One Simple DNA Extraction Device and Its Combination with Modified

Nov 26, 2012 - However, they require expensive instruments with special software, such as ... and Stockyards Administration (USDA/GIPSA) proficiency p...
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One Simple DNA Extraction Device and Its Combination with Modified Visual Loop-Mediated Isothermal Amplification for Rapid On-Field Detection of Genetically Modified Organisms Miao Zhang,†,§ Yinan Liu,†,§ Lili Chen,† Sheng Quan,† Shimeng Jiang,† Dabing Zhang,†,‡ and Litao Yang*,† †

National Center for Molecular Characterization of Genetically Modified Organisms, SJTU-BorLuh Food Safety Center, School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ‡ Institute of Life Sciences and Resources and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Republic of Korea S Supporting Information *

ABSTRACT: Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and crosscontaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

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monitoring and inspection. For GMO inspection and their environmental risk assessment, on-field tests for GM events and/or GM contents need to be done in simple, fast, and inexpensive ways.8,9 The LFS technique based on protein analysis has been mainly used for on-field GMO testing.4,5 However, it is limited to only a small number of GM events due to its protein-specific nature.7 Up to date, there are no more than 15 LFS assays for GMO events. Therefore, development of novel, inexpensive, and highly efficient GMO on-field testing methods targeting at nucleic acid is in great demand. In general, there are three major steps in the GMO detection process using a nucleic acid based analytical method, which are nucleic acid extraction, target amplification, and identification of amplified products. In order to develop a rapid, simple, and effective nucleic acid based method, any improvement or modification aimed at these three steps would be helpful.

n the past 2 decades, the global cultivation area of genetically modified plants has been increasing at a rate of more than 10% each year, reaching 160 million hectares by the end of 2011.1 In the meantime, controversy over potential food and environment risks related to genetically modified organisms (GMOs) has also increased worldwide. To address growing public concerns about GMOs, a series of legislations and regulations have been issued in many countries and regions to strengthen the management of GMOs and label the products containing GM contents.2,3 To implement the GMO labeling regulations, several analytical methods based on nucleic acid or protein detection techniques have been developed and applied, including conventional polymerase chain reaction (PCR), quantitative real-time PCR, DNA chips, enzyme-linked immunosorbent assay (ELISA), and lateral flow strips (LFS), etc.4−6 Because of the high stability of DNA, conventional PCR and real-time PCR methods have become the primarily used approaches for GMO analysis.7 With the rapid development of commercialized GMO events, how to quickly screen and identify these events has become a challenge for GMO © 2012 American Chemical Society

Received: June 13, 2012 Accepted: November 26, 2012 Published: November 26, 2012 75

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Figure 1. Diagram of the developed DNA extraction device and brief protocol for DNA extraction. (A) Detailed diagram of the designed DNA extraction device. (B) Tailor-made filtration column for DNA extraction. (C) Components of the tailor-made column. The silica gel membrane is fixed at the bottom of the column for DNA absorption and elution. Two handles were designed for secure connecting of the column with collection tube. (D) Assembled column collection tube for collection of filtration. (E) The whole assembled DNA extraction device with the syringe (loaded with a piece of yellow cleaning sponge at the tip of the syringe), tailor-made filtration column, and collection tube. (F) Brief work flowchart of DNA extraction from leaf or seed samples using the designed device, including the main steps of sample homogenization, sample lysis with extraction buffer, DNA purification in the filtration column with wash buffers I and II, and DNA eluting with elution buffer.

electrophoresis,24 etc.) with the advantages of high throughput, better specificity, and higher resolution have been developed in addition to agarose gel electrophoresis. However, they require expensive instruments with special software, such as a real-time PCR machine, microarray scanner, and capillary electrophoresis apparatus, etc. To date, most of the reported new GMO detection methods with improved target DNA identification/quantification have been focusing on only one of the three steps and did not address the applicability of these techniques for on-field tests.25−27 Recently, Kiddle et al. reported the development of a new assay called LAMP−BART (LAMP bioluminescent real-time reporter), which can perform real-time on-field quantification of GMOs. In the LAMP-BART assay, a simple DNA extraction method which is suitable for LAMP amplification was developed, and the extracted gDNA could be quantified employing a bioluminescent reporter and a portable commercial instrument.20 In this study, we managed to develop a simple system for GMO detection with modifications to all three steps of DNA extraction, target DNA amplification, and product identification. The system included a low-cost DNA extraction device and modified visual loop-mediated isothermal amplification (vLAMP) assay. It did not require specific instruments. Applicability of this system for on-field detection of GMOs was investigated.

