RT-PCR test for specific indentification of influenzavirus (A/H5N1) in vaccine

Abstract

RT-PCR (Reverse transcription - Polymerase Chain Reaction) is applied to determine the presence of influenza virus A/ H5N1 in vaccine, and to develop an identity process for specific virus strain A/H5N1 in influenza vaccine A/H5N1. Selected samples included: Ivacflu-A/H5N1 vaccine (Institute of Vaccines and Biologicals), Vaxigrip vaccine (Sanofi Pasteur), Influenza virus strain A/ Vietnam/1194/2004(A/H5N1) (NIBSC) was used as positive control; vaccine Varivax (MSD) and DNA/RNA free water was used as negative controls. The results showed that virus strain A/H5N1 was identified as production of RT-PCR that were positive with amplified primer pairs of 2 specific gene sequences of HA whose length 428 and 249 bp. Before starting RT-PCR, it was necessary to eliminate aluminum and the components of RT-PCR reaction included: 5X QIAGEN OneStep RT-PCR Buffer(5µl); dNTP (1µl); forward and reverse primers (1,5 µl); Enzyme (1 µl), H2 O (10 µl), ARN template (5 µl) and thermal cycle of RT- PCR reaction was: 50^oC (30 minutes); 95OC (15 minutes); 94OC (30 seconds); 55OC (30 seconds); 72OC (1 minute); 72OC (10 minutes), 45 cycles.