Event-specific Real-time RPA Detection of Transgenic Rice Kefeng 6  

Chao Xu1 , Liang Li1,2 , Wujun Jin1,2 , Yusong Wan1,2
1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2. Inspection and Testing Center for Environmental Risk Assessment of Genetically Modified Plant-related Microorganisms (Beijing), Ministry of Agriculture, Beijing 100081, China
Author    Correspondence author
GMO Biosafety Research, 2014, Vol. 5, No. 1   doi: 10.5376/gmo.2014.05.0001
Received: 07 Oct., 2014    Accepted: 30 Oct., 2014    Published: 12 Nov., 2014
© 2014 BioPublisher Publishing Platform
This article was first published in Fenzi Zhiwu Yuzhong, 12(5): 875-880 in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Xu et al., 2013, Event-specific Real-time RPA Detection of Transgenic Rice Kefeng 6, GMO Biosafety Research, Vol.4, No.1 1-5 (doi: 10.5376/gmo.2013.04.0001)


GM crops detection commonly uses PCR method currently. It takes too long and needs thermal cycling instruments with accurate temperature control, so it is not suitable for field testing. In this study, event-specific detection method for transgenic insect resistance rice Kefeng 6 and its derivates based on real-time fluorescent recombinase ploymerase amplification (RPA) was established. According to the integration junction sequence of Kefeng 6, we designed and screened a set of RPA primers and probes combination which fit for RPA assays. The results showed that 500 copies of the target molecules in samples could be detected stablely and specifically by the method. The method can finish detection in 10 to 20 min, simplifying the test procedure by real-time fluorescence detection. Detection time was greatly shortened comparing with the conventional PCR detection which needed several hours. It has great application value for field transgenic detection using lithium battery-powered portable apparatus.

GM crops detection; Isothermal amplification; RPA, Rice; Kefeng 6

At the dawn of 21st century, the global genetically modified crops appear overwhelming development trend. Rice as one of the most important staple food crops in China, transgenic rice research and development progress is relatively quickly, and already have a number of excellent lines entered the environmental release phase, applied for commercial planting (Li LiHung et al., 2012, Chinese rice, 18 (6): 1-4). Meanwhile safety issues caused by the proliferation of genetically modified rice (GM rice) attracted considerable public concern, related international trade disputes have also occurred. EU directives issued specifically to enforce the requirement of implementing a comprehensive test to China’s exported rice products. (http://faolex.fao.org/ docs/pdf/eur124539.pdf; http://faolex.fao.org/docs/pdf/ eur124540.pdf). Therefore establishing the quick precise detection method is of vital significance to strengthening the GM rice security management, preventing GM rice without the safe authorization to enter into production, protecting the consumer benefit and the international trade and so on.
Currently PCR technology has been widely used in the detection of genetically modification (Holst-Jensen et al., 2003), but commonly used PCR testing requires precision instruments and complicated test procedures, which is difficult to meet the requirements of field testing in non-laboratory environment. In recent years, isothermal amplification of nucleic acid technology has achieved considerable development. Compared with the commonly used PCR technology, this new technology has no requirement for thermal cycling instrument and can quickly amplify fragments in a short time. It has the advantage of simple, rapid, sensitive and more (Gill and Ghaemi, 2008).
Based on the principle of recombinase polymerase mediated amplification, recombinase polymerase amplification (RPA) analogs the DNA replication in vivo and isothermally amplifies the target fragments at ambient temperature. First, the recombinase initially aggregates with the primers and forms nucleoprotein filaments and the filaments scan the template DNA for homologous sequences. Next, single-stranded DNA binding protein help template DNA melting thus primers and template DNA start pairing (Liu et al., 2010), and extend by DNA polymerase along the 3' hydroxyl end of the primer to form a double-stranded DNA product. At last, a fluorescent probe can be added into the amplification system for real-time detection of the primer extension. There are two T bases in the internal part of the RPA probe, marked with a fluorescent reporter (FAM) and a fluorescent quencher (BHQ1), separately. An abasic nucleotide analogue (dSpacer) is located in the central part of the two fluorescent groups, which can be identified and cleaved by an exonuclease III with a 3'-5' exonuclease activity from E. coli, thereby separating the fluorophore and the quencher and generating a real-time readout (Piepenburg et al.,2006). The use of a quantitative PCR instrument or a portable fluorescence detector can detect the real-time fluorescent signal in 10 to 20 min. Field site inspection can be done by the Twista detector powered by a lithium battery.
The genetically modified (GM) rice Kefeng 6 containing two insect-resistant genes (cry1Ac+SCK) is researched and developed independently by China; currently it has passed the production test and is applying for the Production Application Security Certificate. It has very good commercial prospects (Huang Xin et al, 2010). According to the integration junction sequence of Kefeng 6, we designed and screened a set of RPA primers and probes combination which fit for RPA assays in field transgenic detection.
1 Results and Discussion
1.1 The establishment of the real-time RPA detection method of Kefeng 6
According to the integration junction sequence of Kefeng 6 (GenBank No.: HM124448), we designed 9 RPA primers and 1 probe (Table 1), using the Kefeng 6 rice genomic DNA as template to evaluate the performance of each combinations. 16 primer combinations out of 20 sets of test results successfully produced amplification curves. Finally we selected one primer combination (F4 and R4) based on a low threshold value and ideal plateau fluorescence signal to do the event-specific RPA fluorescence detection (Table 2). Using this combination we tested genomic DNA of the non-genomic rice Minghui 86 and the transgenic rice Kefeng 6. Results showed that significant amplification curve in Kefeng 6 but no detectable amplification signal in Minghui 86 (Figure 1).

