Novel non-radioactive molecular hybridization strategies for the sensitive detection of plant viruses: carnation mottle virus as a case study.

Rapid and sensitive detection of plant viruses is essential for effective disease management. Non-radioactive molecular hybridization using digoxigenin-labelled riboprobes is a robust diagnostic method; however, its sensitivity can limit early virus detection. Here, we report two complementary strategies to improve assay performance using carnation mottle virus (CarMV) as a proof-of-concept model. First, we evaluate the tandem cloning of multiple genomic fragments from a single virus, adapting the polyprobe concept to monovalent detection. Second, we introduce a novel double-hybridization (DH) system in which unlabeled primary probes carrying a common tail hybridize to the target RNA, and a labelled secondary probe binds to the shared tail. When tested on serial dilutions of infected plant extracts, both systems enhance the sensitivity fivefold compared to a conventional single probe. Using full-length CarMV transcripts, the optimized DH system shows the lowest limit of detection: 1.9 × 10⁵ molecules/µL, corresponding to ~400 fg of viral RNA. This represents a 25-fold improvement over a conventional probe and approaches the sensitivity of RT-PCR. Both strategies reported here enhance assay sensitivity, strengthening the diagnostic potential of non-radioactive molecular hybridization. In particular, the DH system provides a streamlined and adaptable platform for developing sensitive polyvalent assays without the constraints of traditional polyprobe engineering. In addition, the estimated cost of molecular hybridization was only 0.03-0.04€ per sample per pathogen, substantially lower than that of other commonly used detection methods. Collectively, these improvements represent a practical advance for diagnostic laboratories and large-scale plant virus surveillance programs.