Technical Note pubs.acs.org/ac
Simple Screening Strategy with Only Water Bath Needed for the Identification of Insect-Resistant Genetically Modified Rice Fang Zhang, Liu Wang, Rui Wang, Yibin Ying, and Jian Wu* College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China S Supporting Information *
ABSTRACT: An informative, with simple instrument needed, rapid and easily updated strategy for the identification of insect-resistant genetically modified (GM) rice has been described. Such strategy is based on a parallel series of loop-mediated isothermal amplification (LAMP) reactions targeting the rice endogenous gene sucrose phosphate synthase (Sps), the top two most frequently used genetic elements (Agrobacterium tumefaciens nopaline synthase terminator (Nos) and Cauliflower mosaic virus 35S promoter (CaMV35S)), and an insect-resistant specific gene (Cry1Ac) and detected visually by phosphate ion (Pi)-induced coloration reaction. After a logical judgment of visible readouts has been obtained, three popular insect-resistant GM rice events in China can be successfully identified within 35 min, using either microwell strips or paper bases.
A
microarrays that meet the demands of the updated GMO database would be long and the operation process is complex. Van de Bulcke et al. described a qPCR-based method for GMO screening through application of a “prime number”-based algorithm, which is, however, rather costly.7,8 We need a new solution to verify GMOs cost-effectively, informatively, simply, and directly. Isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP) and cross-priming amplification (CPA), are attractive because they are easy to operate in the field, more sensitive, and fairly rapid.9−11 But it can rarely be used for multiple genes amplification in one single LAMP or CPA reaction. In this paper, taking the advantage of isothermal amplification, we describe a fundamentally different strategy to identify insect-resistant GM rice that is informative, rapid, and readily used with only one simple heater. The presence of four genes was screened with the naked eye and logically judged for the identification of three popular insect-resistant GM rice events in China.
wide-range screening of target genes within a limited time is always necessary for the identification of genetically modified organisms (GMOs) in many situations. By the end of 2013, 27 genetically modified (GM) crops in 36 countries, involving 336 GM events, have been strictly issued for food use, feed use, and/or environmental release.1 Nevertheless, on occasional, unauthorized GMOs have been released. Thus, there is an urgent need to verify even trace amounts of a particular GMO in products, not only in the countries with mandatory requirement for GMO labeling but also in countries that intend to export agricultural products into countries that have restrictions. The ideal strategy for the detection of GMOs would be informative, sensitive, simple, rapid, reliable, easy to operate on-site, and readily renewable for the updated GMO database. Traditionally, multiple PCRs are favored for the ability of simultaneous detection of multiple DNA targets in one single experiment tube; thus, nowadays, they are frequently used for GMOs screening. However, the problem is that preferential amplification of partial targets and/or nonspecific amplification caused by the interference of different primer pairs exist.2,3 It may take months to years to get a perfect multitargeted primer set. In addition, the detection method for amplified nucleic acid is either time-consuming, because of the need for electrophoresis with cancer-causing staining, or expensive, because of the need for a complicated and bulky optical detection instrument. Although newly developed chiplike oligo microarrays or capillary microarrays for the detection of amplicons were introduced for multi-PCR-based GMO screening, some drawbacks that are inherited from multiple PCR, such as difficulties in designing multiple targeted primer pairs and the requirements of a precise temperature control, still cannot be eliminated.4−6 The development time for the design of new © 2015 American Chemical Society
■
EXPERIMENTAL SECTION Materials. Seeds of GM rice (O. sativa) Huahui 1, Kefeng 6 (KF6), and KMD1 were provided by the Institute of Agriculture Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, China. Nontransgenic ones were purchased from a local market in Hangzhou, China. Rice seeds were unshelled and made into contaminated mixtures with different GM concentrations before they were ground into fine powder. Processed Received: November 24, 2014 Accepted: January 12, 2015 Published: January 12, 2015 1523
DOI: 10.1021/ac504384p Anal. Chem. 2015, 87, 1523−1526
Technical Note
Analytical Chemistry samplesso-called “rice crust”were prepared by steaming and frying in sequence and ground into fine powder for DNA extraction. DNA Extraction. Genomic DNA (gDNA) was extracted with 2% sodium lauryl sulfate from 0.15 g of rice powder, precipitated by 0.6 volume of iso-propanol, washed two times with 70% ethanol and finally eluted in 50 μL of TE buffer. The extracted gDNA was electrophoresed at 90 V for 30 min on a 1% (w/v) agarose gel when necessary, which was presupplemented with GeneGreen (Tiangen Biotech Co., Ltd., Beijing, China) for staining and photographed afterward with a ChemiDoc XRS+ system (BioRad, CA, USA). LAMP assay for Pi-induced Visual Detection. LAMP assays for Cry1Ac and Sps were prepared in a 20 μL reaction mixture containing 1.6 mM dNTPs (Takara Biotechnology Co., Ltd., Dalian, China), 8 mM MgCl2, 1× Gsp Fast DNA polymerase (Ustar Biotech Co., Ltd., Hangzhou, China), 0.8 μM of each inner primer, 0.2 μM of each outer