Detection of Cucumber green mottle mosaic virus in low-concentration virus-infected seeds by improved one-step pre-amplification RT-qPCR

Abstract Background Seeds are an important medium for long-distance transmission of plant viruses; therefore, appropriate, sensitive methods for detecting low concentrations of virus in seeds are crucial to ensure the quality of seed lots. In the present study, we developed a one-step pre-amplification reverse transcription quantitative PCR (RT-qPCR) assay based on the TaqMan technology to detect Cucumber green mottle mosaic virus (CGMMV) in zucchini seeds. Result Seed powder samples with simulated virus infection at a low concentration were prepared (the mass ratio 1:900 and 1:1000), and their uniformity was verified using one-step pre-amplification RT-qPCR. We used one-step pre-amplification RT-qPCR to detect CGMMV in low-concentration virus-infected seeds and compared this method with double antibody sandwich–enzyme-linked immunosorbent assay and universal RT-qPCR, which are the main methods used for virus detection in seeds. The minimum limit of detection (LOD) of the improved one-step pre-amplification RT-qPCR method for simulated CGMMV-infected seeds in a large sample of seeds is 0.1%. Conclusions It can reliably and stably detect a single virus-infected seed in 1000 seeds and demonstrates a higher detection sensitivity than universal RT-qPCR and double antibody sandwich–enzyme-linked immunosorbent assay. Our improved one-step pre-amplification RT-qPCR assay has proved to be very suitable for the analysis of large seed lots.