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

• Published in: Journal of virological methods 1999-08, Vol.81 (1), p.83-90
• Main Authors: Liu, Hung J., Chen, Jen H., Liao, Ming H., Lin, Maw Y., Chang, Gan N.
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Animal viral diseases; Animals; Avian reovirus; Avian reovirus 1; Biological and medical sciences; Blotting, Southern; Chickens; Cloning, Molecular; DNA Restriction Enzymes - genetics; Fundamental and applied biological sciences. Psychology; Genes, Viral - genetics; In Situ Hybridization; Infectious diseases; Medical sciences; Microbiology; Orthoreovirus - genetics; Orthoreovirus - isolation & purification; Polymerase chain reaction; Polymerase Chain Reaction - methods; Quail; Restriction endonuclease; Restriction Mapping - methods; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity; Techniques used in virology; Viral Core Proteins - genetics; Viral diseases; Viral Structural Proteins - genetics; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(99)00063-4

A nested reverse transcription (RT)-polymerase chain reaction with subsequent restriction endonuclease analysis was developed for identification of the σ C-encoded gene of avian reoviruses (ARV). PCR products derived from the σ 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 σ 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 σ 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 TCID 50 of ARV in a ethidium bromide stained gel and could be increased further to 10 −1 to 10 −2 TCID 50 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.