primer (including loop primer for Sps gene assay), 6 U Gsp Fast DNA polymerase (Ustar Biotech Co., Ltd., Hangzhou, China), 1 μL of rice gDNA template, and 1 μM SYTO9 (Invitrogen, Carlsbad, CA, USA) for real-time fluorescent detection or 0.1 U thermostable inorganic pyrophosphatase (New England Biolabs, Ipswich, MA) for Pi-induced visual detection. Assays for the Nos and CaMV35S genes were carried out in the same volume of 20 μL with Loopamp DNA amplification Kit (Deaou Biotechnology Co., Ltd., Guangzhou, China) containing 17 μL of reaction mix, 1 μL of Bst DNA polymerase, 0.1 U thermostable inorganic pyrophosphatase ((New England Biolabs, Ipswich, MA), and 1 μL of rice gDNA template. Templates used for LAMP amplification were prepared by diluting the extracted gDNA 10 times. Detection of LAMP in Microwell/Tube Strips for Identification of Insect-Resistant GM Rice. LAMP assays were carried out with a total volume of 20 μL in microtube or microwell strips and sealed with 50 μL of mineral oil. After an incubation at 63 °C for 5−45 min, coloration reagents (with a final concentration of 0.4% ascorbic acid and 1.68 mM ammonium molybdate, 0.08 mM potassium antimonyl tartrate in 0.4 M sulfuric acid) were added. After ∼10 min at room temperature, visible results were photographed with a camera. Visual Dots Array Detection of LAMP on Paper Base for Identification of Insect-Resistant GM Rice. Four microliters (4 μL) of a Mo−Sb solution (containing 21 mM ammonium molybdate, 2 mM potassium antimonyl tartrate, and 5.4 M sulfuric acid), and 2 μL of ascorbic acid (10%) was added onto the Whatman grade 1 Chr chromatography paper in a array pattern. For GM rice identification, 2 μL of amplified products were added in sequence onto the as-obtained paper strip.
Scheme 1. Schematic of Screening Strategy Described with Only Water Bath Needed for the Identification of InsectResistant GM Rice
amplified samples and a achromatic color in negatively amplified samples. In this way, a resulting readout can be obtained from the visible blue signals of four tracked genes by OR/AND/NOT logical judgment, according to the molecular characteristic of different GM events. In particular, the colorless-to-blue detection of gene amplification was based on the facts that one pyrophosphate ion (PPi) would be released for every nucleotide incorporated into the growing DNA strand and phosphate ions (Pi) would be easily colored by ammonium molybdate in the presence of potassium antimonyl tartrate and ascorbic acid.12−14 Thus, with the preaddition of thermostable inorganic pyrophosphatase (PPase) into the LAMP reaction mixture, genes can be detected visually by tracing Pi hydrolyzed from PPi. This method is called the Pi-based visual detection method, and it was first reported in our previous work.15 Based on this concept, the synchronism of different genes during the same amplification process was first investigated. DNA extracted from samples with 0.9% KF6-contaminated or non-GM rice were employed as the templates. Paralleled LAMP reactions were terminated with different length of incubation and treated with coloration reagent afterward. As shown in Figure 1a, four genes has been amplified in KF6contaminated samples showing four blue readouts. With the time course detection, a significant color change can be observed within 25 min for both the Nos and CaMV35S genes and after ∼10 min more for the Cry1Ac gene. That means, for a simultaneous tracking of Sps, Nos, CaMV35S, and Cry1Ac, an incubation duration of 35 min is needed. Such a lag of the Cry1Ac gene amplification can also be observed in a SYTO 9based fluorescent detection method (see Figure 1b, modified from our previous work, conducted on a Biorad MyiQ2 RealTime PCR Detection System).11 This can be explained by the different efficiency of amplification between Cry1Ac and the other three target genes. Even so, this visual Pi-based method can result in a savings of up to ∼36%, with regard to the time required for GM rice screening, compared with fluorescent methods. Next, we evaluate the performance of this method for the detection of practical GM samples. Mixing levels (by mass fraction) of 0.5%, 0.9%, and 3% were set as the GM contamination to be detected according to the requirements
■
RESULTS AND DISCUSSION To cover all three insect-resistant GM rices present in China, four genes were set to be the targets: the rice endogenous gene sucrose phosphate synthase (Sps), the top two most frequently used genetic elements (Agrobacterium tumefaciens nopaline synthase terminator (Nos) and Cauliflower mosaic virus 35S promoter (CaMV35S)), and insect-resistant specific gene Cry1Ac. As shown in Scheme 1, samples of four different individual targeting genes were prepared in parallel. After a process of isothermal amplification at 63 °C for ∼20−40 min, coloration reagents were added into the microwell/microtube strip on an equal footing, resulting in a blue color in positively 1524
DOI: 10.1021/ac504384p Anal. Chem. 2015, 87, 1523−1526
Technical Note
Analytical Chemistry
insect-resistant GM rice events and the effect induced by processing procedure for the detection of such GM events. Samples contaminated with KF6, TT51-1, and KMD1 at a mixing level of 0.9% were prepared separately in two groups as raw ones and processed ones. Two general types of processing procedures that are frequently used worldwide were employed: steaming and frying. As shown in Figure 3, little difference can
Figure 3. Identification of three insect-resistant GM rice events in real samples with or without food processing in microtube strips. Blank represents samples with H2O as template instead of gDNA from rice. gDNA extracted were separated on a 1% agarose gel.
