Kingfisher flex 96 well robot

The viral RNA was extracted from the pooled swab samples (oropharyngeal and cloacal) using a KingFisher Flex 96-well robot (Thermo Scientific, Waltham, MA) and the MagMAX 96 AI/ND Viral RNA Isolation Kit (Ambion, Inc. Austin, TX) in accordance with the manufacturer’s instructions. Real-time reverse transcriptase PCR (rRT-PCR) was used in conjunction with reference primers and probes to detect the presence of the AIV (InfluA) Matrix (M) gene in viral RNA, as described by the CDC and Spackman (48 (link), 49 ). Then, InfluA (M-gene) positive samples were examined with specific subtypes primers of H5, H7, and H9 as previously described (49 , 50 (link)). The samples were considered as AIV positive for the M-gene if the cycle threshold (Ct) was less than 40 and as H5, H7, and H9 positive if Ct < 37 (51 (link)). Samples that tested positive for the M gene but negative for H5, H7, and H9 were classified as A/untyped.

We applied the magnetic bead-based RNA isolation technology for RNA extraction using the MagMAXTM-96 AI/ND Viral RNA Isolation Kit (Applied Biosystems^™, San Francisco, CA) in a KingFisher^™ Flex 96-well robot (Thermo Scientific^™, Waltham, MA) according to the manufacturer’s procedure. Samples were screened for the presence of AIV by targeting the viral M gene. Firstly, we tested the swab samples for the presence of the M gene using real-time reverse transcription PCR (rRT-PCR) with reference primers and probes, following the procedure described by [35 (link)]. Then, we further subtyped the M gene positive samples for the H5, H9, N1, N2, N6, and N8 utilizing primers and probes in an rRT-PCR test [36 (link)], followed by E Spackman and DL Suarez [37 (link)]. The sample was considered positive if the cycle threshold value was <40 [38 (link)]. Among M gene positive samples, those negative for H5, H9, N1, N2, N6, and N8 were considered AIV/untyped.