International Case Study
Traditional diagnostics applied on samples with typical field symptoms of MLN have often given negative results using ELISA or PCR (polymerase chain reaction) for MCMV and Sugarcane mosaic virus (SCMV). Samples collected in the field with typical MLN symptoms were examined using next generation sequencing (NGS).
SCMV was found to be more prevalent than suggested by targeted diagnostics. Additionally, the panel of samples were found to be infected with a range of other viruses, seven of which are described here for the first time. Although not previously identified in the region, Maize yellow mosaic virus (MYMV) was the most prevalent virus after MCMV.
The development of targeted diagnostics for emerging viruses is complicated when the extent of field variation is unknown, something that can be negated by using NGS methods. As a result, exploration with the MinION technology was introduced and may be more readily deployable in resource poor settings. The results show that this sequencer can diagnose known viruses and future iterations have the potential to identify novel viruses.
Next generation sequencing (NGS) technology, was first used for plant virus detection in 2009 and has since been used a number of times to identify the cause of crop diseases. However, such technology is largely not available in Africa due to high cost of equipment, service support and reagent costs.
The introduction of simpler, table top sequencers such as the MiSeq (Illumina) and Ion Torrent has made this technology easier to access, bringing down both the capital cost of the equipment and per sample cost of the reagents. Despite this, the technology is still only available in specialist centralised diagnostic laboratories.
The current technology relies on detecting the incorporation of nucleotides into a second DNA strand complimentary to the target DNA in a process collectively described as sequencing by synthesis. The detection of these reactions requires a sensitive instrument which contributes to the expense of the systems.
For over 10 years the potential of sequencing DNA by passing it through a nanopore has been discussed. This approach was thought to offer potential benefits in terms of equipment simplicity and hence price, the length of reads generated by the sequencing pores and the preprocessing required for the sample before it could be sequenced.
However, it is only recently that a commercial device based on this concept (MinION, Oxford Nanopore Technology Ltd.) has become available. The MinION sequencer has reduced the cost of next generation sequencing instruments by 50x, in addition since it relies on remote, cloud based analysis there is not a requirement for powerful on-site computing platforms, thus it may be useful for implementation in resource poor settings such as Sub Saharan Africa.
The MinION has recently been used for the sequencing of viral cDNA amplicons during the 2015 Ebola outbreak as well as for the detection and characterisation of isolates of Chikungunya, Ebola and Hepatitis C 19 and poxviruses from human blood.
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