Ambion magmax 96 ai nd viral rna isolation kit

Viral RNA was extracted from 50 μl of the swab sample on a Kingfisher Flex Magnetic Particle Processor (Thermo Fisher Scientific, USA) using the Ambion MagMAX-96 AI/ND Viral RNA Isolation kit (Life Technologies Corporation, Grand Island, NY, USA)^{10 (link)}. RNA was screened using a Bio-Rad CFX96 Real-Time PCR Detection System on a C1000 Thermocycler (Bio-Rad, Hercules, CA, USA), with TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems, Foster City, CA, USA) and primers/probe specific for the influenza M gene¹¹ . Samples with a fluorescence cycle threshold (Ct) value ≤38 were considered positive. Virus isolation was attempted in embryonated chicken eggs on all samples with a Ct ≤35 as previously described^{12 (link)}. Host species were identified using primers designed to amplify a segment of the mitochondrial cytochrome-oxidase I as described ^{13 (link)}.

Viral ribonucleic acid (RNA) was extracted from 50 μL sample using the Ambion MagMAX-96 AI/ND Viral RNA Isolation kit (Life Technologies Corporation, Grand Island, NY, USA) on a Kingfisher Flex Magnetic Particle Processor (Thermo Fisher Scientific, Waltham, MA, USA) as described.^{23 (link)} Influenza matrix (M) gene real-time reverse transcription PCR (RT-qPCR) was performed on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems, Foster City, CA, USA) and primers/probe as described.²⁴ Samples with a cycle threshold value <38 were considered positive^{25 (link)} and viral isolation in embryonated chicken eggs was attempted as described.^{26 (link)} Isolates were confirmed by hemagglutination assay (HA) and RT-qPCR and viral titers determined by Reed and Munch²⁷ with both 50% tissue culture infectious dose (TCID₅₀) in Madin-Darby canine kidney cells (MDCK) and by 50% egg infectious dose (EID₅₀). Viruses were stored at −80 °C.