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Protocol publication

[…] Multiple sequence alignments were performed with the relevant IBV sequences of GenBank in the MEGA software version 5.2.2. The real-time RT-qPCR primers were designed using the online web tool Primer 3 plus. The sequences of the primers for real-time RT-qPCR targeting ORF 1a and amplicon size are listed in Table . Primer-specificity was assessed in silico by use of the Basic Local Alignment Search Tool (BLAST) in public databases and by aligning these primers to ORF 1a genes of pan IBV strains of the worldwide. The risk of primer-dimer formation, and the presence of hairpins at the annealing site were analyzed using OligoAnalyzer 3.1 (Integrated DNA Technologies) with a correction for ion concentrations set at 50 mM for Na+ and 3 mM for Mg2+. All primers used in this study were synthesized by Integrated DNA Technologies and purified by standard desalting. [...] SigmaPlot (Systat Software, Inc.) software was used to analyse the data statistically with one-way analysis of variance (ANOVA). Viral loads in tracheal secretions, plasma and mononuclear cells were log-transformed prior to analysis. The quantification limit of RT-qPCR was 3.4 log10 copies/mL (quantification limit is the concentration/amount of viral RNA copies, which can be quantified reliably). The detection limit of RT-qPCR was 2.4 log10 copies/mL (detection limit is the minimal concentration of viral that can be detected). The results of three independent experiments were shown as mean ± standard deviation (SD). P values of <0.05 were considered to be significant. […]

Pipeline specifications

Software tools BLASTN, OligoAnalyzer, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Infectious bronchitis virus, Gallus gallus