From research to routine – more information about the use of a new virus identification system in plant health inspection
A joint study by the Finnish Food Safety Authority Evira and the University of Helsinki showed that small RNA sequencing, a method widely used in scientific plant virus research, has a sensitivity suitable also for routine diagnostics. In research, small RNA sequencing is used for simultaneous detection of all known and unknown viruses present in a sample. Because the method is broad-based, it would also be suitable for studying plant health inspection survey samples.
"Plant's antiviral defence system targets and processes viral RNA structures leading to accumulation of virus derived small RNAs into the infected cells. These small RNAs can be extracted from plant tissues and sequenced. Millions of individual small RNA sequences are obtained by deep sequencing and can then be reassembled into partial or full viral genomes in silico, allowing simultaneous identification of all viruses present in the plant by comparison with viral sequences available in databases", says Senior Researcher Johanna Santala, Evira.
A step towards routine tests
Small RNA sequencing and other deep sequencing techniques have quickly gained popularity among virus researchers. They can be used to simultaneously reveal all viruses and viroids infecting a plant. This is a major advantage compared to the traditional methods, such as the PCR and ELISA tests, where one test can only detect the occurrence of one virus or virus group.
"A lot of work is, however, still required to adopt small RNA sequencing for rutine diagnostics. For example, a more user-friendly bioinformatics is needed and the analysis needs to be completely validated. In addition, the cost-effectiveness of the analysis must be taken into account, meaning that the costs per sample must remain reasonable", says Santala.
Potato viruses as models
In the study, the sensitivity of a real-time PCR test used at the Finnish Food Safety Authority Evira to detect potato Y and A viruses as well as the sensitivity of small RNA sequencing were tested, i.e. the smallest amount of virus detectable from a sample by each test was determined. The comparison showed that both tests have a similar sensitivity. However, small RNA sequencing also has the added benefit of detecting all possible viruses infecting the plant. The study combined approximately 30 samples into one sequencing run, which resulted in roughly 15 - 20 million small RNA sequences, or approximately half a million sequences per sample.
Long road of research
"This has been a long road of research. The first article on using small RNA sequencing to identify plant viruses was published in 2009, and the application of the method began immediately at the University of Helsinki. The work that was now published began in 2014, and it was concluded in 2018. It is worth noting that nobody has published a similar sensitivity comparison before, even though there has been much discussion on the sensitivity of the method at international meetings in the recent years", says Santala.
The Evira has been involved in the development of small RNA sequencing both internationally and in Finland since 2013.
The results of the study have been published in an expert peer-reviewed scientific publication:
Santala, J., Valkonen, J.P.T.
Sensitivity of small RNA -based detection of plant viruses. Frontiers in Microbiology 2018: Vol. 9, article 939.
The study was carried out by Senior Researcher Johanna Santala from the Plant Analytics Unit of the Finnish Food Safety Authority Evira and Jari Valkonen, Professor of Plant Pathology at the University of Helsinki.
For further information, please contact:
Senior Researcher Johanna Santala, tel. +358 50 410 0235