2015 Volume 21 Issue 4 Pages 517-523
Recently, loop-mediated isothermal amplification (LAMP) method has been rapidly developed and shown promise for direct detection of genetically modified organisms (GMOs). We developed a LAMP method for detecting GM soybean DP-356043-5. The optimum amplification was performed at 65°C within 60min, with a set of four specific primers that recognize six distinct sequences on the target gene. The result verdict was based on SYBR Green I and agarose gel electrophoresis. Different plant materials were utilized to ensure the specificity of developed LAMP assay, and the result indicated high specificity. The sensitivity of LAMP was detected at low concentration (1% – 0.005%), and the limit of detection (LOD) was 0.05%, corresponding to about 20 copies. The results revealed the LAMP assay developed can provide a reliable and efficient method to detect the GM soybean DP-356043-5, which was a rapid and high-throughput analysis.
Genetic modified (GM) technique has been broadly used in plant genetic research, and genetically modified organisms (GMOs) have been significantly increased globally in the past years. Between 1996 and 2013, GM crop hectares have increased by more than 100 fold—from 1.7 million hectares in 1996 to over 175 million hectares in 2013—representing 18 consecutive years of successful commercialization of GM crops (James, 2014). 27 GM soybean events have been granted regulatory approvals in 26 countries since 1996 (James, 2014). The majority were glyphosate-tolerant GM soy, and the herbicide glyphosate was the most widely used herbicide, with a production of 620,000 tons in 2008 (Pollak et al., 2011). With the commercialization of GMOs, the issues of food safety and biosafety highlight increasingly. Many countries and regions strictly lay down the regulations and labeling on GMOs and their products (Moon and Shin, 2012; Song et al., 2014). In China, the mandatory labeling regulation set zero tolerance as the threshold level of GM ingredients; while in Australia, European Union and Japan, their mandatory labeling regulations have the threshold levels of GM ingredients to certain ranges (from 0.9% to 5%)(Zhang and Guo, 2011).
To date, many testing methods have been developed and combined for GMOs detection. They mainly contained DNA-based detection methods and protein-based detection. Among these, PCR (polymerase chain reaction), one kind of DNA-based analytical methods, is commonly used, including singleplex PCR, multiplex PCR, nested PCR, competitive PCR, and real-time quantitative PCR etc (Zhang and Guo, 2011; Kamle and Ali, 2013). Detections of GM soybean DP-356043-5 have been reported, which contained regulation PCR, multiplex PCR, inverse PCR, real-time PCR, and other high-throughput analytical system (Querci et al., 2009; Kluga et al., 2011; Xu et al., 2011; Kim et al., 2013; Mazzara et al., 2013). However, PCR-based methods are strict in equipment and operation, and time-consuming, moreover, inconvenient on field (Gill and Ghaemi, 2008).
Loop-mediated isothermal amplification (LAMP) (Notomi et al., 2000) was an outstanding gene amplification procedure, in which the reaction can be processed at a constant temperature by one type of enzyme, and its rapid and simple features make it clearly different from the existing genetic tests (Tomita et al., 2008). LAMP is a one-step amplification in isothermal environment of 60°C–65°C, taking 30min–60min. It needs a set of four special primers that recognize a total of six distinct sequences on the target DNA, so that the specificity is extremely high. LAMP is not only rapid and simple, but is also a very robust, innovative and powerful molecular diagnostic method (Francois et al., 2011). During the past years, LAMP has been widely used and improved, especially in the event-specific detection, including GM maize T25 (Xu et al., 2013), GM canola MS8 and RF3 (Lee et al., 2009), GM soybean GTS40-3-2, MON89788 (Liu et al., 2009; Guan et al., 2010; Zhang et al., 2013), A2704-12 (Shao et al., 2013; Shao et al., 2014) and DP-305423 (Tang et al., 2013), GM rice KF6, KMD1 and TT51-1 (Chen et al., 2012; Li et al., 2013), GM wheat B73-6-1 (Cheng et al., 2013).
