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Virus Isolation- genome detection

Different IB virus types are idenyified via glycoprotein or nucleic acid sequencing or by using real time PCR.

IBV typing by RT-PCR and RFLP

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. In addition, it can identify more than one virus type in a sample.

IBV RT-PCR/RFLP

RFLP

IBV typing by nucleic acid sequencing

Molecular typing of IBV is also possible through nucleic acid sequencing. Typically the hypervariable regions of the spike glycoprotein gene, which are highly variable sequence regions that correlate with IBV type, are RT-PCR amplified. The amplified product can be directly sequenced to determine the type of IBV in the sample. In addition, the sequence data can be used to search 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.

Chromatogram of IB sequence data

IBV typing by real-time RT-PCR

Real-time RT-PCR is a typical RT-PCR reaction that includes a fluorescent dye or labeled probe that is used to detect the amplified DNA as it is being made. The advantage of real-time RT-PCR is that it can be conducted in a matter of minutes and it is quantitative. Specific real-time RT-PCR tests have been developed for IBV (Callison et al. J. Virol. Methods 138:60-65, 2006).

Ct = cycle threshold or the cycle number where the amount of fluorescence indicates the sample is positive. The lower the Ct value, the more virus in the sample.