title: Methods in virus diagnostics: from ELISA to next generation sequencing.
abstract: Despite the seemingly continuous development of newer and ever more elaborate methods for detecting and identifying viruses, very few of these new methods get adopted for routine use in testing laboratories, often despite the many and varied claimed advantages they possess. To understand why the rate of uptake of new technologies is so low, requires a strong understanding of what makes a good routine diagnostic tool to begin. This can be done by looking at the two most successfully established plant virus detection methods: enzyme-linked immunosorbant assay (ELISA) and more recently introduced real-time polymerase chain reaction (PCR). By examining the characteristics of this pair of technologies, it becomes clear that they share many benefits, such as an industry standard format and high levels of repeatability and reproducibility. These combine to make methods that are accessible to testing labs, which are easy to establish and robust in their use, even with new and inexperienced users. Hence, to ensure the establishment of new techniques it is necessary to not only provide benefits not found with ELISA or real-time PCR, but also to provide a platform that is easy to establish and use. In plant virus diagnostics, recent developments can be clustered into three core areas: (1) techniques that can be performed in the field or resource poor locations (e.g., loop-mediated isothermal amplification LAMP); (2) multiplex methods that are able to detect many viruses in a single test (e.g., Luminex bead arrays); and (3) methods suited to virus discovery (e.g., next generation sequencing, NGS). Field based methods are not new, with Lateral Flow Devices (LFDs) for the detection being available for a number of years now. However, the widespread uptake of this technology remains poor. LAMP does offer significant advantages over LFDs, in terms of sensitivity and generic application, but still faces challenges in terms of establishment. It is likely that the main barrier to the uptake of field-based technologies is behavioural influences, rather than specific concerns about the performance of the technologies themselves. To overcome this, a new relationship will need to develop between centralised testing laboratories offering services and those requiring tests; a relationship which is currently in its infancy. Looking further into the future, virus discovery and multiplex methods seem to converge as NGS becomes ever cheaper, easier to perform and can provide high levels of multiplexing without the use of virus specific reagents. So ultimately the key challenge from a routine testing lab perspective will not be one of investment in platforms-which could even be outsourced to commercial sequencing services-but one of having the skills and expertise to analyse the large datasets generated and their subsequent interpretation. In conclusion, only time will tell which of the next-generation of methods currently in development will become the routine diagnostics of the future. This will be determined through a combination of factors. And while the technology itself will have to offer performance advantages over existing methods in order to supplant them, it is likely to be human factors e.g., the behaviours of end users, laboratories and policy makers, the availability of appropriate expertise, that ultimately determine which ones become established. Hence factors cannot be ignored and early engagement with diagnostic stakeholders is essential.
authors.affiliation: The Food & Environment Research Agency (FERA), Sand Hutton, York YO41 1LZ, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Boonham
authors.affiliation: International Potato Center (CIP), PO Box 1558, Lima 12, Peru.
authors.forename: Jan
authors.initials: J
authors.lastname: Kreuze
authors.affiliation: Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; in German), Plant Virus Department, Inhoffenstrasse 7B, 38124 Braunschweig, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Winter
authors.affiliation: Plant Research International, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: René
authors.initials: R
authors.lastname: van der Vlugt
authors.affiliation: Plant Research International, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Bergervoet
authors.affiliation: The Food & Environment Research Agency (FERA), Sand Hutton, York YO41 1LZ, UK.
authors.forename: Jenny
authors.initials: J
authors.lastname: Tomlinson
authors.affiliation: The Food & Environment Research Agency (FERA), Sand Hutton, York YO41 1LZ, UK. Electronic address: rick.mumford@fera.gsi.gov.uk.
authors.forename: Rick
authors.initials: R
authors.lastname: Mumford
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay; D059014:High-Throughput Nucleotide Sequencing; D058889:Molecular Typing / Q000295:instrumentation / Q000379:methods*; D009691:Nucleic Acid Denaturation; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D010944:Plants / Q000821:virology*; D012150:Polymorphism, Restriction Fragment Length; D060888:Real-Time Polymerase Chain Reaction
keywords: Field testing; LAMP assays; Molecular diagnostics; Multiplex, Luminex; Next generation sequencing; Real-time PCR