Simplified sample processing combined with a sensitive nested polymerase chain reaction assay for detection of infectious bursal disease virus in the bursa of Fabricus.

Abstract

A rapid and sensitive protocol for the detection and amplification of infectious bursal disease virus (IBDV) RNA in the bursa of Fabricius was developed. By digestion with proteinase K and subsequent extraction with phenol and chloroform, IBDV RNA was efficiently released from a single bursa. IBDV RNA extraction time was shortened to 4 hr, compared with 24 hr with the traditional method, and only one bursa was needed instead of five. This more simplified procedure resulted in a significant reduction in costs due to less labor and the reduction in expensive chemicals and reagents. Four primers were selected from the sequence of a hypervariable region in VP2 genes. For the amplification of genomic IBDV RNA, the product (643 bp) of reverse transcriptase-polymerase chain reaction (RT-PCR) was reamplified and double checked by a nested PCR amplifying a 491-bp cDNA. The sensitivity of nested PCR was at least 100 times greater than RT-PCR as determined by dilution of the bursal homogenate. The fidelity of the nested PCR product was confirmed by Southern blotting, demonstrating specificity to the VP2 gene of IBDV. The simplified sample processing, shortened procedure time, and technical ease of this nested PCR render it more suitable for implementation in routine RT-PCR with restriction fragment length polymorphism testing for the detection and differentiation of IBDV RNA, especially for studies of IBDV infections of individual chickens.