Biotechnol Lett (2015) 37:211–218 DOI 10.1007/s10529-014-1658-3
ORIGINAL RESEARCH PAPER
Application of carboxyphenylboronic acid-functionalized magnetic nanoparticles for extracting nucleic acid from seeds Ning Sun • Congliang Deng • Guanglu Ge Qiang Xia
•
Received: 20 July 2014 / Accepted: 27 August 2014 / Published online: 12 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Magnetic iron oxide nanoparticles functionalized with 4-carboxyphenylboronic acid (CPBAMNPs) were developed for extracting genomic DNA, total RNA and nucleic acids from seeds. The seed samples were genetically-modified maize seeds and unmodified soybean seeds infected by bean pod mottle virus and tobacco ringspot virus. The total nucleic acids, genomic DNA, and RNA could be separately extracted from these seeds with high qualities using CPBA-MNPs under different conditions. Furthermore, the results of real-time quantitative qPCR and real-time reverse transcription (RT)-PCR indicated that the nucleic acids extracted from these seeds using CPBA-MNPs were suitable for the detection of genetically-modified seeds and seed-borne viruses.
Ning Sun and Congliang Deng have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1658-3) contains supplementary material, which is available to authorized users. N. Sun Q. Xia (&) State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Sipailou No. 2, Nanjing 210096, China e-mail: [email protected] N. Sun Q. Xia Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou 215123, China
Keywords 4-Carboxyphenylboronic acid DNA extraction Magnetic nanoparticles Maize seeds RNA extraction Seed-borne virus Soybean seeds Introduction During the past two decades, genetically-modified organisms (GMOs) have been approved for cultivation in several countries. However, controversy continues to surround all aspects of GMOs, regardless of their rapidly growing planting areas and importance in trade. As more and more foods and feeds are produced from GMOs, the public has paid greater attention on GMOs than ever before. On the other hand, the qualities of maize and soybean seeds are of great importance to agricultural production, because the health and vigor of the seeds greatly affect crop growth. Seed-borne viruses may cause serious plant diseases which can result in enormous crop yield losses (Skakiba et al. 2012). Therefore, the detection of genetically-modified seeds and seed-borne viruses is necessary for the proper labeling of geneticallymodified seeds and foods and for protecting the security N. Sun C. Deng Beijing Entry-exit Inspection and Quarantine Bureau, Beijing 100026, China N. Sun G. Ge CAS Key Laboratory of Measurement and Standardization for Nanotechnology, National Center for Nanoscience and Technology, Beijing 100190, China
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of crop production. A number of PCR-based methods have been developed for the detection of GMOs (Nageswara-Rao et al. 2013; Peano et al. 2004) and plant viruses (Deng et al. 2013; Liu et al. 2010; Sun et al. 2014). The attainment of high-quality nucleic acids from seed samples is indispensable for the nucleic acid amplification-based detection method. However, the challenges in the conventional extraction methods remain unresolved because the abundant amounts of polysaccharides and polyphenols contained in seed samples strongly inhibit the extraction of nucleic acid (Demeke and Jenkins 2010). Therefore, the development of suitable nucleic acid extraction methods has been intensively pursued. Phenylboronic acid affinity separation has attracted increasing attention because of the reverse binding ability of phenylboronic acid to glycoproteins (Xu et al. 2008), RNA (Gomes et al. 2011; Singh and Willson 1999), ¨ zdemir and Tuncel 2000), and nucleotides (C¸lc¸ek 2005; O other compounds. The relationship between phenylboronic acid agarose and RNA has been investigated and the results suggest that the amount of RNA adsorption can be enhanced in the presence of divalent cations such as Mg2?, Ca2?, or Ba2? (Singh and Willson 1999). Both RNA and gDNA can be adsorbed on to phenylboronic acid for chromatography under alkaline lysis-derived Escherichia coli lysates (Gomes et al. 2011). Moreover, it is possible that nucleic acids can be isolated from biological samples using phenylboronic acid-functionalized magnetic nanoparticles, although such a study has not yet been reported. Therefore, it is necessary to investigate the feasibility and specificity of phenylboronic acid-functionalized magnetic nanoparticles for the extraction of nucleic acids. Herein, we developed a novel nucleic acid extraction method based on the use of 4-carboxyphenylboronic acid-functionalized magnetic nanoparticles (CPBA-MNPs) and applied the method for the detection of genetically-modified maize seeds and two types of seed-borne viruses, i.e., bean pod mottle virus and tobacco ringspot virus.
