There is limited information on the compared performance of currently used biological, serological and molecular assays with high-throughput sequencing (HTS) for viral indexing in temperate fruit crops. Here, using a range of samples of predetermined virological status, we compared two performance criteria (inclusivity and analytical sensitivity) of ELISA, molecular hybridization, RT-PCR and double-stranded RNA (dsRNA) HTS for the detection of a total of 14 viruses (10 genera) and four viroids (three genera). Using undiluted samples from individual plants, ELISA had the lowest performance, with an overall detection rate of 68.7%, followed by RT-PCR (82.5%) and HTS (90.7%, and 100% if considering only viruses). The lower performance of RT-PCR reflected the inability to amplify some isolates as a consequence of point mutations affecting primer-binding sites. In addition, HTS identified viruses that had not been identified by others assays in close to two-thirds of samples. Analysis of serial dilutions of fruit tree samples allowed to compare analytical sensitivity for various viruses. ELISA showed the lowest analytical sensitivity but RT-PCR showed higher analytical sensitivity than HTS for a majority of samples. Overall, these results confirm the superiority of HTS over biological indexing in terms of speed, and inclusivity and show that while absolute analytical sensitivity of RT-PCR tends to be higher than that of HTS, PCR inclusivity is affected by viral genetic diversity. Taken together these results make a strong case for the implementation of HTS-based approaches in fruit tree viral testing protocols supporting quarantine and certification programs.
Keywords: Pathogen Detection; Virology.