For DNA extraction and purification, the cetyltrimethylammonium bromide (CTAB) method, salt extraction method,10 and commercial kits (DNeasy Plant Mini Kit, Qiagen; Wizard Magnetic DNA Purification System for Food, Promega) are commonly used. During DNA extraction and purification processes using these methods, laboratory instruments such as centrifuges are generally needed, and the whole process could take up to 1−1.5 h. Although many commercial DNA extraction kits have been using novel DNA extraction techniques, such as the most widely used silica-based techniques invented by Boom et al. (silica membrane, silica gel member, and silica membrane spin columns),11 to improve the time and efficiency of DNA extraction, they still require specific laboratory instruments,12,13 which would limit their applications for on-field tests. Some apparatus based on a vacuum manifold (such as QuickGene-Mini80, FUJIFILM) can get rid of the requirement for centrifuges, but a vacuum source is still needed. For target DNA amplification and/or quantification, PCR techniques such as conventional PCR for qualitative analysis, competitive PCR for semiquantitative analysis, and real-time PCR for quantitative analysis have been widely used.14 All of these PCR techniques require various kinds of expensive thermal cyclers. The invention of in vitro isothermal DNA amplification techniques has made it possible to amplify target DNA without using these thermal cyclers, thus dramatically lowering the cost and time for target DNA amplification. Several isothermal DNA amplification techniques such as loopmediated isothermal amplification (LAMP),15−17 nucleic acid sequence-based amplification (NASBA),18 and rolling-circle amplification (RCA)19 have been successfully used in GMO detection. As a novel, rapid, and sensitive amplification technique, LAMP can tolerate several known inhibitors of conventional PCR amplification, particularly acidic polysaccharides.20 Several published reports indicated that Taq DNA polymerase is more prone to inhibition by plant acidic polysaccharides than Bst DNA polymerase.16,21 For identification of amplified products, agarose gel electrophoresis, which separates DNA fragments based on their size differences, is generally used. Some new DNA detection techniques (melt-curves,22 DNA chip,23 and capillary gel

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EXPERIMENTAL SECTION Plant Materials. The non-GMO seeds of maize, rice, soybean, cotton, wheat, and canola used in this study were purchased from local supermarkets in Shanghai, China and tested to be free of GMO contents in our lab. The genetically modified (GM) soybean seed samples of MON89788 and GTS 40-3-2 were developed and kindly provided by Monsanto Company. A single copy of intact exogenous DNA was inserted into the host genome in both MON89788 and GTS 40-3-2. The seed samples of homozygous GM rice T1c-19 and TT51-1 were kindly provided by Huazhong Agricultural University, China. Seeds of homozygous GM rice Kefeng no. 6 were provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Both T1c-1928 and Kefeng no. 629 have a single copy of inserted exogenous DNA 76

