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

Plant Methods

Li Xin¹  Yin Xinying²  Cao Jijuan²  Piao Yongzhe²  Yang Lili²  Zheng Qiuyue² 

[1] Dalian Customs Technology Center;Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, Dalian Minzu University;

关键词: Low concentration virus-infected seed;    Cucumber green mottle mosaic virus (CGMMV);    One-step pre-amplification RT-qPCR;    Sensitivity;    Detection;   

DOI  :  10.1186/s13007-022-00901-2

来源: DOAJ

【 摘 要 】

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

【 授权许可】

Unknown