A COMPARISON OF 2 RNA ISOLATION METHODS FOR DOUBLE-STRANDED-RNA OF INFECTIOUS BURSAL DISEASE VIRUS

Infectious bursal disease virus (IBDV), a member of the birnaviridae f amily, contains a bisegmented double-stranded RNA (dsRNA) genome. The segments are linked covalently at 5' termini by a large (90 kDa) viral genomic protein that migrates similar to viral RNA dependent RNA poly merase of IBDV. A proteinase K digestion based approach and acid-guani dium-phenol-chloroform (AGPC) RNA extraction method were used to extra ct dsRNA of IBDV from infected bursae. After extraction, dsRNA of IBDV was purified by precipitation with lithium chloride. The yield and pu rity of dsRNA of IBDV extracted by AGPC method was lower than that of proteinase K digestion based approach. This observation correlates wit h the presence of a genome-linked protein in IBDV. Although dsRNA obta ined by both methods are suitable for reverse transcription-polymerase chain reaction (RT-PCR) amplification of at least up to 1201 base pai rs (bp) of cDNA, dsRNA extracted by the proteinase K digestion method is more suitable than that by AGPC method for the amplification of lon ger fragments (1958 bp) of IBDV cDNA by PCR. (C) 1998 Elsevier Science B.V. All rights reserved.