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Genomic DNA Extraction Using Commercial Kits. Two commercial gDNA extraction kits, the DNeasy Plant Mini Kit (Qiagen) and Wizard Magnetic DNA Purification System for Food (Promega), were used in comparison to evaluate the performance of our gDNA extraction device. Plant gDNA from seed and leaf samples was extracted using these two kits according to manufacturer’s protocols. Briefly, for the DNeasy Plant Mini Kit, ground tissues were lysed by addition of lysis buffer containing RNase A. Proteins and polysaccharides in the lysate were precipitated by salt precipitation. Tissue debris and precipitates were removed in a single step by filtration through the QIAshredder column. Binding buffer and ethanol were added to the cleared lysate to promote binding of DNA to the DNeasy membrane. gDNAs were then eluted in a small volume of elution buffer or distilled water by centrifugation. For the Wizard Magnetic DNA Purification System for Food, paramagnetic particles were used to bind nucleic acids, contaminants were removed in several washing steps, and gDNAs were then released from the paramagnetic particles. DNA Quantification and Purity Analysis. The qualities and quantities of extracted gDNAs were evaluated and calculated using a NanoDrop 1000 UV−vis spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, U.S.A.). The A260/280 ratio and measured DNA concentrations were recorded. The qualities of extracted gDNA were further analyzed by gel electrophoresis in 1% (w/v) agarose gels (120 V for 30 min). The gels were run in 0.5× TBE running buffer (0.045 mol/L Tris−boric acid, 0.001 mol/L EDTA, pH 8.0) and stained with 100× GelRed fluorescent dye.31 Gel images were then filmed and visually inspected for DNA quality evaluation. Oligonucleotide Primers and Probes. For conventional PCR and quantitative real-time PCR, oligonucleotide primers and TaqMan probes for maize, cotton, soybean, rice, canola, and wheat endogenous reference genes were designed according to information from the Chinese National Standards and GMO Detection Method database (GMDD, http://gmdd. shgmo.org/).32 For LAMP assays, the primer set included two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (loop F and loop B). LAMP primers for CaMV35S promoter, NOS terminator, and Bar gene were newly designed using online software Primer Explorer V4 (Eiken Chemical Co. Ltd., Japan, http://primerexplorer.jp/ elamp4.0.0/index.html). LAMP primers for GM events MON89788, GTS 40-3-2, 59122, MON863, and Tc1507 were described in previous publications.33,34 Sequences of all primers and probes are listed in Supporting Information Table S1. The primers and TaqMan probes were synthesized by Invitrogen Co. Ltd. in Shanghai, China. Primers were polyacrylamide gel electrophoresis (PAGE)-purified, and the TaqMan probes were purified by high-performance liquid chromatography (HPLC). Conventional PCR Assays. Conventional PCR assays were performed on a Veriti thermal cycler (Applied Biosystem, U.S.A.) in a volume of 25 μL. Each reaction mixture contained the following reagents: 10 mM Tris−HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each of dNTPs, 0.2 μM of each primer, 1.25 units of Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd.), and 2 μL of extracted plant gDNA. The PCR program was one cycle of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C, and a final step of 7 min at 72 °C. The amplified products were analyzed by gel electrophoresis in 2% (w/v) agarose gels that

in the host genome, while two copies of exogenous DNA were inserted into the host genome of TT51-1.30 Fresh leaf samples of maize, rice, soybean, cotton, wheat, and canola were collected from plants grown in the greenhouse of our laboratory. Maize and soybean ground seed powder samples from the U.S. Department of Agriculture/Grain Inspection, Packers, and Stockyards Administration (USDA/GIPSA) proficiency program in April 2010 were also used to evaluate the applicability of the developed system for GMO analysis. For the on-field test of blind samples, 100 individual plants were randomly sampled, and one leaf was collected from each plant. The 100 sampled leaves from 100 individual plants were grouped into one test sample. Genomic DNA Extraction Using the Simple DNA Extraction Device. The design concept for our genomic DNA (gDNA) extraction device was based on air pressure filtration, the nucleic acid binding properties of a silica gel membrane, lysing, and the nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate.11 The device was manufactured according to our design (Figure 1A) by Nantian Biotechnology Company (Haimen, China). The silica gel membrane in the filtration column was purchased from Viogene BioTek Company (Shanghai, China). For DNA extraction in the laboratory, seed and leaf samples were homogenized using either a SPEX Freezer Mill 6870 (SPEX SamplePrep, U.S.A.) or mortar/pestle in liquid nitrogen. For the on-field test of leaf samples, plant leaves were manually homogenized in the following procedure: 100 mg of fresh plant leaves was cut into small pieces and put into a 2 mL Eppendorf tube. Am amount of 10 mg of quartz sand and 1 mL of DNA extraction buffer (5 M guanidine thiocyanate, 50 mM Tris, 20 mM EDTA, 21.3 mM Triton X-100, pH 6.4) was then added into the tube. The mixture was ground using a disposable plastic pestle for 1 min. After sample homogenization, gDNA was extracted in the following procedure: (1) Homogenized mixture was shaken for 2 min by hand. (2) The homogenized mixture was then transferred into the barrel of a specially designed syringe that was filled with a piece of sponge at the tip (manufactured by Henghong Company, Shanghai, China). (3) The mixture was filtered through the sponge and into the filtration column by slowly pressing the plunger, and the filtrate in the column was incubated at room temperature for 1 min. (4) A new syringe was connected to the column, and the filtrate was passed through the silica gel membrane by pressing the plunger slowly (gDNA was retained on the silica gel membrane). (5) The column was connected onto a new 2 mL collection tube, 400 μL wash buffer I (5 M guanidine thiocyanate, 50 mM Tris, pH 6.4) was added to the column, the mixture was incubated at room temperature for 1 min and filtered through the silica gel membrane by applying air pressure using the syringe. (6) An amount of 200 μL of wash buffer II (10 mM Tris, 100 mM NaCl, pH 8.0) was added to the column, the mixture was incubated at room temperature for 1 min, filtered through the silica gel membrane again using the syringe, and the silica gel membrane was air-dried at room temperature. (7) The column was connected onto a new 2 mL collection tube;. 50 μL of DNA elution buffer (10 mM Tris− HCl, pH 8.0; 1 mM EDTA, pH 8.0) was added to the column, and the mixture was incubated at room temperature for 1 min. gDNA was finally eluted from the silica gel membrane into the collection tube by air pressure. The brief procedure of DNA extraction using the designed device is also shown in Figure 1F. 77

