Development and validation of RT-PCR tests for the detection and S1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples.

Two tests were developed that allow the detection and genotyping of infectious bronchitis virus (IBV) and other closely related gammacoronaviruses. The first test employs a one-step, reverse transcription-polymerase chain reaction (RT-PCR) assay in which the amplification is monitored in real time using a TaqMan(®) probe. This real-time RT-PCR test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls' eggs. A total of 323 field samples were tested; 176 samples were positive using the real-time RT-PCR method, but only three were positive by virus isolation. Sequencing was used to confirm the positive real-time RT-PCR results for a subset of samples. The test is suitable for swabs and post-mortem samples and has been shown to be highly sensitive and specific. The second test, a genotyping method, was developed for identification of the strain of IBV present in field samples based on nucleotide variations within the gene encoding the S1 subunit of the surface spike (S) glycoprotein. This method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of IBV within the UK. The performance of the test was evaluated using laboratory isolates of IBV and field samples. Both tests are suitable for use in a high-throughput diagnostic laboratory.