Figure 1 Event-specific RPA detection for 1 transgenic rice Kefeng 6 and 2 non-transgenic rice Minghui 8

Table 1 Primers and probe for RPA assay

Table 2 The result of primers screening

Table 3 Test samples for the RPA assay

1.2 Sensitivity of the RPA assays of Kefeng 6
The 47.5 ng/μL genomic DNA isolated from the GM rice (Kefeng 6) was serially diluted with 47.5 ng/μL non-GM rice DNA Minghui 86. The concentration of Kefeng 6 genomic DNA was 10,000, 2,000, 500, 100, 50 and 0 copies/μL at each dilution. In each dilution primer combination F4 and R4 was used to do the RPA fluorescence detection. Results displayed that threshold time value increased and the fluorescent signal value decreased with the decreasing the copy of the template. 100 copies of the target molecules in samples could still produce amplification curve, but 50 and 0 could not; 500 copies or above could produce very distinctive curve and the thresh hold value and the fluorescent signal value fully meet the field inspection requirements (Figure 2).

Figure 2 Sensitivity test of the RPA method. 1-6, copy number of a series of 10,000; 2,000; 500; 100; 50; 0

1.3 Examination specificity analysis
Using the above fluorescent RPA detection method, the specificity of the assay was evaluated at different content of the GM rice Kefeng 6 containing varying amounts of other ingredients (A was 5% GM rice Kefeng 6; B was 1% GM rice Kefeng 6; C was 0.5% GM rice Kefeng 6; D was 25% for each of GM rice TT51-1, M12, Kefeng 8, and Kemingdao 1; E was 25% for each of Non-GM rice Jigeng 88, Nipponbare, Yangdao 6, and Minghui 86; F was 5% for each of GM maize Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, 59122, MIR604, and MON88017; G was 5% for each of GM cotton MON1445, MON531, MON15985, MON88913, LLCOTTON25, sGK321, and sGK9708; H was 5% for each of GM soybean 356043, 305423, CV127, MON89788, A5547-127, A2704-12, and GTS40-3-2). Test results showed that only the samples containing Kefeng 6 (A~C) produced amplification signals, while no amplification signals were observed for the samples not containing Kefeng 6 (D~H) (Figure 3), indicating that this method has a very strong specificity to the GM rice Kefeng 6.

Figure 3 Specificity test of the RPA detection method. A-H: Mix samples as described in Table 3

