Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

GONZALO TOMÁS; MARTÍN HERNÁNDEZ; ANA MARANDINO; YANINA PANZERA; LETICIA MAYA; DIEGO HERNÁNDEZ; ARIEL PEREDA; ALEJANDRO BANDA; PEDRO VILLEGAS; SEBASTIÁN AGUIRRE; RUBEN PÉREZ

JOURNAL OF VIROLOGICAL METHODS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2012 vol. 185 p. 101 - 107

0166-0934

Rapid and reliable detection and classification of infectious bursal disease viruses (IBDVs) is of crucial importance for disease surveillance and control. This study presents the development and validation of a real-time RT-PCR assay to detect and discriminate very virulent (vv) from non-vv (classic and variant) IBDV strains. The assay uses two fluorogenic, minor groove-binding (MGB) TaqMan probes targeted to a single nucleotide polymorphism (SNP) embedded in a highly conserved genomic region. The analyti- cal sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA. The assay demonstrated a wide dynamic range between 102 and 108 standard RNA copies per reaction. Good repro- ducibility was also detected, with intra- and inter-assay coefficients of variation ranging from 0.13% to 2.23% and 0.26% to 1.92%, respectively. The assay detected successfully all the assessed vv, classical, and variant field and vaccine strains and correctly discriminated all vvIBDV strains from non-vvIBDV strains. Other common avian RNA viruses tested negative, indicating high specificity of the assay. The high sensi- tivity, rapidity, reproducibility, and specificity of the real-time RT-PCR assay make this method suitable for general and genotype-specific detection and quantitation.