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Rapid identification of transgenic black pepper using loop-mediated isothermal amplification (LAMP) and real-time LAMP assays

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Abstract

A loop-mediated isothermal amplification (LAMP) and real-time LAMP based assays were developed for quick and sensitive detection of transgenic black pepper plants. Primers (six each) were designed based on the nucleotide sequence of two target regions [kanamycin and Cauliflower mosaic virus (CaMV) 35S promoter] integrated into the genome of transgenic black pepper. Both assays successfully detected the transgenic plants and no cross-reaction was recorded with non-transgenic plants. The sensitivity of LAMP was up to 104 times that of conventional PCR while real-time LAMP was up to 103 times that of LAMP and 107 times to that of PCR. The addition of 6 mM magnesium sulphate and 0.4 M betaine with 1 h reaction time proved optimal for amplification through LAMP assay. The assays were validated by testing putative transformants of black pepper. The present study clearly established that LAMP and real-time LAMP assays can provide a rapid and simple approach for screening transgenic black pepper and other plants transformed by using the above target gene sequences.

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Fig. 1
Fig. 2

Abbreviations

CaMV 35S:

Cauliflower mosaic virus 35S promoter

LAMP:

Loop-mediated isothermal amplification

PCR:

Polymerase chain reaction

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Acknowledgments

We thank Department of Biotechnology, Government of India for financial support, Distributed Information Sub Centre and Director, ICAR-Indian Institute of Spices Research, Kozhikode for providing facilities.

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Authors and Affiliations

  1. Division of Crop Protection, ICAR-Indian Institute of Spices Research, Marikunnu, Kozhikode, 673012, Kerala, India

    Shina Sasi, K. A. Revathy & A. I. Bhat

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  1. Shina Sasi
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  2. K. A. Revathy
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  3. A. I. Bhat
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Corresponding author

Correspondence to A. I. Bhat.

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Fig. S1

Validation of LAMP and real-time LAMP assays for the detection of transgenic black pepper plants by amplifying kanamycin sequences inserted into the transgenic plants. Lane M: molecular size markers; Lane 1–20: test samples of putative transgenic black pepper; Lane NC: negative control; Lane PC: positive control. (a) PCR, (b) LAMP, (c) real-time LAMP. (TIFF 6565 kb)

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Sasi, S., Revathy, K.A. & Bhat, A.I. Rapid identification of transgenic black pepper using loop-mediated isothermal amplification (LAMP) and real-time LAMP assays. J. Plant Biochem. Biotechnol. 24, 466–469 (2015). https://doi.org/10.1007/s13562-015-0302-1

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  • DOI: https://doi.org/10.1007/s13562-015-0302-1

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