AbstractThe importance of seeds as virus vehicles for long‐distance dissemination makes essential the availability of adequate methods of analysis to guarantee the quality of seed lots. To improve the repertoire of sensitive methods for seed diagnosis, we have developed quantitative real‐time RT‐PCR assays (RT‐qPCR) based on the TaqMan technology to detect three viruses which are seed transmitted in cucurbits, namely, cucumber green mottle mosaic virus (CGMMV), squash mosaic virus (SqMV) and melon necrotic spot virus (MNSV), and compared these assays with DAS‐ELISA, the main method used for virus detection in seeds. The estimated RT‐qPCR limits of detection were 96, 97 and 740 RNA target molecules for CGMMV, SqMV and MNSV, respectively. The estimated RT‐qPCR analytical sensitivity (highest dilution capable of generating a detectable amplification signal) ranged between 10 and 1 pg/μL for the three viruses. Using RT‐qPCR, we could reliably detect a single SqMV‐ or CGMMV‐contaminated seed among 999 uncontaminated seeds in a seed lot, and sensitivities were 1,000 and 10,000 times of those provided by DAS‐ELISA for SqMV and CGMMV, respectively. Our RT‐qPCR assays have proved to be highly suitable for the analysis of seed lots, and the possibility of their implementation into certification programme should be taken into consideration.