Detection of IBV-derived RNA by qRT-PCR in tracheal samples from birds used in the protection study.

Notes: Tracheas from three birds per group were removed 4, 5 and 6 days after challenge and kept in RNAlater®. After disruption and homogenisation, RNA was extracted and used as a template for IBV-specific qRT-PCR. Trachea samples were taken from three different birds each day (n = 9), for the control group (mock:4/91) tracheas were taken from one bird per day (n = 3) for assessment. All reactions were performed in triplicate in a 7500FAST Taqman machine. Amplification plots were analysed using Applied Biosystems SDS software. The qRT-PCR method used in this manuscript has previously been assessed for sensitivity, maximum sensitivity being of 100 copies [40].