Materials and methods Chemicals and reagents Iron(III) acetylacetonate [Fe(Acac)3] and 4-carboxyphenylboronic acid (CPBA) were purchased from Sigma-Aldrich. Plant Genomic DNA kit was
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purchased from Tiangen Biotech Co., Ltd. RNeasy Plant mini-kit was obtained from Qiagen GmbH. RNase A1, One-step PrimeScript RT-PCR kit (Perfect Real-time), Premix Ex Taq (Probe qPCR), k-HindIII digest DNA Marker, DL2000 DNA marker, DL500 DNA marker, and 100 bp DNA ladder were purchased from TaKaRa Biotech. Co. Ltd. The geneticallymodified maize seeds and non-genetically-modified soybean seeds infected with bean pod mottle virus (BPMV) and tobacco ringspot virus (TRSV) were kindly provided by Beijing Entry–Exit Inspection and Quarantine Bureau. Preparation and characterization of MNPs Fe3O4 nanoparticles were synthesized by following the protocol of Li et al. (2011). The particles were washed three times with ethanol and lyophilized using a vacuum freeze-drying apparatus for 48 h. Fe3O4 nanoparticles, 10 lg, were added to 2 ml ethanol containing 30 mg CPBA. The mixture was sonicated for 45 min until it became transparent and uniform, and was then left overnight at room temperature. After the addition of 2 ml saturated NaCl solution into the mixture to destroy the stability of the solution, the particles were collected with an external magnet and washed several times with ethanol and deionized water. The nanoparticles modified with CPBA (CPBA-MNPs) were then dispersed in the deionized water at 5 mg/ml. Afterward, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy and dynamic light scattering (DLS; Malvern Zetasizer NanoZS) analyses were performed to characterize the particles. Extraction of nucleic acid The procedures for the extraction of gDNA (method A), total nucleic acid (method B) and RNA (method C) from seeds are shown in Fig. 1. In method A, 900 ll CTAB lysis buffer (2 % CTAB and 1.4 M NaCl, pH 8.0) was added into a tube containing 50 mg seeds, held at 65 °C for 15 min and then cooled to room temperature. After the addition of 2 ll RNase A1 (50 mg/ll), the tube was incubated at 37 °C for 15 min. After the addition of 100 ll potassium acetate (5 M, pH 5.2), the tube was placed on ice for 5 min and centrifuged at 12,0009g for 2 min. Subsequently, 200 ll supernatant (more than 800 ll in total) was
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method A. Finally, 10 ll extracted nucleic acid was analyzed by 1.5 % agarose gel electrophoresis. In addition, three commercial kits, Plant Genomic DNA kit, TRIzol and RNeasy Plant mini-kit, were used for the extraction of gDNA or RNA for comparing the efficiencies of different extraction methods. To produce an accurate comparison, the nucleic acid was extracted from 50 mg of the seeds using these three commercial kits, and finally, 200 ll elution buffer was used to dissolve the nucleic acid in these experiments. Ten microlitre extracted nucleic acid was analyzed by agarose gel electrophoresis, and the concentrations were measured at 260 nm. Real-time PCR and reverse transcription (RT)PCR
Total nucleic acid method A
method B
method C
Fig. 1 Schematic procedure for the extraction of gDNA (method A), total nucleic acid (method B) or RNA (method C) from seed using CPBA-MNPs
transferred into a new tube containing 1 mg CPBAMNPs and 200 ll ethanol was added then vortexed for 15 s and incubated for 3 min at room temperature. The particles were collected by DynaMag-2 Magnet and the remaining supernatant was discarded. The collected particles were washed twice with 70 % (v/v) ethanol and dried at room temperature. Fifty microlitre of TE buffer (50 mM Tris/HCl and 5 mM EDTA, pH 8.8) were added into the tube and held at room temperature for 3 min. The supernatant was transferred into a new tube after the CPBA-MNPs were collected. Ten microlitre extracted nucleic acid was analyzed by 1 % agarose gel electrophoresis. In the extraction of total nucleic acids (method B), the step of using RNase A to digest RNA was omitted from method A. In method C, 1 ml of SDS lysis buffer (2 % SDS, pH 8.0) was added into a tube containing 50 mg seeds. The tube was incubated at 65 °C for 15 min and placed on ice for 5 min, followed by centrifugation at 12,0009g for 2 min. [If the supernatant was not clear, one vol phenyl/chloroform (1:1, v/v) was added to 200 ll supernatant, followed by centrifugation at 12,0009g for 5 min]. Then 200 ll supernatant was transferred into a new tube containing 1 mg CPBA-MNPs, 200 ll ethanol was added followed by the steps similar to those described in