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Table 1. Comparison of the Quality of Extracted DNA DNA qualitya species

ploidy

leaves

seeds

soybean rice canola cotton maize wheat

2 2 4 2 2 6

1 1 1 1 3 2

1 1 1 1 3 1

soybean rice canola cotton maize wheat

2 2 4 2 2 6

1 1 1 1 2 2

1 2 1 1 1 2

soybean rice canola cotton maize wheat

2 2 4 2 2 6

2 1 2 1 1 1

1 1 1 1 1 1

A260/280b leaves

yield (ng/mg)c

seeds

leaves

seeds

DNeasy Plant Mini Kit 2 1 5 3 3 3 3 3 3 1 2 1 Wizard Magnetic DNA Purification 3 1 1 2 3 2 4 3 2 2 2 3 3 3 1 3 3 1 DNA Extraction Device Developed in This Work 3 3 2 1 2 3 3 4 3 3 2 3 3 2 3 3 1 2 3 3 2 1 2 2

PCR leaves

2 positive 3 positive 2 positive 1 positive 1 positive 1 positive System for Food 2 positive 2 positive 2 positive 1 positive 2 positive 1 positive 2 3 3 2 1 2

positive positive positive positive positive positive

seeds

timed (min) ∼45

∼120

∼15

1 = no DNA degradation; 2 = slight DNA degradation; 3 = moderate DNA degradation. b1 = 1.40 ≤ A ≤ 1.64; 2 = 1.65 ≤ A ≤ 1.79; 3 = 1.80 ≤ A ≤ 1.99; 4 = 2.0 ≤ A ≤ 2.09; 5 = A ≥ 2.1. c1 = yield ≥ 50 ng/mg; 2 = 20 ng/mg ≤ yield ≤ 50 ng/mg; 3 = yield ≤ 20 ng/mg. dApproximate time needed to finish the DNA extraction process. a

were run in 0.5× TBE and stained with GelRed fluorescent dye. Each PCR assay was performed in two replicates for each template gDNA, and each assay was repeated three times. TaqMan Real-Time PCR Assays. TaqMan real-time PCR assays were carried out on a Rotor-Gene 3000A (Corbett Research, Australia) fluorescent thermal cycler. The real-time PCR reaction was 25 μL and contained the following reagents: 1× PCR buffer (10 mM Tris−HCl pH 8.3, 50 mM KCl), 0.1 mM each of dNTPs, 320 nM of each primer, 100 nM TaqMan probe, 1.5 units of Taq DNA polymerase, 6.5 mM MgCl2, and 5 μL of extracted gDNA. Real-time PCR reactions were performed using the following program: one cycle of 10 min at 94 °C, followed by 45 cycles of 15 s at 94 °C and 1 min at 60 °C. The fluorescent signal was monitored during every PCR cycle at the annealing step. PCR data was analyzed using Rotor Gene 3000 software (version 6.0). All PCR reagents were purchased from Biocolor Co. (Shanghai, China). Each real-time PCR reaction was repeated three times and each time with three replicates. Preparation of Microcrystalline Wax Encapsulated Detection Beads. The microcrystalline wax encapsulated detection beads were made from microcrystalline wax (no. 85, melting point = 85 °C. Sinopec, Nanyang, P. R. China). To prepare the detection beads, 100 μL of heat-melted wax was added into a capsule mold and solidified when the temperature cooled down after 1 min. An amount of 1 μL of SYBR green I (1000×) fluorescent dye (Generay Biotech Co., Ltd. Shanghai, China) was then added onto the solidified wax. Another 100 μL of heat-melted wax was poured on top of the SYBR green I dye and cooled down to solidify, making the final detection bead embedded with SYBR green I in the middle. Visual Loop-Mediated Isothermal Amplification Assays. The vLAMP reaction mixture consisted of 20 mM Tris− HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 4 mM

