Hj. Liu et al., Identification of the sigma C-encoded gene of avian reovirus by nested PCRand restriction endonuclease analysis, J VIROL MET, 81(1-2), 1999, pp. 83-90
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.