Amplification and Cloning by Long RT-PCR of Full-length Genome of Larger Segment of Chicken Infectious Bursal Disease Virus.

ABSTRACT

To develop the genetic rescue techniques for infectious bursal disease virus (IBDV), Birnaviridae family, the full-length cDNA of the larger segment of the chicken IBDV was amplified and cloned by long RT-PCR. A comparison of four purification and extraction methods of RNA from IBDV infected chicken embryoid fibroblasts (CEF) showed that the ultracentrifugation followed by proteinase Kdigestion extracted dsRNA more effectively. Then reverse transcription was carried out at 50 degrees using Superscipt II enzyme, followed by RNase H digestion. Amplification of single stranded cDNA in a single step resulted in the synthesis of the full-length segment A of 3 259 bp. The amplified product was cloned and sequenced, identifying that it was an IBDV. This method is superior to other methods based on amplifying different parts of the genome many times, therefore the cloning procedure was simplified.