INVESTIGADORES
GIRI Adriana Angelica
congresos y reuniones científicas
Título:
Desarrollo de un ensayo para el tamizaje molecular del virus de hepatitis B en donantes de sangre.
Autor/es:
CASAL, PABLO E; PEREZ, GERMÁN R.; BOLATTI, ELISA M; CHOUHY, DIEGO; TABORDA, MIGUEL A.; GARDIOL, DANIELA; GIRI ADRIANA A
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.