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

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.

Recombinase Polymerase Amplification for Diagnostic Applications

“…RPA was successfully used to detect major human pathogens including bacteria (6,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), viruses (9, 24 -32 ), fungi (33 ), and parasites (34,35 ), as well as genetically modified organisms (36,37 ) and genetic alterations observed in cancer cells (38,39 ). RPA was also used for HIV diagnosis in low-resource settings (40,41 ) (Tables 2 and 3).…”

Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection

“…also, Listeria monocytogenes, and other viral and fungal pathogens [33]. Regression analysis of threshold -times against DNA template copy numbers of several Real-time RPA assays, showed good correlation expressed as R 2 values between 0.8 and 0.9 [26,34].…”

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

“…The exponential amplification is carried out on the target fragment by initiating a chain exchange reaction to form DNA synthesis [14]. The amplified products can typically be detected after operating only 5-10 min optimally at 37-40 • C. Since the first report in 2006, RPA has been emerging in medical diagnosis [15][16][17], foodborne pathogens [18,19], and genetically modified crops [20,21], as well as research on viruses [22,23]. For the detection of Salmonella, the fimY gene has been reported as unique to the Salmonella species and hence is suitable to use as an appropriate target gene for detecting Salmonella [24].…”