Amplification and cloning of infectious bursal disease virus genomic RNA segments by long and accurate PCR

Abstract

Improved methods of reverse transcription, polymerase chain reaction (PCR) amplification, and cloning of full-length coding region of both strands of infectious bursal disease virus (IBDV) variant strain E genome were developed. Denaturation of IBDV RNA by heat in the presence of primers and use of a reverse transcriptase lacking RNase-H activity produced full-length coding region and partial non-coding region cDNA copies of the viral genomic segments. Digestion of the RNA component of RNA-cDNA hybrids by RNase-H followed by long and accurate PCR (LA-PCR) amplification of IBDV cDNA in a single step resulted in the synthesis of 3182 base-pairs (bp) of segment A and 2777 bp of segment B cDNA copies of IBDV genome. The resulting amplicons were successfully cloned and sequenced revealing their identity of IBDV. The LA-PCR method can be utilized for the amplification and cloning of the other IBDV strains or isolates and will greatly enhance the availability of sequence information or infectious cDNA copies of IBDV.