Abstract
To determine the complete nucleotide sequence of Infectious Bursal Disease virus (IBDV) isolates, an efficient method was developed to generate full-length cDNA of both the genomic A- and B-segments. Reverse transcription was carried out at the highest possible temperature (50°C) for the reverse transcriptase enzyme, and the single stranded cDNA was subsequently amplified by using an optimized PCR. The double stranded, full-length cDNA was efficiently cloned into a high copy number plasmid. Our results show that the entire cDNA of both the A- and B-segment of a classical attenuated isolate (CEF94), and a very virulent field isolate (D6948), can be cloned. The method will simplify greatly the procedure to generate full-length cDNA and determine the nucleotide sequence of the entire genome of IBDV isolates.
Original language | English |
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Pages (from-to) | 49-58 |
Journal | Journal of Virological Methods |
Volume | 84 |
Issue number | 1 |
DOIs | |
Publication status | Published - 2000 |
Keywords
- Full length cDNA
- Infectious Bursal Disease virus
- Reverse transcription