Measuring infectious bursal disease virus RNA in blood by multiplex real-time quantitative RT-PCR

Abstract

A quantitative reverse transcription polymerase chain reaction (RT-PCR) protocol for assessing infectious bursal disease virus (IBDV) RNA levels in blood was developed using the ABI PRISM 7700 Sequence Detection System coupled with TaqMan chemistry. To control for variations in sampling and processing between samples 28S rRNA was co-amplified in a multiplex reaction and used to quantify total RNA. Relative quantification and standardisation was achieved using a log10 dilution series of RNA extracted from IBDV stock. A linear relationship was observed between input RNA and cycle threshold values (C(T)) over 5 log10 dilutions for the IBDV-specific product and 6 log10 dilutions for the 28S rRNA-specific product. As a test of the assay it was used to determine whether differences in susceptibility to IBDV observed between inbred lines of chickens could be detected at the level of viral load in the blood. Viral RNA levels peaked 2 days post-infection when there was significantly less viral RNA in the blood of resistant line 6(1) chickens compared with the more susceptible Brown Leghorns (P = 0.01). These results demonstrate that the course of IBDV infection can be monitored by quantifying IBDV RNA extracted from blood of infected chickens using TaqMan technology.