Development of a Real-time Turbidimeter-based LAMP Method for Detection of NPTⅡ Gene in the Genetically Modified Plant

Abstract

Abstract: According to NPTⅡ gene(HE582394.1), the common selective marker gene of genetically modified plant(GMP), six specific primers were designed. A loop-mediated isothermal amplification(LAMP)method for screening GMP was established. Real-time monitoring of the LAMP reaction was achieved by a turbidimeter. Specificity, sensitivity, stability and repeatability of this method were tested. The results showed that the method can specifically detect NPTⅡ gene, and the detection sensitivity of the method was 0.5%. With the genetically modified maize MON863(NPTⅡ positive)samples of 10%, 1%, 0.5%, 0%(W/W)as templates, stability and repeatability testing was conducted, and false negative rate was 0. The results showed that the LAMP method is suitable for specifically detecting NPTⅡ gene in GMP.

Key words: Loop-mediated isothermal amplification(LAMP),real-time turbidimeter, NPTII, Genetically modified organisms, Detection

Kuang Xiaoshan, Hu Songnan, Wang Xiaoyu, Tang Shiming, Cheng Xiaowei, Feng Jiawang. Development of a Real-time Turbidimeter-based LAMP Method for Detection of NPTⅡ Gene in the Genetically Modified Plant[J]. Biotechnology Bulletin, 2014, 0(3): 60-64.

References

[1] 郭桦, 郭祀远.转基因食品安全性的探讨[J].现代食品科技, 2007, 23(8):71-73.
[2] 李俊, 郑秀丽, 邓平建, 等.商品化转基因植物的外源基因及其检测技术[J].中国农业科技导刊, 2008, 10(3):31-39.
[3] Fuchs RL, Ream JE, Hammond BG, et al. Safety assessment of the neomycin phosphotransferaseII(NPTII)protein[J].Biotechnology, 1993, 11(13):1293-1543.
[4] Asensio L, Gonzalez I, Garcia T, et al. Determination of food authenticity by enzme-linked immunosorbent assay(ELISA)[J].Food Control, 2008, 19:1-8
[5] 丁耀魁, 沈娟, 马黎黎.快速检测试纸条法在大豆转基因检测中的应用[J] . 粮油食品科技, 2010, 18(2):45-46.
[6] Morisset D, Stebih D, Cankar K, et al. Alternative DNA amplification methods PCR and their application in GMO detection:a review[J]. Eur Food Res Technol, 2008, 277:1287-1297.
[7] Hernandez M, Esteve T, Prat S, et al. Development of real-time PCR systems based on SYBR? Green I, Amplifluor^TM and TaqMan? techno-logies for specific quantitative detection of the transgenic maize event GA21[J]. Journal of Cereal Science, 2004, 39(1):99-107.
[8] Liu G, Su W, Xu Q, et al.Liquid-phase hybridization based PCR ELISA for detection of genetically modified organisms in food[J].Food Control, 2004, 15(4):303-306.
[9] Volpe G, Ammid NH, Moscone D, et al. Development of an immuno-magnetic electrochemical sensor for detection of BT CRY1AB/CRY-1AC proteins in genetically modified corn samples[J].Anal Lett, 2006, 39(8):1599-1609
[10] Leimanis S, Hernandez M, Fernandez S, et al. A microarray based detection system for genetically modified(GM)food ingredients[J].Plant Molecular Biology, 2006, 61(1):123-139.
[11] Notomi T, Okayama H, Masubuchi H, et al.Loop-mediated isothe-rmal amplification of DNA[J].Nucleic Acids Research, 2000, 28(12):e63.
[12] Clive J. 2011年全球生物技术／转基因作物商业化发展态势[J]. 中国生物工程杂志, 2012, 32(1):1-14.
[13] 李全芬, 李志辉, 王慧翌, 等.转基因生物检测技术研究进展[J].中国畜牧兽医, 2011, 38(1):152-156.
[14] 陈超, 展进涛.国外转基因标识政策的比较及其对中国转基因标识政策制定的思考[J].世界农业, 2007, 11(7):17-20.

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