Real-time PCR was used for detecting the geneticallymodified maize seeds, while real-time RT-PCR was performed for the detection of BPMV and TRSV. Both detection processes were performed on an ABI 7900HT real-time PCR system (Applied Biosystems). In the detection of genetically-modified maize, the cauliflower mosaic virus 35S (CaMV35S) promoter region and the nopaline synthase (NOS) terminator region were amplified by real-time PCR according to the instructions of entry-exit inspection and quarantine standards of the People’s Republic of China (SN/T 1024–2003. Protocol of real-time PCR for detecting genetically-modified plants and their products, China, 2003). The detection of BPMV and TRSV by real-time RT-PCR was conducted by referring to the procedures described in previous studies (Deng et al. 2013; Liu et al. 2010). The primers and probes used in this study are given in Supplementary Table 1. Mg2? can be used to improve the activity of enzymes due to the complexation of EDTA in the elution buffer.
Results and discussion Characterization The morphologies of Fe3O4 nanoparticles with or without CPBA modification were characterized by TEM after the samples were dried using ethanol (Fig. 2). The Fe3O4 nanoparticles without any modifications are not dispersible in either water or hexane (Li et al. 2011) and can maintain their stability in
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Fig. 3 FT-IR spectra of Fe3O4 nanoparticles (a), CPBA-MNPs (b), and CPBA (c). The absorption peaks at *1,345 cm-1 and at*1,710 cm-1 are assigned to the carboxyl group, demonstrating that CPBA was successfully immobilized onto the iron oxide nanoparticles
Fig. 2 TEM images of Fe3O4 nanoparticles (a) and CPBAMNPs (b) dried through ethanol
ethanol for 10 min precipitating due to aggregation. However, CPBA-MNPs after surface functionalization can maintain their stability in ethanol for a prolonged time. This indicates that Fe3O4 nanoparticles can be functionalized with CPBA, which can significantly improve the dispersibility of the functionalized nanoparticles (Fig. 2b). FT-IR spectra of Fe3O4 nanoparticles, CPBAMNPs, and CPBA are shown in Fig. 3. As compared with the spectrum of Fe3O4 nanoparticles, a
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characteristic band (1,340 cm-1), which is ascribed to the B–O bond, can be observed from the spectrum of CPBA-MNPs (Xu et al. 2008). As compared with CPBA, CBPA-MNPs do not exhibit the characteristic band of COOH at 1,710 cm-1. These results provide strong evidence for the attachment of the carboxyl group of CPBA to Fe3O4 nanoparticles and indicate the success of using CPBA for particle modification. In our previous study, Fe3O4 nanoparticles dispersed in deionized water immediately aggregated together or attached to the sidewalls of the tube (Li et al. 2011), indicating the hydrophobicity of the particles. The hydrodynamic size and f-potential of CPBA-MNPs were characterized by using the Malvern Zetasizer NanoZS system. The characterization results revealed an average particle size of approximately 11 nm (Fig. 4a). CPBA-MNPs were positively charged below pH 9, above which the particles were slightly negatively-charged (Fig. 4b), and only a slight difference was observed in the pKa value from that reported for CPBA (Yan et al. 2004). The reason might be that the immobilization of CPBA on the surface of Fe3O4 nanoparticles affected the particle ionization. Extraction of gDNA and detection of geneticallymodified seeds To confirm the efficiency of the method, CPBA-MNPs were used to extract gDNAs from geneticallymodified maize seeds and non-genetically-modified
