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.
Liu
H J
HJ
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Taiwan, ROC. hjliu@mail.npust.edu.tw
Chen
J H
JH
Liao
M H
MH
Lin
M Y
MY
Chang
G N
GN
D000818:Animals; D015139:Blotting, Southern; D002645:Chickens; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D017403:In Situ Hybridization; D018165:Orthoreovirus / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D011784:Quail; D015183:Restriction Mapping / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
D014758:Viral Core Proteins; D015678:Viral Structural Proteins; D004262:DNA Restriction Enzymes