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Comparison and Evaluation of Two Diagnostic Methods for Detection of npt II and GUS Genes in Nicotiana tabacum

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Abstract

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.

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Authors and Affiliations

  1. Young Researchers and Elite Club, North Tehran Branch, Islamic Azad University, Tehran, Iran

    Mohammad Amin Almasi & Mehdi Aghapour-ojaghkandi

  2. Department of Biotechnology, Research Institute of Physiology and Biotechnology, University of Zanjan, Zanjan, Iran

    Khadijeh Bagheri & Mohammadreza Ghazvini

  3. Young Researchers and Elite Club, Meybod Branch, Islamic Azad University, Meybod, Iran

    Seyed Mohammad Hosseyni-dehabadi

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  1. Mohammad Amin Almasi
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  2. Mehdi Aghapour-ojaghkandi
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  3. Khadijeh Bagheri
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  4. Mohammadreza Ghazvini
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  5. Seyed Mohammad Hosseyni-dehabadi
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Correspondence to Mohammad Amin Almasi.

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Almasi, M.A., Aghapour-ojaghkandi, M., Bagheri, K. et al. Comparison and Evaluation of Two Diagnostic Methods for Detection of npt II and GUS Genes in Nicotiana tabacum . Appl Biochem Biotechnol 175, 3599–3616 (2015). https://doi.org/10.1007/s12010-015-1529-y

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