Identification of the sigma C-encoded gene of avian reovirus by nested PCR and restriction endonuclease analysis

A nested reverse transcription (RT)-polymerase chain reaction with subsequent restriction endonuclease analysis was developed for identification of the sigma C-encoded gene of avian reoviruses (ARV). PCR products derived from the sigma C-encoded gene of all tested ARVs resulted in a specific DNA band of 1023 bp, indicating that there were no apparent insertions or deletions in this region. Amplification with the nested primer pairs S1M-S1N and S1P-S1N generated 330 and 239 bp, respectively. PCR products amplified from the sigma C-encoded of all tested ARVs isolates were further confirmed by Southern blot hybridization and restriction endonuclease analysis. PCR amplified cDNA fragment (1023 bp) cleaved with Pst I generated two fragments of 565 and 458 bp. The amplified sigma C-encoded gene of ARV was subcloned into PQE 32 vector for further study of its antigenicity and immunogenicity. The sensitivity of RT-PCR was examined on nucleic acids from the ARV infected cell cultures. The detection limit was 10(0) to 10(-1) TCID50 of ARV in a ethidium bromide stained gel and could be increased further to 10(-1) to 10(-2) TCID50 of ARV by Southern blot hybridization using a digoxigenin-labeled cDNA probe. The sensitivity increased approximately 10(3) to 10(4) folds when the cDNA was reamplified with two sets of nested primers.