Dust RNA was extracted from approximately 5 mg of sample using ISOLATE II RNA Mini kit (Bioline, Australia) according to the manufacturer’s recommendations. Dust samples weighing less than 5 mg (2/64) were not processed. Swabs were cut using clean scissors into a 1.5 ml microtube containing 800 μl of sterile buffered phosphate saline. After vortexing for 10 seconds, 200 μl of swab wash was taken and RNA was extracted using GeneJET Viral DNA and RNA Purification Kit (Thermo Fisher Scientific, Australia) following manufacturer’s recommendations providing a final eluted volume of 60 μl. Extracted RNA were stored at -20°C until needed for reverse transcriptase (RT)-PCR analysis.

Extracts were tested for IBV and NDV RNA by a one-step duplex real-time RT-PCR targeting the matrix (M) gene of NDV [33] and the 5′ untranslated region (UTR) gene of IBV [34]. Standards used for quantification of IBV and NDV genome copies (GC) were constructed using target DNA templates transcribed to RNA using TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific, Australia) following the manufacturer’s protocol and used to create standard curves. Viral GC were expressed as log10 GC per milligram of dust for dust samples and log10 GC per swab for swabs. The concentration per swab was calculated by multiplying the viral load per reaction by 80, i.e. ×20 (corresponding to 3μl of 60μl elution volume was used as template for PCR) then × 4 (corresponding to 200 μl of 800 μl total volume of PBS from swabs used for nucleic acid extraction).

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