be noticed between samples with or without food processing, which suggests that processing procedures such as steaming and frying can hardly affect the performance of this LAMPbased Pi-tracking detection method. This is because the target chosen for LAMP reaction is always at an optimal length of ∼200 bp, which is still detectable even after gDNA has degraded (as gel electrophoresis, shown in Figure 3).19,20 In addition, by assigning every blue readout as “1” and every colorless readout as “0”, samples can be assigned serial numbers, such as “1111” or “1101” and so on. On the basis of sequenced numbers obtained, the exact events of testing samples can be identified. Since the described method is generally dependent on the individual LAMP reactions and a logical permutation of different target genes, it can be easily updated and get a largest coverage with a least number of genes to be amplified. To further explore the possible application of this strategy described, a paper-based colorimetric assay was conducted. Whatman grade 1 Chr chromatography paper was chosen to be the matrix, according to Carrilho et al., and pretreated with Pi coloration reagents in an array pattern.21 After incubation at 63 °C for ∼35 min, samples can be added onto the paper base; as a result, an array of blue dots appears. (See Figure 4.) This asobtained test strip provides a rapid and sensitive alternative for gel electrophoresis as needed.
Figure 1. Time-course detection of four LAMP amplified targets. (a) Visible readouts from Pi-based detection method; reactions were terminated at 5, 15, 25, 35, and 45 min separately and colored by Mo− Sb and Vc reagents. (Targeted genes, from left to right: Sps, Nos, CaMV35S, and Cry1Ac.) (b) Curves obtained from traditional fluorescent detection method ((▲) Sps, (◆) Nos, (■) CaMV35S, and (×) Cry1Ac amplification). Both 0.9% GM rice KF6-containing samples (red) and non-GM rice samples (blue) were investigated.
of worldwide labeling regulations.16−18 Reactions were conducted with a negative control (non-GM rice as the template) and also a blank control (H2O as the template). As shown in Figure 2, with an incubation of 35 min at 63 °C, decreasing the contents of GM contamination resulted in a slightly lighter blue color. Samples with a contamination level of 0.5% can be detected visually and specifically. Encouraged by the initial analysis described above, we examined the feasibility of this method to identify different
■
CONCLUSION In conclusion, here we have described a new screening strategy for multiple genes that is based on a series of paralleled LAMP reactions targeting different genes and detected visually by a phosphate ion (Pi)-induced coloration reaction. As a consequence, such a method can be easily carried out with only one simple heater in resource-limited areas, and it can be modified or updated as desired in any laboratory without extra operator training. As displayed in this paper, this method can be successfully applied for the identification of insect-resistant genetically modified (GM) rice. With four genes tracked and a logical judgment of visible readouts, three popular insect-
Figure 2. Detection of the KF6 event in samples with different mixing levels of contamination. Three levels of contaminated GM rice samples (by mass fraction) were investigated: 0.5%, 0.9%, and 3%. Blank represents samples with H2O as the template, instead of gDNA from rice. 1525
DOI: 10.1021/ac504384p Anal. Chem. 2015, 87, 1523−1526
Technical Note
Analytical Chemistry