MgSO4, 0.1% Triton X-100, 0.5 M betaine (Sigma, U.S.A.), 0.8 μM each of inner primers FIP and BIP, 0.4 μM each of outer primers F3 and B3, 0.2 μM each of loop primers F and B, 400 μM each of dNTPs, 8 units of Bst DNA polymerase large fragment (New England Biolabs, U.S.A.), and 2 μL of template gDNA. Before the starting of LAMP amplification, one detection bead embedded with 1 μL of 1000× SYBR green I fluorescent dye was placed in the LAMP reaction tube without touching the reaction mixture at the bottom. The LAMP reaction mixture was incubated at 65 °C for 40 min in a thermostatic water bath. After the vLAMP amplification, the reaction was incubated at 85 °C for 2 min to melt the detection bead. vLAMP products could be visually observed in real-time for the color change (green indicates a positive result, and orange indicates a negative result) or by agarose gel electrophoresis analysis.16 Each vLAMP assay was performed in two replicates for each template gDNA, and the assay was repeated three times.

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RESULTS AND DISCUSSION Design of the DNA Extraction Device. In general, a centrifuge is required for commonly used DNA extraction methods such as the CTAB method or commercial DNA isolation kits. This requirement for the specific laboratory instrument has limited the applicability of these DNA extraction methods for on-field sample analysis. In this study, we designed a simple device for DNA extraction without using a centrifuge or any other specific laboratory instruments. The schematic illustration and detailed size measurement of the device is shown in Figure 1A. The device consisted of two parts: a tailor-made filtration column using a silica gel membrane and a modified medical syringe equipped with a sponge filter. For the filtration column (Figure 1B), the connecting interface between the column and the syringe was 78

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amplifications using gDNA extracted by the two commercial kits (Figure 2, parts C and D). The quantitative real-time PCR assays were used to check for presence of potential PCR inhibitors in the extracted gDNA. In real-time PCR assays, four DNA dilutions (serially diluted with a 4× dilution factor) were used to construct amplification standard curves. Linearity of the standard curves and PCR efficiency were calculated to evaluate the presence of PCR inhibitors in extracted gDNA.35,36 The linearity and PCR efficiency of real-time PCR assay of endogenous reference genes from wheat, soybean, rice, canola, cotton, and maize are shown in Table 3. We found that almost all real-time PCR assays have good linearity (>0.99) and high PCR efficiency (>0.80), except for assays of wheat leaf DNA (0.72), wheat seed DNA (0.73), and canola seed DNA (0.74). Also, the linearity and PCR efficiency of the real-time PCR assays with gDNA extracted from our device were comparable to those using the Qiagen and Promega commercial kits. All these data indicated that there were few PCR inhibitors in gDNA extracted from our device, and the quality of the gDNA was good enough for DNA amplification. On the basis of the summarized testing results (Table 1), the designed DNA extraction device and its standard operating procedure have advantages of low cost, time-saving (10−15 min), no need of specific lab instruments, and yet good DNA quality. Despite of the lower DNA yields compared with those using the two commercial DNA extraction kits, this device should have good applicability in rapid on-field nucleic acids extraction, particularly for on-field gDNA extraction where access to specific laboratory instruments (such as centrifuges) is not available. In addition, the DNA extraction device is very versatile, and many combinations of resins, membranes, and different extraction buffers can be used with this device. Modification of vLAMP Assays. In general vLAMP assays, amplified products are visually inspected for color change by adding fluorescent dye into the reaction mixture after the vLAMP reaction is finished. Because of the high sensitivity and high DNA yields of vLAMP amplification, template contaminations in post-LAMP operations are easy to happen, which in turn could cause false positive results in subsequent vLAMP assays. One solution to avoid opening the reaction tube at the end of a vLAMP reaction is to add fluorescent dyes directly into the vLAMP reaction mix, but one potential problem with this practice is that fluorescent dyes such as SYBR green I can greatly decrease the efficiency of vLAMP amplification.37 To solve this problem, we modified the vLAMP method by introducing a microcrystalline wax encapsulated detection bead in the reaction mixture. Instead of adding fluorescent dye after LAMP amplification, the detection bead containing fluorescent dye was directly added into the reaction tube at the beginning of vLAMP amplification. The fluorescent dye in the detection bead was not in touch with the reaction mixture during vLAMP amplification and was released into the reaction mixture by melting the detection bead at 85 °C after the amplification was finished. The released SYBR green I dye binds to the amplified DNA products, so the amplification results could be visually inspected by monitoring color change. Analytical Sensitivity of the Integrated Simple DNA Extraction/Detection System. By combining the simple DNA extraction device with the modified vLAMP assay described above, we have developed a quick, simple, and effective system for target DNA analysis, which would be very useful for on-field testing of GMOs. Because sensitivity is one

