATTI; DIEGO CHOUHY; MIGUEL A. TABORDA; DANIELA GARDIOL; ADRIANA A. GIRI
Lugar:
Rosario
Reunión:
Congreso; XVI Congreso y XXXIV Reunión Anual de la Sociedad de Biología de Rosario; 2014
Institución organizadora:
Sociedad de Biología de Rosario
Resumen:
In developing countries, the inaccessible costs of commercial molecular assays and the lack of regional biotechnological developments, has delayed the implementation of nucleic acid testing (NAT) in public blood banks. In this sense, transmission of hepatitis B virus (HBV) during the serological window period is an important concern in transfusional medicine. In this work we show the development of a highly sensitive assay for the molecular screening of HBV that includes a universal internal control (UIC) to verify every analytical step. The assay format comprises the following steps: a fixed amount of the UIC is added to the plasma sample followed by viral DNA extraction; DNA amplification by multiplex PCR with biotinylated reverse primers specific 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 multiplex-PCR has been optimized for the simultaneous amplification of HBV and bacteriophage P1 (UIC) in the same reaction. Assay limit of detection was 15 copies of HBV DNA/reaction. Test specificity was evaluated using plasma samples derived from acute and chronic HBV patients, and healthy donors. The definitive validation of our NAT method will be carry on with the ?Second WHO International Standard for Hepatitis B Virus DNA?, available in our lab. In conclusion, the methodology developed provides a highly sensitive, low cost molecular assay that could be evaluated for its incorporation in the molecular screening of HBV infection of blood donors attending public blood banks of Santa Fe province, Argentina.