Event DP-356043-5 is a genetically modified soybean that imparts tolerance to glyphosate, developed by Pioneer Company. DP-356043-5 was produced by insertion of the glyphosate acetyltransferase (glyat) gene from Bacillus licheniformis and a modified version of the soybean acetolactate synthase gene (gm- hra) (Appenzeller et al., 2008; Kim et al., 2013). The insertion of the gm-hra gene produces a modified form of the acetolactate synthase (ALS) enzyme. ALS is essential for branched chain amino acid biosynthesis and inhibited by certain herbicides. The modification in the gm-hra gene overcomes this inhibition and thus provides tolerance to a wide range of ALS-inhibiting (Guida et al., 2011). Being tolerant to glyphosate and at least one ALS-inhibiting herbicide, the soybean event DP-356043-5 has been approved for any use in 13 countries since 2007(i). And in early 2009, soybean DP-356043-5 was already one of the new GM soybean events in the commercial pipline in US (Stein and Rodríguez-Cerezo 2010). Consequently, the development of easy and effective detection method for soybean DP-356043-5 has become increasingly important. Only a few publications reported about PCR-based detection of the DP-356043-5 soybean (Xu et al., 2011; Mazzara et al., 2013), but there is no report on the LAMP-based event-specific detection of DP-356043-5 soybean. In present study, we established a simple, rapid, specific and cost-sensitive LAMP assay for detecting DP-356043-5 soybean.
Plant material Event soybean DP-356043-5 seed powder was purchased from IRMM (Institute for Reference Materials and Measurements, Geel, Belgium). The samples of GM soybean (DP-305423, MON87769, CV127, A5547-127, MON87705) and other plants with the transgenic events (GM maize Bt176, GM rice KF6, GM cotton MON1445, GM canola RF1, GM alfalfa J163) were kindly provided by the Center of Science and Technology Development, Ministry of Agriculture of the People's Republic of China (Beijing, China). Non-GM seeds of soybean, wheat and bean were stored in the Agro-Environmental Protection institute, Ministry of Agriculture (Tianjin, China).
DNA extraction The genomic DNA from 100 mg of finely ground material was extracted by the cetyltrimethylammonium bromide (CTAB) method reported by Dellaporta et al. (1983). The quantity and quality of extracted genomic DNA were measured and evaluated by the ultraviolet (UV) spectrophotometer (BioPhotometer plus, Eppendorf), and further characterized by 1% (w/v) agarose gel electrophoresis in 1×TAE buffer.
Primer design The DP-356043-5 soybean event-specific sequences (described in US patent 20080051288) were selected according to Free Patents Online (ii), and the target fragment covered the soybean genome and exogenous sequences. A set of four primers for LAMP were designed by online software of Primer Explorer version 4 (iii). Forward inner primer (FIP) consists of the F1c (complementary sequence of F1) and the F2 sequence. Backward inner primer (BIP) contains the B1c (complementary to B1) and the B2 sequence. The outer primers F3 and B3 locate outside of the F2 and B2 regions and inside of the positive control. The conventional PCR primers (DP-356043-5-F/R), which have been applied in the national standards of China (National Standard of the People's Republic China-the Agriculture Ministry Announcement No. 1782-1-2012 “Detection of genetically modified plants and derived products-Qualitative PCR method for herbicide-tolerant soybean 356043 and its derivates”), were used. The size of target sequence was 145bp. All primers used in this study were detailed in Fig. 1, synthesized and HPLC purified by Sangon Biotechnology Co. Ltd. (Shanghai, China).
Primers' design for GM soybean DP-356043-5 conventional PCR and LAMP assay. Locations and nucleotide sequence for conventional PCR and LAMP primers; conventional PCR primers are in therectangular box and LAMP primers are underlined.
Conventional PCR To compare sensitivity of the established LAMP assays, conventional PCR was performed. The reaction was carried out in 25 µL volume of 2.5 µL of 10 × buffer, 1.5 mM of MgCl2, 0.2 mM of each dNTP, 0.4 µM of each primer, 0.025 U/µL of Hot Start Taq polymerase (Takara, Dalian, China) and 50 ng of genomic DNA. The PCR program included an initial denaturation of 3 min at 94°C, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, with a final extension of 5 min at 72°C. The PCR products were analyzed by agarose gel (2%) electrophoresis.
LAMP assay LAMP assay was performed in a 25 µL total reaction mixture containing 1×ThermoPol Buffer, 2.0 mM MgSO4, 0.6 M betaine (Sigma Aldrich Co.), 3.2 µM each FIP and BIP, 0.2 µM each F3 and B3, 0.8 mM each dNTP mix, 50 ng of genomic DNA, 0.32 U/µL Bst DNA Polymerase (New England Biolabs). The mixture was incubated at 65°C for 60 min, and terminated by heating at 80°C for 10 min. LAMP assay was verified by triplicate, and ddH2O was instead of template DNA as no template control (NTC). At the end of reaction, 0.5 µL of 10000×SYBR Green I was added to the mixture to visualize the LAMP amplification. With the presence of LAMP products, the color will turn green, otherwise it remains orange. Alternatively, 5 µL of each product was analyzed by 2% agarose gel electrophoresis in 1×TAE stained with ethidium bromide.
