Pengembangan Sejumlah Primer untuk Reverse Transcriptase Polymerase Chain Reaction Guna Melacak Virus Flu Burung di Indonesia (DEVELOPMENt OF PRIMERS FOR REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION TO DETECT AVIAN INFLUENZA VIRUS IN INDONESIA)

Avian Influenza Virus Endemicity During the Covid-19 Pandemic in Indonesia

“…Swab samples were placed in transport medium (Dulbecco's modified Eagle's medium; GIBCO, Thermo Fisher Scientific, USA) and portable refrigerator freezer (−20 °C) was used to store samples during the trip to the Indonesian Research Center for Veterinary Science, Bogor, Indonesia. Field samples were then processed in the IRCVS virology laboratory to identify the presence of AI virus using the RT-PCR technique [16][17][18][19]). Positive PCR results will be followed by virus isolation in SPF eggs aged 9-11 days.…”

“…Viral RNA obtained from infected allantoid fluid was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) as per the manufacturer's instructions. AIV subtyping was performed on samples that indicated positive for influenza A [17], but negative with the three H5-specific primers designed by Lee et al [18]; Dharmayanti et al [19] which were then identified with specific primers H9, H3 and H10. RT-PCR was carried out using a 9800 Fast Thermal Cycler Applied Biosystems machine (Qiagen, Hilden, Germany) with the Superscript III One-Step RT-PCR system by Life Technologies (Waltham, MA, USA), using a 10 µL RNA reaction mixture as a template, 2 µL for each primer, 1 µL Taq Polymerase enzyme, 25 µL PCR Master Mix (2X), and NFW (nuclease-free water) up to 50 µL.. RT-PCR was performed using thermal cycling conditions at an initial denaturing temperature at 42 °C for 45 min; 95 °C for 3 min; denaturation at 95 °C for 30 s; annealing at 50 °C for 40 s; extension at 72 °C for 40 s (35 cycles); and final extension at 72 °C for 10 min.…”