Molecular identification of plant viruses by Sanger sequencing
In Plant Health ‘barcoding’, i.e. identifying and classifying an organism based on its unique DNA sequence of a PCR product generated by a set of conserved primers, has become a standard for the identification of many micro-organisms and pathogens. Plant viruses, however, in general show too little nucleotide sequence conservation between different and even within genera to use such ‘barcoding’ approach for virus identification.
Still, virus species are among others, distinguished by the level of nucleotide or amino acid sequence identity of a particular genomic region. These levels and genomic regions vary between different genera. These criteria are set by individual working groups within the International Committee on Taxonomy of Viruses (ICTV) and can be consulted in the online ICTV Virus taxonomy report.
Given their generally small size, virus genomes contain different compact motifs with varying biological functions. Often these motifs share significant sequence homologies even between unrelated viruses. But in other genome regions clear differences exist between viruses, down to the species levels. These regions can serve as substitute for the ‘barcoding’ regions of other organisms. By using either genus-specific primer sets or virus-specific primer sets to amplify such regions, following Sanger sequencing of these RT-PCR products and analysis of the obtained sequence, one can distinguish between species, . However, given the sometimes significant sequence differences between viruses within a genus or even within a species, there is no guarantee that particular primer sets will always work for all species they are intended for.
In addition, viral sequences can be obtained by high throughput sequencing (HTS), which allows to obtain near complete genomes. However, this technique requires considerable additional technical efforts and no standards are available yet.
In general the methodology can be summarized as follows:
Following amplification of a particular (reverse-transcriptase [RT])-PCR product and checking for its expected size through gel-electrophoresis, the DNA-product is cleaned from the reaction mixture by standard PCR clean-up methods. The PCR product is then sequenced through Sanger sequencing using both RT-PCR primers or, depending on the size of the PCR product, using additional internal primers. Following assembly of the obtained trace files and generation of the consensus sequence, the sequence can then be compared with known sequences of the virus or related viruses through standard BLAST sequence comparison in the ID section of EPPO-Q-bank in combination with GenBank, since EPPO-Q-bank is restricted in number of sequences. For details see EPPO PM7/129(2) DNA barcoding as an identification tool for a number of regulated pests, Appendix 7 Sanger sequencing, consensus preparation and data-analysis (https://gd.eppo.int/standards/PM7/). The last updated version of this standard is also available in EPPO Global database.