(8) Querci, M.; Van den Bulcke, M.; Ž el, J.; Van den Eede, G.; Broll, H. Anal. Bioanal. Chem. 2010, 396, 1991−2002. (9) Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Nucleic Acids Res. 2000, 28, e63. (10) Gaolian, X.; Lin, H.; Huayan, Z.; Hongying, W.; Sei-ichi, Y.; Tristen, C. W.; Paul, J. R.; Sam, P.; Qimin, Y. Sci. Rep. 2012, 2. (11) Zhang, F.; Wang, L.; Fan, K.; Wu, J.; Ying, Y. Anal. Bioanal. Chem. 2014, 406, 3069−3078. (12) Gibson, N. J.; Newton, C. R.; Little, S. Anal. Biochem. 1997, 254, 18−22. (13) Motomizu, S.; Li, Z. H. Talanta 2005, 66, 332−340. (14) Shiddiky, M. J. A.; Rahman, M. A.; Park, J.-S.; Shim, Y.-B. Electrophoresis 2006, 27, 2951−2959. (15) Zhang, F.; Wang, R.; Wang, L.; Wu, J.; Ying, Y. Chem. Commun. 2014, 50, 14382−14385. (16) Matsuoka, T. In APEC-JIRCAS Joint Symposium and Workshop on Agricultural Biotechnology, Bangkok, Thailand, Sept. 3-7, 2001. (17) South Korean Ministry of Agriculture and Forestry, Gwacheon, Gyeonggi Province, South Korea, 2000. (18) Regulation (EC) 1830/2003, Directive 2001/18/EC. Off. J. Eur. Communities: Legis. 2003, L 268, 24−28. (19) Zhang, W.; Xing, F.; Selvaraj, J. N.; Liu, Y. J. Food Sci. 2014, 79, T1055−T1065. (20) Song, S.; Zhou, G.; Gao, F.; Zhang, W.; Qiu, L.; Dai, S.; Xu, X.; Xiao, H. Food Chem. Toxicol. 2011, 49, 3174−3182. (21) Carrilho, E.; Phillips, S. T.; Vella, S. J.; Martinez, A. W.; Whitesides, G. M. Anal. Chem. 2009, 81, 5990−5998.
Figure 4. Identification of insect-resistant GM rice with visual dots on a paper base. Blank represents samples with H2O as the template, instead of gDNA from rice.
resistant GM rice events in China can be distinguished reliably, within 35 min, from the events involving rice-resourced but nongenetically modified materials and those of other species with or without genetic modification. In addition, such a method can also be applied for a paper-based detection process, which would be cost-effective, simple, and instructive for the development of other forms of gene screening in the future.
■
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.
■
AUTHOR INFORMATION
Corresponding Author
*Tel./Fax: 0086-571-88982180. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work is supported by the Synergistic Innovation Center of Modern Agricultural Equipment and Technology (No. NZXT01201402) and National Natural Science Foundation of China (No. 31271617). The authors would also like to thanks Institute of Agriculture Quality and Standard for Agroproducts, Zhejiang Academy of Agricultural Sciences, Hangzhou, China, for providing insect-resistant GM rice materials.
■
REFERENCES
(1) James, C. Global Status of Commercialized Biotech/GM Crops: 2013, ISAAA Brief No. 46; International Service for the Acquisition of Agri-biotech Applications (ISAAA): Ithaca, NY, 2013. (2) Brownie, J.; Shawcross, S.; Theaker, J.; Whitcombe, D.; Ferrie, R.; Newton, C.; Little, S. Nucleic Acids Res. 1997, 25, 3235−3241. (3) Chaouachi, M.; Chupeau, G.; Berard, A.; McKhann, H.; Romaniuk, M.; Giancola, S.; Laval, V.; Bertheau, Y.; Brunel, D. J. Agric. Food Chem. 2008, 56, 11596−11606. (4) Shao, N.; Jiang, S.-M.; Zhang, M.; Wang, J.; Guo, S.-J.; Li, Y.; Jiang, H.-W.; Liu, C.-X.; Zhang, D.-B.; Yang, L.-T.; Tao, S.-C. Anal. Chem. 2013, 86, 1269−1276. (5) Guo, L.; Yang, H.; Qiu, B.; Xiao, X.; Xue, L.; Kim, D.; Chen, G. Anal. Chem. 2009, 81, 9578−9584. (6) Guo, J.; Yang, L.; Chen, L.; Morisset, D.; Li, X.; Pan, L.; Zhang, D. Anal. Chem. 2011, 83, 1579−1586. (7) Van den Bulcke, M.; Lievens, A.; Barbau-Piednoir, E.; MbongoloMbella, G.; Roosens, N.; Sneyers, M.; Casi, A. Anal. Bioanal. Chem. 2010, 396, 2113−2123. 1526
DOI: 10.1021/ac504384p Anal. Chem. 2015, 87, 1523−1526
