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Int. J. Mol. Sci. 2014, 15(10), 18197-18205; doi:10.3390/ijms151018197

Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

1,†
,
1,2,†
,
1,2
and
1,2,*
1
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2
Inspection and Testing Center for Environmental Risk Assessment of Genetic Modified Plant-Related Microorganisms (Beijing), Ministry of Agriculture, Beijing 100081, China
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 14 August 2014 / Revised: 20 September 2014 / Accepted: 29 September 2014 / Published: 10 October 2014
(This article belongs to the Special Issue Detection and Safety Assessment of Genetically Modified Organisms)
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Abstract

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15–25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops. View Full-Text
Keywords: recombinase polymerase amplification (RPA); isothermal amplification; CaMV-35S promoter (P-35S); nos terminator (T-nos); genetically modified crops (GMCs) recombinase polymerase amplification (RPA); isothermal amplification; CaMV-35S promoter (P-35S); nos terminator (T-nos); genetically modified crops (GMCs)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Xu, C.; Li, L.; Jin, W.; Wan, Y. Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops. Int. J. Mol. Sci. 2014, 15, 18197-18205.

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