Interlaboratory evaluation of two Reverse-transcriptase Polymeric Chain Reaction-based methods for detection of four fruit tree viruses

Recent technological development of molecular methods has led to the prolifer- ation of new rapid PCR or reverse-transcriptase (RT)-PCR-derived diagnostic tests for plant viruses. Nevertheless, for routine use, the reliability of all these new methods is not widely established and there is still an apprehension to adopt them in official diagnostic for certification of plant material. This is partly because of the lack of confidence in the obtained results and the poor knowledge on the reproducibility and limits of the RT-PCR protocols. There is a lack of information on the adequate risk assessment in the use of this new technology. An interlabor- atory evaluation of two RT-PCR duplex protocols for the detection of four different fruit tree viruses was performed to address these questions. Identical samples were sent as crude extract preparation to each of the participant laboratories. Samples were coded to ensure a double-blind test. General principles of result analysis are described, for example calculation of parameters such as specificity, sensitivity, repeatability, reproducibility, likelihood ratios and post-test probabilities. These parameters and the integration of the protocols within official certification scheme are discussed. Finally, guidelines for researchers desirous of validating their new plant virus diagnostic protocols through interlaboratory evaluation are suggested.