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.
Highlights
The International Service for the Acquisition of Agri-biotech Applications (ISAAA) estimates that millions of farmers cultivated genetically modified (GM) crops over more than 170 million hectares across 27 countries in 2013; the major GM crop species were canola, maize, cotton, and soybean [1].Due to the constant emergence of new GM crops and their derivatives, consumers are becoming increasingly concerned regarding risks posed by GM crops and products
We describe the initial development of a real-time Recombinase polymerase amplification (RPA) assay to detect P-35S and the nopaline synthase gene (T-nos) sequences for purposes of genetically modified organisms (GMOs) screening and detection
We tested 24 and 16 primer combinations for the target elements of P-35S and T-nos, respectively (Table 1), using 100 copies of GM rice (Kefeng 6 strain) genomic DNA to evaluate the performance of each combination based on a short time to amplification onset and ideal plateau fluorescence signal
Summary
The International Service for the Acquisition of Agri-biotech Applications (ISAAA) estimates that millions of farmers cultivated genetically modified (GM) crops over more than 170 million hectares across 27 countries in 2013; the major GM crop species were canola, maize, cotton, and soybean [1]. DNA-based GMO detection methods can be classified as screening, gene-specific, construct-specific, and event-specific detection according to their level of specificity [2]. Recombinase polymerase amplification (RPA) offers a portable, rapid, and highly specific isothermal alternative to PCR and is ideally suited to point-of-use molecular assays for GMO detection. This technique can be combined with a fluorescent probe for real-time detection, and assays can be completed in a short period of time (within 30 min) at a constant temperature (37–42 °C) by simulating in vivo DNA recombination. Initial development of a real-time RPA assay to detect P-35S and T-nos sequences for purposes of GMO screening and detection
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