Sensitive Real-Time RT-PCR for IBV Detection

The oligonucleotides and the probe used for initial screening of IBV were described previously [31 (link)]. These oligonucleotides and the probe target the relatively conserved IBV N gene at nucleotide positions 741–1077 of the IBV Massachusetts H120 reference strain (GenBank accession number: AM260960). RNase free water and Newcastle disease virus strain (HB1) were included as negative controls. One-step RT-PCR was performed with 12.5 μl of 2× RT-PCR buffer mix, 0.5 μlof MgSO₄ (50 mmol/l), 0.5 μl of Rox reference dye (25 mmol/l, Life Technologies), 0.5 μl of Moloneymurine leukemia virus reverse transcriptase (200 U/μl), 0.5 μlof Taq DNA polymerase (Life Technologies), 0.5 μl of primers (0.2 μmol/l), 0.25 μl of probe (0.1 μmol/l), and 5 μl of RNA template to make a final volume of 25 μl.The reaction was carried out using a StepOne Plus real-time PCR system (Smart Cycler, Cepheid, Sunnyvale, CA) at 50 °C for 15 min, 95 °C for 5 min, and followed by 40 cycles of 9 °C for 15 s; 60 °C for 45 s; 72 °C for 30 s, and a final extension step of 74 °C for 5 min. Amplifications were recorded and analyzed, and the threshold cycle (C_t) was determined with StepOne software (Smart Cycler).