Development of loop-mediated isothermal amplification technique for LM canola T45, 73496, and MON88302 and its field application

IF 1.7 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Wonkyun Choi, A-Mi Yoon, Hye Song Lim, Jung Ro Lee
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Abstract

Brassica is an essential genus in agriculture, and gene flow from living modified (LM) organisms to native relatives or non-modified cultivars of the crucifer family is a critical issue in Asia. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, and accurate method for identifying LM crops. This study aimed to develop a LAMP assay for the laboratory diagnosis and field analysis of three novel LM canola events: T45, 73496, and MON88302. The genomic DNA of each LM canola was amplified by incubating at 63 °C for 30 min with a newly developed 2 × LAMP premix containing Bst DNA polymerase and event-specific primer sets. The sensitivity of the LAMP assay for the three canola events in our condition ranged from 100 pg·μL−1 to 1 ng·μL−1. Genetic elements of the three LM events were identified through a combination of the LAMP assay, a syringe-type DNA extraction kit, and a portable isothermal amplifier. This study proposes a novel, simple system based on isothermal amplification for the DNA detection of the three LM canola events in a survey field.

Abstract Image

LM油菜T45、73496和MON88302环介导等温扩增技术的建立及其田间应用
芸苔属(Brassica)是一个重要的农业属,在亚洲,基因从改性活植物(LM)向十字花科的本地亲缘或非改性品种流动是一个关键问题。环介导等温扩增(LAMP)是一种简单、快速、准确的LM作物鉴定方法。本研究旨在开发一种LAMP检测方法,用于实验室诊断和现场分析三种新型LM油菜籽事件:T45、73496和MON88302。用新开发的含有Bst DNA聚合酶和事件特异性引物的2 × LAMP预混料在63°C下孵育30分钟,扩增每个LM油菜的基因组DNA。LAMP法对三种油菜籽事件的敏感性为100 pg·μL−1 ~ 1 ng·μL−1。三个LM事件的遗传因子通过LAMP分析、注射器式DNA提取试剂盒和便携式等温放大器进行鉴定。本研究提出了一种新的、简单的基于等温扩增的系统来检测一个调查领域的三个LM油菜事件。
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来源期刊
Plant Biotechnology Reports
Plant Biotechnology Reports 生物-生物工程与应用微生物
CiteScore
4.10
自引率
4.20%
发文量
72
审稿时长
>12 weeks
期刊介绍: Plant Biotechnology Reports publishes original, peer-reviewed articles dealing with all aspects of fundamental and applied research in the field of plant biotechnology, which includes molecular biology, genetics, biochemistry, cell and tissue culture, production of secondary metabolites, metabolic engineering, genomics, proteomics, and metabolomics. Plant Biotechnology Reports emphasizes studies on plants indigenous to the Asia-Pacific region and studies related to commercialization of plant biotechnology. Plant Biotechnology Reports does not exclude studies on lower plants including algae and cyanobacteria if studies are carried out within the aspects described above.
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