2 Discussion
Usually the target serial amplification is used in current DNA-based GMO detection methods. According to their level of specificity of target sequence, such methods can be classified as screening (target elements such as 35S promoter and Nos terminator), gene-specific (detection of extraneous source gene), construct-specific (detection of vector components, genes and vector backbone connection sequence), and event-specific detection (detection of inserting DNA exogenous and plant genome integrated area serial). According to the integration junction sequence of Kefeng 6 and the insertion point side serial, we designed a set of RPA primer and probe, so this method is an event-specific detection, which can be used to specifically distinct Kefeng 6 from other transgenic rice strains. At the same time our lab established the screening methods and gene-specific detection methods used in RPA, which will be published in near future.
Currently some nucleic acid isothermal amplification technologies have been used for nucleic acid testing, such as loop-mediated isothermal amplification (LAMP) (Guan et al., 2010; Chen et al., 2011; 2012), nucleic acid sequence-based amplification (NASBA) (Morisset et al., 2008) and more. Compared with those isothermal amplification technologies, the most significant characteristic of RPA is the greatly shortened detection time and the reduced reaction temperature. Ever since this technical being published, it has been applied in pathogenic micro-organisms detection area (Euler et al., 2012a; 2012b; Boyle et al., 2012), but no domestic or foreign publication in GM crop detection.
Because of the high amplification efficiency of the RPA, gel electrophoresis product contamination is more likely happen and the need for delicate equipment limits the point-of-use in field settings. However, with the combinations of primers and probes and using the using lithium battery-powered portable fluorescence detector, the whole process can be finished in 10 to 20 min. In this study the application of fluorescent probes require the testing process completed with closed tubes, thereby avoiding product contamination in RPA and false-positive test results. So it is more suitable for rapid and accurate detection in field settings. The results showed that 500 copies of the target molecules in samples could produce very distinctive amplification curve. So this method requests lowly to the DNA template density and has great application value for field transgenic detection combined with fast DNA extraction methods.
3 Experimental Section
3.1 Materials
The RPA TwistAmp DNA Amplification Exo Kits and the portable fluorescence detector Twista were all purchased from the TwistDX, UK. Plant genomic DNA extraction Kit was purchased from TIANGEN Agro-tech Co., Beijing, China. The RPA primers and probes were synthesized by Sangon Biotech Co., Shanghai, China.
Genuine seeds from GM rice Kefeng 6 (cry1Ac/SCK), TT-51-1 (cry1Ac/cry1Ab), M12 (Xa21), Kefeng 8 (cry1Ac/SCK), Kemingdao 1 (cry1Ab) and the non-GM rice Minghui 86 were collected by our laboratory. GM maize (Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, 59122, MIR604, and MON88017), GM soybean (356043, 305423, CV127, MON89788, A5547-127, A2704-12, and GTS40-3-2), GM cotton (MON1445, MON531, MON15985, MON88913, LLCOTTON25, sGK321, and sGK9708)and non-GM rice (Jigeng 88, Nipponbare, Yangdao 6, and Minghui 86) were provided by Jilin Academy of Agricultural Science.
3.2 Test sample preparation for the RPA assay
GM rice Kefeng 6 and Non-GM rice Minghui 86were mixedaccording to the GM rice quality 5 %, 1 % and 0.5 %, numbered A~C. GM rice TT51-1M12Kefeng 8Kemingdao 1 and other were equal quality mixed to make test sample D. The specific test samples A~H were shown as Table 3.
3.3 Extraction of plant genomic DNA
Plant genomic DNA was extracted from seeds powder and purified using a plant genomic DNA extraction kit (TIANGEN Agro-tech Co., Beijing, China) according to the manufacturer’s instructions. The quality and quantity of the extracted DNA samples were measured with agarose gel electrophoresis and a NanoDrop 1000 UV/Vis spectrophotometer. Deionized double distilled water was used to adjust DNA concentration to about 50ng/µL, and final concentration was measured by NanoDrop 1000 UV/Vis spectrophotometer.
3.4 RPA Assays
RPA reactions were performed in a total volume of 50 μL. 29.5 μL of TwistAmp rehydration buffer, deionized double distilled water 12.5 μL, 2 μL (10 μmol/L) of each RPA primer, 0.5 μL (10 μmol/L) of RPA probe, 1 μL of plant genomic DNA were added into the 0.2 mL TwistAmp Exo reaction tube containing freeze-dried enzyme powder. At last 2.5 μL of magnesium acetate (280 mmol/L) was added to the tube. Mix well and the tubes were immediately placed in the Twista tube scanner device TwistDX to start the reaction at 39 °C for 20 min (for a low template copy number, the strip was removed after 4 min, centrifuge 3~5s and then placed back in the device). For positive samples, the fluorescence signal increased markedly but there was no increase in the negative control samples.
3.5 Primers design and screening
RPA primer length requirement is for 30~35nt. In this research a probe was designed according to the integration junction sequence of Kefeng 6 (GenBank No.: HM124448). 4 forward primers and 5 reverse primers were designed on the both sides of the probe for screening and establishing the detection method.
3.6 Sensitivity of the RPA Assays
The 47.5 ng/μL genomic DNA isolated from the GM rice (Kefeng 6) was serially diluted with 47.5 ng/μL non-GM rice DNA Minghui 86. The concentration of Kefeng 6 genomic DNA was 10,000, 2000, 500, 100, 50 and 0 copies/μL at each dilution. 1 μL of each dilution was used to do the RPA fluorescence detection.
3.7 Specificity analysis of RPA assays
The plant genomic DNA of each sample (A~H) was extracted and diluted to the concentration of 50 ng/μL with deionized double distilled water. RPA assays were conducted as 3.4 RPA Assays to verify the specificity of this method.
Author Contributions
Chao Xu performed experiment design, experiments, data analysis and manuscript drafting. Liang Li and Wujun Jin participated in experiment design, data analysis and manuscript drafting. Yusong Wan conceived and funded the study, and participated in its design and coordination and revised the manuscript. All authors read and approved of the final manuscript.
This research was funded by Genetically Modified Organisms (GMOs) New Species Cultivating Major Special Projects (2011ZX08012-001; 2013ZX08012-001).
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