Polymerase chain reaction methods for the detection of grapevine viruses and viroids

Abstract

The need to diagnose and manage viral pathogens that have been accumulating in grapevines across most of recorded history has become a central focus of modern viticulture. In recent times, the polymerase chain reaction (PCR) has replaced other diagnostic methods, such as biological indexing assays and enzyme-linked immunosorbent assays (ELISA), in most applications. For virus detection, the PCR reaction now provides the highest possible level of sensitivity and specificity in virus identification. Advances in primer production have made degenerate primers routinely available for the detection of broad generic groups of distantly related viruses. The more diverse members of those groups had previously been invisible to PCR reactions primed by specific sequence primers. The PCR process has benefited from declines in the costs of primers, as well as from improved procedures for sample preparation, improvements in the fidelity of thermostable polymerases, and from the integration of computer data processing capabilities into thermocyclers. Real-time fluorescent detection of the progress of the amplification reaction has significantly boosted the precision and accuracy of quantitative PCR analysis. Reverse transcription quantitative PCR (RT-qPCR) has been combined with multiplex combinations of primers each labeled with different fluorescent dyes to allow for the simultaneous detection of specific members of broad groups of viruses in single reactions. The PCR assay in its many forms has become the primary diagnostic tool in plant virus control programs for grapevine.