carefully designed to make sure the connection was tightly sealed, which would prevent air leakage or spilling of filtrate. The silica gel membrane was fixed at the bottom of the column, and the column was designed to fit onto 2 mL collection tubes (Figure 1D). For the modified syringe (Figure 1E), a piece of yellow cleaning sponge that served as a molecular sieve was packed into the tip of the syringe barrel. This piece of yellow cleaning sponge could separate solid debris from soluble lysates in the crude extract to avoid obstruction of the silica gel membrane. Lysate filtration, DNA binding, washing, and elution were carried out manually by applying air pressure using the syringe, eliminating the need for a centrifuge or other laboratory instruments. The whole DNA extraction process was rather quick, which can be finished within 10−15 min (Figure 1F). The simplicity, quickness, and portability of this device made it well-adapted for on-field DNA extraction and molecular testing. Evaluation of the Applicability of the Designed DNA Extraction Device. To evaluate the applicability of this DNA extraction device on various plant species and different tissue types, we used this device to extract gDNA from seeds and leaves of six crops (maize, rice, wheat, canola, soybean, and cotton). DNA yield, quality, and presence of possible PCR inhibitors in the DNA preparation were examined. For comparison, each DNA extraction was also carried out using two commonly used commercial DNA extraction kits (DNeasy Plant Mini Kit, Qiagen, and Wizard Magnetic DNA Purification System for Food, Promega). The OD260/280 ratios of gDNA extracted from leaves and seeds using our designed device were between 1.8 and 2.1 (Table 1), which were similar to that of the DNA extracted using the two commercial kits. DNA yield from 12 extractions (six seed and six leaf samples) using our device ranged from 10 to 70 ng/mg tissue, which were generally lower compared to those extracted using commercial kits but were enough for on-field testing and routine laboratory analysis (Table 2). We also checked the DNA quality by agarose gel Table 2. Comparison of DNA Yields of Three DNA Extraction Systems Qiagen (ng/mg)

Promega (ng/mg)

our device (ng/ mg)

species

leaves

seeds

leaves

seeds

leaves

seeds

soybean rice canola cotton maize wheat

56.0 19.9 19.1 18.4 72.6 98.0

34.8 19.7 26.2 126.5 50.0 71.6

67.7 36.3 26.1 18.9 61.5 122.1

25.7 33.7 33.2 52.4 46.4 200.8

21.7 15.2 12.2 10.1 18.5 20.0

23.6 9.4 13.6 21.9 69.0 21.6

electrophoresis analysis, and the results showed that the quality and integrity of gDNA extracted using our device were comparable to those extracted using the two commercial kits (Figure 2, parts A and B), although there are some differences of DNA band intensity on gel, which might be attributed to unreliable quantification using the UV absorption spectra method.20 We further tested the extracted gDNA as templates in conventional PCR assay of endogenous reference genes in six different crops. In all PCR amplifications with gDNA extracted using our designed device, amplified DNA fragments with expected sizes of the endogenous reference genes could be observed, and the results were the same with those PCR 79

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Figure 2. Agarose gel electrophoresis of extracted gDNAs from six crops and PCR amplicons performed on these gDNAs. The gDNAs were extracted using a Qiagen kit, Promega kit, and our developed device, respectively. (A and B) Gel loading of 50 ng of extracted gDNAs from leaf samples and seed samples, respectively. Lanes 1−6 are the six crops used in the study (1 = soybean, 2 = rice, 3 = canola, 4 = cotton, 5 = maize, and 6 = wheat); lane M is DL8000 DNA marker. (C and D) Conventional PCR amplification of endogenous reference genes from extracted leaf gDNA and seed gDNAs. Lanes 1−6 are the endogenous gene amplicons of six crops (1 = soybean Lectin gene, 2 = rice SPS gene, 3 = canola HMG I/Y gene, 4 = cotton Sad 1 gene, 5 = maize zSSIIb gene, and 6 = wheat Waxy-D1 gene); lane M is DL8000 DNA marker.

