Amplification and
Cloning by Long RT-PCR of Full-length Genome of Larger Segment of Chicken
Infectious Bursal Disease Virus
HUANG Yao-Wei1,2,
YU Lian1*, DING Hong-Mei1, LI Jian-Rong1,2,
SONG Kun-Hua1
( 1Institute of Preventive Veterinary Medicine, Zhejiang
University, Hangzhou 310029, China； �お┆ �2Department of Biomedical
Engineering, Zhejiang University, Hangzhou 310027, China )
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 ℃ 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.
Key words Segment A of infectious bursal
disease virus； Long RT-PCR； full-length cDNA cloning
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