Viral RNA Extraction and Detection

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)}.

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Jiménez-Bluhm P., Karlsson E.A., Freiden P., Sharp B., Di Pillo F., Osorio J.E., Hamilton-West C, & Schultz-Cherry S. (2018). Wild birds in Chile Harbor diverse avian influenza A viruses. Emerging Microbes & Infections, 7, 44.

Publication 2018

Kingfisher Flex Magnetic Particle Processor Ambion MagMAX-96 AI/ND Viral RNA Isolation kit CFX96 Real-Time PCR Detection System C1000 Thermocycler TaqMan Fast Virus 1-Step Master Mix

Chicken Cytochrome oxidase Eggs Fluorescence Host species Influenza Isolation Mitochondrial Primers Viral rna Virus

Corresponding Organization :

Other organizations : University of Chile, Institut Pasteur du Cambodge, St. Jude Children's Research Hospital, University of Wisconsin–Madison

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Variable analysis

independent variables

Extraction of viral RNA from 50 μl of the swab sample using the Kingfisher Flex Magnetic Particle Processor and the Ambion MagMAX-96 AI/ND Viral RNA Isolation kit

dependent variables

Fluorescence cycle threshold (Ct) value of the samples
Positive or negative status of the samples based on the Ct value (≤38 considered positive)
Successful virus isolation in embryonated chicken eggs for samples with Ct ≤35
Host species identification using primers designed to amplify a segment of the mitochondrial cytochrome-oxidase I

control variables

Not explicitly mentioned

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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