Almasi Mohammad Amin, Aghapour-Ojaghkandi Mehdi, Bagheri Khadijeh, Ghazvini Mohammadreza, Hosseyni-Dehabadi Seyed Mohammad
Young Researchers and Elite Club, North Tehran Branch, Islamic Azad University, Tehran, Iran,
Appl Biochem Biotechnol. 2015 Apr;175(8):3599-616. doi: 10.1007/s12010-015-1529-y. Epub 2015 Feb 10.
To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and also a visual detection protocol on the basis of npt II and GUS genes in transgenic tobacco plants were used. Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum leaf discs was performed with plant transformation vector of pBI 121. From kanamycin-resistant plants selected by their antibiotic resistance, four plants were selected for DNA isolation. Presence of the transgene was confirmed in the transformants by PCR and LAMP. In this regard, all LAMP and PCR primers were designed on the basis of the gene sequences of npt II and GUS. The LAMP assay was applied for direct detection of gene marker from plant samples without DNA extraction steps (direct LAMP assay). Also, a novel colorimetric LAMP assay for rapid and easy detection of npt II and GUS genes was developed here, its potential compared with PCR assay. The LAMP method, on the whole, had the following advantages over the PCR method: easy detection, high sensitivity, high efficiency, simple manipulation, safety, low cost, and user friendly.
为了缩短包括聚合酶链反应（PCR）、环介导等温扩增（LAMP）等一些诊断检测所需的时间，还采用了基于转基因烟草植物中npt II和GUS基因的视觉检测方案。使用植物转化载体pBI 121通过根癌农杆菌介导对烟草叶片圆盘进行转化。从通过抗生素抗性筛选出的卡那霉素抗性植物中，挑选出四株植物用于DNA提取。通过PCR和LAMP在转化体中确认转基因的存在。在这方面，所有LAMP和PCR引物均基于npt II和GUS的基因序列设计。LAMP检测用于直接从植物样品中检测基因标记，无需DNA提取步骤（直接LAMP检测）。此外，本文还开发了一种用于快速简便检测npt II和GUS基因的新型比色LAMP检测方法，并将其与PCR检测方法的潜力进行了比较。总体而言，LAMP方法相对于PCR方法具有以下优点：检测简便、灵敏度高、效率高、操作简单、安全、成本低且用户友好。