Primer design for LAMP assay The loop-mediated isothermal amplification (LAMP) employs a set of four specially designed primers (two inner and two outer primer) that recognize a total of six distinct sites (F1c, F2c, F3c sites on the 3′ side and B1, B2, B3 sites on the 5′ side) flanking the amplified DNA sequences on the target (Gill and Ghaemi, 2008; Fu et al., 2011).The inner primers are called the forward inner primer (FIP) and the backward inner primer (BIP), respectively, and each contains two distinct sequences corresponding to the sense and antisense sequences of the target DNA, one for priming in the first stage and the other for self-priming in later stages (Notomi et al., 2000).The ordinary and single-domain primers, F3 and B3, which each recognizes one of the six sites and so amplifies the entire target region, are supplemented by FIP and BIP (Tomita et al., 2008). The DP-356043-5 LAMP primers were designed according to the event-specific sequence of the 5′ end sequence of exogenous integration from US patent 20080051288 (Guida et al., 2011). The detailed locations of LAMP primers in the target DNA sequences are shown in Fig. 1.
Optimization of the LAMP reactions A series of reactions was performed to determine conditions of LAMP. The reaction was conducted with varied concentrations of dNTPs, betaine, Mg2+ ions, and different ratio of outer primers to inner primers. dNTPs concentrations used were 0.2 mM–1.2 mM, and 0.8 mM was found to be satisfactory (Fig 2(a)). Betaine may significantly improve the reaction as known PCR additive (Henke et al., 1997; Ralser et al., 2006; Lajin et al., 2013). 0 M–1.0 M betaine was utilized and the intensity of the typical ladder-like pattern products increased from 0 M to 0.6 M betaine, and remained the same until the concentration reached 1.0 M (Fig 2(b)). Accordingly, combined with others' researches, 0.6 M betaine was determined as optimal concentration (Li et al., 2013; Huang et al., 2014). The LAMP reaction produced large amounts of the insoluble product magnesium pyrophosphate, which was generated by pyrophosphate ions (a byproduct in LAMP) and Mg2+ ions (Chen et al., 2012). The concentration of added Mg2+ was 1.0 mM–8.0 mM, and 2.0 mM was optimum (Fig 2(c)). In addition, inner primers (FIP and BIP) are used in excess, compared to outer primers (F3 and B3) (Tomita et al., 2008). The ratio of outer primers to inner primers was also optimized from 1:2 to 1:20. The result showed that the intensity of the typical ladder-like pattern products increased from 1:2 to 1:16, and remained the same until the ratio reached 1:20. Consequently, the result showed that the optimal ratio of outer primers to inner primers was 1:16 (Fig 2(d)). The most significant advantage of LAMP is the ability to amplify specific sequences of DNA under isothermal conditions between 60°C and 65°C, the suitable temperature for Bst polymerase (Notomi et al., 2000; Cheng et al., 2013). In this experiment, the test temperatures were 61°C, 63°C, and 65°C. No significant difference was observed (Fig 2(e)). The isothermal condition under 65°C was determined as commonly used (Chen et al., 2011; Randhawa et al., 2013; Huang et al., 2014).
Optimization of conditions of LAMP assay. a Different final concentration of dNTPs in LAMP assays. Lane M, 50 bp ladder marker; lane1–6, the final concentration of dNTPs with 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM and 1.2 mM. b Different final concentration of betaine in LAMP assays. Lane M, 50 bp ladder marker; lane1–6, the final concentration of betaine with 0 M, 0.2 M, 0.4 M, 0.6 M, 0.8 M and 1.0 M. c Different final concentration of Mg2+ in LAMP assays. Lane M, 50 bp ladder marker; lane1–8, 1.0 mM, 2.0 mM, 3.0 mM, 4.0 mM, 5. 0 mM, 6.0 mM, 7.0 mM and 8.0 mM. d Different ratio of outer primers to inner primers concentration in LAMP assays. Lane M, 50 bp ladder marker; lane1–8, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18 and 1:20. e Different reaction temperature of LAMP assays. Lane M, 50 bp ladder marker; lane1–3, the reaction temperatures of 61°C, 63°C and 65°C.
