Accurate and fast identification is critical to maximising biosecurity response opportunities. Accuracy relies on both the precision of the differential diagnostic process and the robustness of the reference database, irrespective of the technology (e.g. microscope/LUCID key or PCR/GenBank). Time is critical in biosecurity decisions: first-find, border or trade decisions have substantial economic (and political) consequences. Additionally, both false positive and false negative identifications undermine confidence in the biosecurity system. As knowledge of microbial diversity increases, the boundaries that differentiate taxa have become harder to define: horizontal gene transfer, phage activity and non-chromosomal diagnostic loci have all contributed to inaccurate identifications, taxonomic uncertainty and biosecurity conundrums.
Massively parallel and other recent sequencing technologies, combined with sophisticated analytical software, underpin the genomic age of diagnostics. Comparative genomics has revealed new loci, in particular from unculturable taxa, as potential targets for differential diagnostics. Importantly, many of these loci discriminate at the sub-species level. Bioinformatic-informed analyses have identified hypothetical protein coding regions as useful differential diagnostic loci. Many genomes contain hypothetical proteins: putative protein coding regions with no known analogues or predicted function. Ten of the 15 genomic loci identified for sub-species differentiation between clades of Ca. Liberibacter solanacearum were located within hypothetical proteins encoded in the bacterial genomes.
Using these new identification assays in isothermal-based assays provides both fast and accurate in situ identification (point of contact diagnostics). LAMP and RPA are just two of the isothermal techniques available for in-field/orchard or at-border identification of biosecurity pests. Definitive results are available within 25 min of sampling, using battery-powered, communication-enabled isothermal instruments. Depending on the biosecurity mandate, available decisions range from pre-determined response-initiation to limiting site access whilst a sample is prioritised for diagnosis at an accredited laboratory. Biosecurity diagnostics is entering a new phase: accurate and fast point-of-contact identification is the new paradigm.