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Loop-mediated isothermal amplification (LAMP) method for rapid detection of cry1Ab gene in transgenic rice (Oryza sativa L.)

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Abstract

The cry1Ab gene is a foreign gene which encodes Bt insecticidal Cry1Ab protein and was transferred into genomic DNA of plants to acquire insect resistance. Here a loop-mediated isothermal amplification (LAMP) assay with high specificity and rapidity under isothermal conditions was developed for detecting cry1Ab gene in transgenic rice. Partial sequence of cry1Ab gene was used as the target template to design LAMP primers. The reaction conditions were optimized as follows: 60 min of reaction time, 1:3 of outer primer and inner primer concentration ratio, 25 μL of reaction volume and 0.6 μM of betaine. The results of detection could be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with that of regular PCR and real-time PCR. The results showed that the LAMP assay exhibited high specificity and the sensitivity of 3 × 102 copies number of the positive control plasmid, and of 0.5 % genetically modified (GM) contents. In comparison with real-time PCR method, LAMP showed the same results with simple instruments. The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for detecting cry1Ab gene from transgenic rice plants and rice ingredients in processed foods aimed at screening the growing transgenic crops in the fields and detecting genetically modified (GM) ingredients in imported and domestic foods.

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Abbreviations

LAMP:

Loop-mediated isothermal amplification

GMO:

Genetically modified organism

GM:

Genetically modified

EU:

European Union

ELISA:

Enzyme-linked immunosorbent assay

FIP:

Forward inner primer

F1c:

Complementary sequence of F1

BIP:

Backward inner primer

B1c:

Complementary sequence of B1

EB:

Ethidium bromide

PC:

Positive control

NC:

Negative control

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Acknowledgments

This work was supported by the Natural Science Foundation Programs of Zhejiang Province (Y3090617, Y304463), the Innovation Team of Zhejiang Province (No. 2010R50028) and the Key Science and Technological Achievement Transformation Project of Zhejiang Province (No. 2011E61018), China. The authors thank Dr. Mingzhe Zhang (Zhejiang Entry-exit Inspection and Quarantine Institute of Science and Technology, China) for providing GM samples.

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  1. Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Science, China Jiliang University, Xueyuan Street, Xiasha Higher Education Zone, Hangzhou, 310018, China

    Qingchao Li, Jiehong Fang, Xu Liu, Xi Xi, Mujie Li, Yunfei Gong & Mingzhou Zhang

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  1. Qingchao Li
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  2. Jiehong Fang
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Correspondence to Jiehong Fang.

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Li, Q., Fang, J., Liu, X. et al. Loop-mediated isothermal amplification (LAMP) method for rapid detection of cry1Ab gene in transgenic rice (Oryza sativa L.). Eur Food Res Technol 236, 589–598 (2013). https://doi.org/10.1007/s00217-013-1911-3

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