Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus |
| |
Authors: | Tomás Gonzalo Hernández Martín Marandino Ana Panzera Yanina Maya Leticia Hernández Diego Pereda Ariel Banda Alejandro Villegas Pedro Aguirre Sebastián Pérez Ruben |
| |
Institution: | Sección Genética Evolutiva, Instituto de Biología, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. |
| |
Abstract: | 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 analytical sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA. The assay demonstrated a wide dynamic range between 10(2) and 10(8) standard RNA copies per reaction. Good reproducibility 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 sensitivity, rapidity, reproducibility, and specificity of the real-time RT-PCR assay make this method suitable for general and genotype-specific detection and quantitation. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|