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

A nested reverse transcription (RT)-polymerase chain reaction with subseque nt restriction endonuclease analysis was developed for identification of th e 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 ban d of 1023 bp, indicating that there were no apparent insertions or deletion s in this region. Amplification with the nested primer pairs S1M-S1N and S1 P-S1N generated 330 and 239 bp, respectively. PCR products amplified from t he sigma C-encoded of all tested ARVs isolates were further confirmed by So uthern blot hybridization and restriction endonuclease analysis. PCR amplif ied cDNA fragment (1023 bp) cleaved with Pst I generated two fragments of 5 65 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. Th e sensitivity of RT-PCR was examined on nucleic acids from the ARV infected cell cultures. The detection limit was 100 to 10(-1) TCID50 of ARV in a et hidium 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 w hen the cDNA was reamplified with two sets of nested primers. (C) 1999 Else vier Science B.V. All rights reserved.