Comparative performance evaluation of double-stranded RNA high-throughput sequencing for the detection of viral infection in temperate fruit crops
Résumé
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.