trategies for simultaneous detection of multiple plant viruses

Simultaneous detection of Brazilian isolates of grapevine viruses by TaqMan real-time RT-PCR

“…Each individual amplification reaction in a duplex assay must work under identical conditions, avoiding competition for reagents or inhibition between the individual reactions. In an optimized multiplex reaction the results of multiplex PCR are similar to those obtained from individual reactions for each target (James et al, 2006), such as was verified in this work.…”

“…Especially for RNA viruses, probe-based assays can fail to recognize all characterized virus variants (isolates, strains or subgroups). This is particularly true when mutation or nucleotide substitution occurs within the probe pairing site, resulting in false-negative (James et al, 2006). Marbot et al (2004) demonstrated that a mismatch of a single nucleotide between probe and the target viral sequence may prevent the fluorogenic reaction from proceeding.…”

“…As a consequence, failures in detecting some isolates from a specific geographical region can occur. This emphasizes the need of more sensitive techniques to avoid false negative results (James et al, 2006;Pallas et al, 2009). The aims of this study were to evaluate the efficiency of TaqMan real-time RT-PCR in individual and simultaneous (duplex) reactions for detection of different isolates of ten important viral species that infect grapevines in Brazil.…”

“…Real-time RT-PCR has been used for detection of viruses that infect different host plants (James et al, 2006), including grapevines (Osman et al, 2007;Pacifico et al, 2011), having higher sensitivity than conventional RT-PCR, overcoming limitations of conventional virus detection systems affected by the erratic distribution of viruses in the host, variations in virus titre throughout the season and type of sampling tissue, as observed in grapevines. Additionally TaqMan probes can be labeled with different fluorophores, excited at different wavelengths, allowing the simultaneous detection of more than one virus species in a single reaction.…”

Inter-laboratory evaluation of a duplex RT-PCR method using crude extracts for the simultaneous detection of Prune dwarf virus and Prunus necrotic ringspot virus

“…The main advantage of RT-PCR in the detection of fruit tree viruses is its high sensitivity. Another great advantage is the possibility to simultaneously detect several targets in a single reaction (James et al 2006). This property is particularly interesting for PDV and PNRSV as they are often present in mixed infections and their absence has to be controlled in phytosanitary certification programmes.…”