Table 3. Linearity and PCR Efficiency of Real-Time PCR Assays of Extracted DNAs leaf DNA species soybean rice canola cotton maize wheat soybean rice canola cotton maize wheat soybean rice canola cotton maize wheat

endogenous reference gene

R2

PCR efficiency

seed DNA R2

DNeasy Plant Mini Kit Lectin 0.9941 1.07 0.9990 SPS 0.9923 0.87 0.9941 HMG I/Y 0.9973 0.97 0.9981 SadI 0.9925 0.92 0.9943 zSSIIb 0.9971 1.02 0.9994 Waxy 0.9958 0.95 0.9921 Wizard Magnetic DNA Purification System for Food Lectin 0.9962 0.89 0.9965 SPS 0.9928 1.03 0.9987 HMG I/Y 0.9952 1.09 0.9913 SadI 0.9985 0.94 0.9965 zSSIIb 0.9922 0.97 0.9970 Waxy 0.9942 0.96 0.9974 DNA Extraction Device Developed in This Work Lectin 0.9980 0.90 0.9960 SPS 0.9913 0.90 0.9969 HMG I/Y 0.9914 0.91 0.9901 SadI 0.9908 0.88 0.9970 zSSIIb 0.9957 0.97 0.9907 Waxy 0.9905 0.72 0.9954

PCR efficiency 0.95 0.93 0.98 0.91 0.99 1.07 0.92 1.06 1.02 0.87 0.93 0.96

Figure 3. Sensitivity of GTS 40-3-2 and MON89788 event-specific assays using gDNAs extracted with our designed DNA extraction device: (A) MON89788 assay; (B) GTS 40-3-2 assay. Top is agarose gel showing different amounts of gDNA. Middle is LAMP amplification products on agarose gel. Bottom is visual LAMP assay. Lanes 1−7: 20 000, 2000, 200, 20, 10, 5, and 2.5 copies of soybean haploid gDNA, respectively. Lane M: DL2000 DNA marker.

0.93 1.04 0.74 1.02 0.83 0.73

time PCR assays of rice, wheat, cotton, canola, and maize (data not shown). The results demonstrated that the purified gDNA using our DNA extraction device was suitable for not only LAMP assays, but also conventional PCR amplifications. Application of the System in GIPSA Proficiency Testing of GM Maize and Soybean Events. For semiannual GIPSA proficiency testing of GMOs organized by the USDA, the highly recommended analytical techniques are qualitative PCR, quantitative real-time PCR, and ELISA. We explored the possibility to apply the developed DNA extraction device and modified vLAMP assay system to analyze GM maize and soybean samples from the GIPSA proficiency test. Three maize samples (C4.1, C4.4, and C4.6) and two soybean samples (S4.2 and S4.3) from the GIPSA proficiency test in April 2010 were selected and tested. LAMP primers for GM maize (TC1507, Mon863, and DAS-59122-7) and GM soybean GTS 40-3-2 were described in previous publications.29,30 In these samples, the content of each GM event ranged from 0% to 2.0%. Test results using our system showed that selected endogenous

of the most important criteria for any new analytical system, we repeated two previously reported LAMP assays for GM soybean events GTS 40-3-2 and MON89788 to test the limit of detection (LOD) using our system. Serially diluted gDNAs of GTS 40-3-2 and MON89788 soybean at concentrations of 20 000, 2000, 200, 20, 10, 5, and 2.5 copies of haploid genome per reaction were used in the sensitivity test. The results showed that the lowest detection level was 5 copies of haploid genome per reaction in GTS 40-3-2 and MON89788 analysis, which was comparable to the LODs in previously reported LAMP assays34,38 and more sensitive than the LODs of conventional PCR assays (Figure 3). An LOD of 10 copies haploid genome per reaction could be obtained for MON89788 soybean PCR assay, and similar LODs were obtained in real80

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Table 4. Test Results of the GIPSA Proficiency Test Samples Using Our Developed Systema TC1507

a

Mon863

DAS-59122-7

GTS 40-3-2

sample name

official values (%)

PCR results

LAMP results

official values (%)

PCR results

LAMP results

official values (%)

PCR results

LAMP results

official values (%)

