Faster, cheaper identification of emerging virus problems (VIRFAST)

Plant pest diagnosis is performed by official laboratories upon request of National Plant Protection Organizations (NPPOs), growers or traders, in samples that inspectors have collected in situ (a consignment, a place of production, an outbreak area, a buffer zone, etc.). Resources allocated to official laboratories have decreased over time, while trade in plants and plant products, and consequently the material to be tested, have increased steadily. As indicated in the Euphresco Strategic Research Agenda (priority R-6), on-site detection and identification tests, that are both high throughput and scalable at contained costs should be developed and validated to accelerate diagnosis (especially in the case of perishable goods) and to relieve pressure on laboratories. In particular, the application of on-site detection and surveillance methods for plant viruses and viroids is needed for the quick assessment of the health status of plant material. New protocols and technologies based on sequencing are under development (such as Oxford nanopore direct RNA sequencing - cDNA sequencing), but need to be tested, optimised and validated in order to be applicable in routine on-site testing. Furthermore, current barriers for the use of novel on-site technologies need to be addressed.

The VIRFAST project explored the possibility of on-site virus diagnosis with the MinION technology (Oxford Nanopore sequencing). Early results indicated that the traditional RNA extraction protocol could be bypassed resulting in a quicker and low-tech solution for practical diagnostics on-site. However, skipping the extraction needs additional development for diagnostic purposes, as the sequencing threshold and, consequently, the sensitivity of the test is greatly reduced. However, it should be noted that validation was performed using dehydrated samples and moreover some participating laboratories used the Oxford nanopore sequencer on crude extract material for the first time. Better analytical sensitivity is expected when working with fresh tissue and if the users are familiar with the use of crude extract material.

Two sample preparation kits were also compared starting from purified RNA extracts. In summary, better results for virus/viroid detection when using cDNA-PCR library preparation approach, compared to direct RNA sequencing approach, due to the higher relative amount of sequencing reads of viral origin. The increased analytical sensitivity when using the cDNA-PCR library preparation opens the possibility of using this method to monitor an outbreak in the field, in a middle-tech laboratory or in remote locations with limited access to plant diagnostics facilities.

A survey of 26 NPPO laboratories showed that while only 15% were using high-throughput sequencing (HTS) in their diagnostic workflows in 2019, almost all wished to adopt it and that improved staff training and reduced costs would be essential for this uptake.

The results of a survey on on-site testing allowed also to observe interesting trends. Twelve laboratories were already using on-site testing with an estimated volume ranging from 25 to 2,500 tests per year. The main context for using on-site testing kits was field inspection (63%), followed by screening test in the laboratory (19%). Time saving was the most important advantage for use. The laboratories also highlighted the ease of use. The three main limitations for a wider use of HTS were the lack of validation data, the limited sensitivity and the cost.