IBV typing by nucleic acid sequencing
Molecular typing of IBV is routinely conducted by sequence analysis of the spike glycoprotein gene. Typically the hypervariable regions of the spike gene, which are highly variable sequence regions that correlate with IBV serotype, are RT‐PCR amplified. The amplified product can be directly sequenced to determine the type of IBV in the sample. In addition, a BLAST or Basic Local Alignment Search Tool, can be used to find similar sequences in GenBank (http://www.ncbi.nlm.nih.gov/), a database of nucleic acid sequences from all over the world. Phylogenetic analysis conducted with the viruses in the database will show how closely the viruses are related. See the Phylogenetic tree.
Chromatogram of IB sequence data.
IBV typing by real‐time RT‐PCR
The spike glycoprotein of IBV contains unique sequences that can be used to identify different types of the virus. First RT‐PCR is conducted to amplify the spike gene then restriction enzymes that act like molecular scissors, specifically cut the amplified product into unique lengths called restriction fragments. When visualized on an agarose gel, the restriction fragments form a pattern that can be used to identify the type of IBV in the sample. This technique can identify all known serotypes and variant viruses of IBV. More importantly, it can be used to identify more than one virus type in a single sample.