PCR results

LAMP results

C4.1 C4.4 C4.6 S4.2 S4.3

0.8 1.5 0.1 0.0 0.0

+ + + − −

+ + + − −

0.4 0.0 0.8 0.0 0.0

+ − + − −

+ − + − −

0.1 0.1 2.0 0.0 0.0

+ + + − −

+ + + − −

0.0 0.0 0.0 0.2 1.5

− − − + +

− − − + +

“−” = negative; “+” = positive; “official values” = officially decrypted results.

reference genes could be detected in all five samples. TC1507 maize event was detected in samples C4.1, C4.4, and C4.6. MON863 maize event was detected in samples C4.1 and C4.6. DAS-59122-7 maize event was detected in samples C4.1, C4.4, and C4.6, and GTS 40-3-2 soybean event was detected in samples S4.2 and S4.3 (Table 4). These results were consistent with those obtained using conventional PCR methods and correctly matched the officially decrypted results (http:// archive.gipsa.usda.gov/biotech/quarterly reports/april_2010_ final_report.pdf). This demonstrated that our system was well-suited for practical analysis of GM maize and soybean samples. Application of the System for On-Field Testing of GM Rice. Since the developed DNA extraction and analysis system was designed to have quick DNA extraction without using a centrifuge, LAMP amplification without using a commercial thermal cycler, and visual inspection without gel electrophoresis, we tested the applicability of this system for on-field analysis of GM rice T1c-19, TT51-1, and Kefeng no. 6. Three sets of LAMP primers were designed to amplify common transgenic elements (CaMV35S promoter, NOS terminator, and bar gene), and the LODs of these three developed LAMP assays (for CaMV35S promoter, NOS terminator, and bar gene) were 10, 15, and 10 copies haploid genome per reaction, respectively. For on-field tests, all three GM rice events could be successfully detected using our developed system: CaMV35s promoter was detected in the field samples of T1C-19 and Kefeng no. 6, NOS terminator was detected in all three GM rice samples, and bar gene was detected in a field sample of T1C-19 (Figure 4). These results indicated that the developed DNA

extraction and analysis system could be successfully applied for on-field testing of GMOs. It took several minutes for sample homogenization (2−3 min for leaves), 10−15 min for DNA extraction, and 40 min for LAMP amplification and result visualization. So the whole detection process could be completed in about 1 h. Previously, the most commonly used on-field test technique for GMOs was LFS, which is a proteinbased method targeting exogenous proteins.4 Although LFS has been used for rapid field screening of GM events in several plant species, there are some limitations of LFS.7 First, it is protein-specific, which means for each specific GM event, a specific LFS assay has to be developed to target the specific exogenous protein (such as Cry1Ac, Cry9C, and Cry3A in GM crops). Second, LFS assay is not event-specific, because the same exogenous protein can be expressed in different crops or different events of the same crop. Third, the development of a specific LFS assay can be very difficult and time-consuming, particularly due to difficulties to generate monoclonal antibodies. Considering these limitations, it is simply not possible to develop a specific LFS assay for every GM event currently available in the market. To date, no more than 15 LFS assays have been developed for GMO analysis. Therefore, we believe that our developed DNA extraction and analysis system, which combines a simple, cheap, and fast DNA extraction device with a modified vLAMP assay, will help greatly in meeting the demand for new techniques in GMO on-field testing.

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CONCLUSIONS The simple GMO detection system combining a rapid and inexpensive DNA extraction device with a modified vLAMP assay in this study was validated as a useful on-field GMO detection tool. It was successfully tested for both GIPSA proficiency test samples and field GM rice samples. The system is at low cost, easily operated, sensitive, and specific to target DNA. For future improvement of this system, we are testing possibilities of combining the DNA extraction device and vLAMP assay into one integrated device, which will make it more practical for on-field detection of GMOs.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 21 34207174. Fax: +86 21 34204869. E-mail: [email protected].

Figure 4. Results of on-field testing using the developed system. The transgene targets are NOS terminator (A), CaMV35s promoter (B), and bar gene (C): lanes 1−2, T1c-19; lanes 3−4, Kefeng no. 6; lanes 5−6, TT51-1; lanes 7−8, Minhui63; lane M, DL2000 DNA marker.

Author Contributions §

The first two authors contributed equally to this work.

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Analytical Chemistry

Article

Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Transgenic Plant Special Fund (China, 2011ZX08012-002 and 2011ZX08012003) and the Shanghai Rising-Star Program (11QA1403300).

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