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

Abstract

Recent technological development of molecular methods has led to the proliferation 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 interlaboratory 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.

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

“…In other models, this value can be increased to up to 65 cycles of amplification without increasing the number of positive results (Osman & Rowhani, ). In any case, diagnostic parameters such as likelihood ratios can clarify false positive and negative rates, allowing risk assessment associated with any method (Massart et al ., ; Vidal et al ., 2012a). In this study, the print of a single pedicel from a leaf with HLB symptoms always tested positive with C t values ranging from 32·7 to 39·9.…”

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

“…ASGV and ApMV belong to the most frequently emerged pathogens encounters in pome fruit trees. Several molecular-based methods have been elaborated to perform routine detection [7,8,11,12]. However, a highly sensitive real-time based detection and quantification assay for these viruses is missing so far.…”

Current impact and future directions of high throughput sequencing in plant virus diagnostics

“…Other strategies may help to resolve contamination/low representation and detection threshold issues, for example the detailed analysis of the coverage of the viral genomes by the sequences obtained, since a low but distributed coverage is likely to reflect the presence of a virus while the same number of reads mapping to a single genomic region are more likely to reflect a contamination problem. Associated with these aspects will come questions about the need to validate the NGS-based approaches and therefore about their reproducibility and repeatability both within a given laboratory and between laboratories using the same protocol (Massart et al, 2008(Massart et al, , 2009. Another question concerns the adaptation of the protocols and techniques to particular plant species or tissues or to particular target virus(es) or diagnostic needs.…”