Specificity of LAMP assay LAMP can amplify a few copies of DNA to 109 in less than an hour under isothermal conditions and with greater specificity (Notomi et al., 2000). To evaluate the specificity of the developed visual LAMP assay, genomic DNA from GM soybeans (DP-356043-5, DP-305423, MON87769, CV127, A5547-127, MON87705), other plants with the transgenic events (Bt176 maize, KF6 rice, MON1445 cotton, RF1 canola, J163 alfalfa), and non-GM plants (soybean, wheat and bean) were isolated and analyzed. Fig. 3 indicated that the green color of the reaction was only obtained in the sample containing GM soybean DP-356043-5. No color change occurred in other reaction mixture. The specificity of the amplification was further confirmed from gel electrophoresis, with the presence of typical ladder-like pattern only in lane1 (GM soybean DP-356043-5). Nevertheless other samples did not amplify from this primer pair. Furthermore, as shown, the consequence of visual observation by SYBR Green I was consistent with gel electrophoresis. These results declared the high specificity of developed LAMP assay for detecting genomic DNA from GM soybean DP-356043-5.
LAMP specificity for different crops. Lane M, 100 bp ladder marker; lane1, GM soybean DP-356043-5; lane2–6, GM soybeans DP-305423, MON87769, CV127, A5547-127, MON87705; lane7, non-GM soybean; lane8, GM maize Bt 176; lane9, GM rice KF6; lane10, GM cotton MON1445; lane11, GM canola RF1; lane12, GM alfalfa J163; lane13, non-GM wheat; lane14, non-GM bean.
Limit of detection of LAMP assay The limit of detection (LOD) is the lowest amount or concentration of analyte in a sample, which can be reliably detected (Chen et al., 2011; Mazzara et al., 2013). It is an important index to validate a detection method. Many reports located on the LAMP reaction to detect GMOs (Fu et al., 2011). In present study, the sample of GM soybean DP-35604-3-5 was added to non-GM soybean DNA to different final concentrations of 1%, 0.5%, 0.1%, 0.05%, 0.01% and 0.005%. A total of 50 ng genomic DNA was amplified as template in each reaction, and the corresponding copy number of the transformation event DP-35604-3-5 were 400, 200, 40, 20, 4 and 2 copies, respectively. In the light of JRC Scientific and Technical Reports—EUR 24790 EN-2011 “Verification of analytical methods for GMO testing when implementing interlaboratory validated methods”, to calculate the procedure for absolute LOD (LODabs) of a method with 95% confidence it is necessary to analyse 60 PCR replicates for each tested concentration. As this may not be a practical approach, it is proposed to calculate the false negative rate on a lower number of replicates e.g. 10 replicates. The false negative rate has to be below 5% (i.e. all 10 PCR replicates have to be positive). This approach allows an approximate estimation of the LODabs. In present study, amplification reactions were performed in 10 replicates of each diluted concentration (replicates data not shown). As shown in Fig 4(a), the amplified LAMP fragments and green color were steadily observed from 1% level to 0.05% level. The LAMP assay was able to detect 0.05% reliably in 10 out of 10 replicates, so the LODabs of LAMP assay was as low as 20 copies. Meanwhile, the sensitivity of the developed LAMP assays was the same as conventional PCR (Fig4(b)). In addition, there have been some researches on detection of GM soybean DP-35604-3-5 by multiplex PCR, inverse PCR, real-time PCR, and other high-throughput analytical system, and the LOD was 0.04% – 0.05% (Querci et al., 2009; Kluga et al., 2011; Xu et al., 2011; Kim et al., 2013; Mazzara et al., 2013). Comparably, LAMP assay is more rapid and simple. Moreover, the results via visual observation by SYBR Green I was quite consistent with gel eletrophoresis analysis. In brief, the LAMP assay is of high efficiency.
Limit of detection of LAMP assay and conventional PCR. a The sensitivity of LAMP assay. Lane M, 50 bp ladder marker; lane1, NTC; lane2–7, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%; lane8, non-GM soybean. b The sensitivity of conventional PCR. Lane M, 50 bp ladder marker; lane1–9, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%; lane10, NTC.
In this study, we successfully developed the visual and rapid detection for soybean DP-356043-5 with optimized LAMP method. LAMP assay, one isothermal nucleic acids amplification technique, was more efficient and convenient than regular PCR. Adding SYBR Green I dye into the reaction mixture, the amplified products can be directly detected by naked eye instead of conventional gel electrophoresis analysis or fluorescent detection. The developed visual LAMP method was so highly specific and sensitive, and cost effective, that made it to be useful in GM soybean DP-356043-5 samples analysis.
Acknowledgments This work was supported by Central Public Research Institutes Basic Funds for Research and Development (Agro-Environmental Protection Institute, Ministry of Agriculture).
