Methods in virus diagnostics: From ELISA to next generation sequencing

• Published in: Virus research 2014-06, Vol.186, p.20-31
• Main Authors: Boonham, Neil, Kreuze, Jan, Winter, Stephan, van der Vlugt, René, Bergervoet, Jan, Tomlinson, Jenny, Mumford, Rick
• Format: Article
• Language: English
• Subjects: Enzyme-Linked Immunosorbent Assay; Field testing; High-Throughput Nucleotide Sequencing; LAMP assays; Molecular diagnostics; Molecular Typing - instrumentation; Molecular Typing - methods; Multiplex, Luminex; Next generation sequencing; Nucleic Acid Denaturation; Plant Diseases - virology; Plant Viruses - genetics; Plant Viruses - growth & development; Plant Viruses - isolation & purification; Plants - virology; Polymorphism, Restriction Fragment Length; Real-time PCR; Real-Time Polymerase Chain Reaction
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2013.12.007

•We describe the current state-of-the-art in plant virus diagnostics.•We compared established technologies including ELISA and real-time PCR with new technologies currently in development. These include:.•Field-based testing methods including those utilising LAMP.•Multiplex methods including those based upon Luminex bead arrays.•Methods for virus discovery based upon next-generation sequencing. 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 p