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

Abstract

Background Seeds were an important medium for long-distance transmission of plant viruses. Therefore, appropriate, more sensitive methods for detecting low concentrations of virus-infected in seeds were crucial to ensure the quality of seed lots. In this study, we have 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 CGMMV-infected at a low concentration were prepared (the mass ratio 1:900 and 1:1000), and their uniformity were 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 universal RT-qPCR and double antibody sandwich–enzyme-linked immunosorbent (DAS–ELISA) assay, the main methods used for virus detection in seeds. The minimum limit of detection (LOD) of the improved one-step pre-amplification RT-qPCR assays for simulated CGMMV-infected seeds in large lots seeds samples were 0.1%. Conclusions One-step pre-amplification RT-qPCR assays could reliably and stably detected a single CGMMV-infected seed in 1000 seeds and demonstrated a higher detection sensitivity than universal RT-qPCR (infected seeds versus healthy seeds 1:900) and DAS–ELISA assay (infected seeds versus healthy seeds 1:500). Our improved one-step pre-amplification RT-qPCR assay have proved to be very suitable for the analysis of large seed lots. Graphical Abstract

Magnetic biosensors for identification of SARS-CoV-2, Influenza, HIV, and Ebola viruses: a review

“…However, in some cases a positive result is not achieved until 28 d after illness onset [18] For SARS-CoV-2 serum, anti-SARS-CoV-2 IgA, IgG, or IgM antibodies against SARS-CoV-2 have been used to detect the Spike (S) or the Nucleocapsid (N) proteins in samples. Searchers have reported various limit of detection (LOD) for this method such as 0.111% LOD for cucumber green mottle mosaic virus [19] 6.25 × 10 -9 (g ml −1 ) and 0.125 (HAU [Hemagglutinating Unit]/50 μl) LOD for H7N9 and live virus, respectively [20] 0.0065 (IU/assay) LOD for human immunodeficiency virus-1 (HIV-1) p24 [21] 25 × 10 -9 g LOD for crimeancongo hemorrhagic fever virus [22] 10 4 (plaque-forming units-PFU) LOD for Dengue virus [23] 10 1.08 , 10 0.74 , and 10 0.92 (IU ml −1 ) for HIV1, HCV, HIV2, HBV, respectively [24] etc.…”