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Fig. 5 Agarose gel electrophoresis analysis of nucleic acids extracted from genetically-modified maize seeds and nongenetically-modified soybean seeds by method A and a commercial kit (Plant Genomic DNA kit). Lane 1 k-Hind IIIdigested DNA marker; lanes 2–3 and lanes 6–7 nucleic acids extracted from genetically-modified maize seeds and soybean seeds, respectively, using method A; lanes 4–5 and lanes 8–9: nucleic acids extracted from genetically-modified maize seeds and soybean seeds, respectively, using the commercial kit. All of the nucleic acids which were analyzed by agarose gel electrophoresis were extracted from independent samples
result indicates that gDNAs extracted by CPBAMNPs are suitable for PCR-based detection methods with demonstrated good performance. Total nucleic acid and RNA extracted from maize and soybean seeds Fig. 4 Hydrodynamic size distribution (a) and f-potential (b) of CPBA-MNPs in water measured by the Malvern Zetasizer NanoZS system
soybean seeds. The obtained results were compared with those of the commercial extraction kit (Plant Genomic DNA kit; Tiangen). Genomic DNAs were analyzed by 1 % agarose gel electrophoresis (Fig. 5). The concentration was determined from the absorbance at 260 nm, and the A260/280 ratio was between 1.68 and 1.82. Approximately 11 lg gDNA was extracted from 50 mg seeds using CPBA-MNPs, whereas only 7.5 lg gDNA was obtained using the commercial kit, indicating a higher efficiency of the CPBA-MNPs-based method. The nucleic acids extracted from genetically-modified maize seeds were detected by real-time PCR. As shown in Table 1, the threshold cycle (Ct) values obtained from CPBA-MNPs were slightly lower than those from Plant Genomic DNA kit, although the difference between the two methods was not statistically significant (Student’s t test, P = 0.208). This
Figure 6a and b show the electrophoresis results of the nucleic acids extracted from the maize and soybean seeds, respectively. Total nucleic acids were successfully extracted from the seeds with potassium acetate (KA) and gave an enhanced quality with an average A260/280 ratio of 1.697, as compared with the extraction without KA. Although the extraction of gDNA or total nucleic acids from seed samples has been successfully demonstrated, the extraction of RNA remains as a challenging task. In magnetic particlebased nucleic acid extraction, gDNA and RNA are usually co-extracted because of their chemical similarities (Sun et al. 2014). In the present study, 2 % SDS, pH 8.0, was used as the lysis buffer to extract nucleic acids from the independent samples. The extracted nucleic acids were determined by 1.5 % agarose gel electrophoresis. As shown in Fig. 6a and b, the total RNA could be successfully extracted from the samples without visible gDNA bands (Fig. 6a, lanes 5–7; Fig. 6b, lanes 3–4). Then, the performance of CPBA-MNPs for RNA extraction from maize or soybean seeds was compared with that of the
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Fig. 6 Gel agarose electrophoresis analysis of nucleic acids extracted using CPBA-MNPs, TRIzol, and RNeasy Plant minikit. a Nucleic acids extracted from soybean seeds by method B (lanes 2–4) and method C (lanes 5–7), and k-Hind III-digested DNA marker (lane 1); b nucleic acids extracted from maize seeds by method B (lanes 1–2) and method C (lanes 3–4); c nucleic acids extracted from maize seeds (lanes 2–3) and
soybean seeds (lanes 4–5) using TRIzol, and 100-bp DNA ladder (lane 1); d nucleic acids extracted from maize seeds (lanes 2–3) and soybean seeds (lanes 4–5) using RNeasy Plant mini-kit, and 100-bp DNA ladder (lane 1). All of the nucleic acids which were analyzed by agarose gel electrophoresis were extracted from independent samples
commercial kits (TRIzol and RNeasy Plant minikit) (Fig. 6c and d). Only a minute amount of RNA was extracted from the maize seeds by both commercial kits (Fig. 6c, lanes 1–2; Fig. 6d, lanes 1–2), and the resulting RNA integrity was inferior to that obtained from the CPBA-MNPs method (Fig. 6a). In addition, a large amount of degraded RNA was obtained from the soybean seeds using TRIzol (Fig. 6c, lanes 4–5), and high-quality RNA containing much gDNA contamination was extracted by using RNeasy Plant mini-kit (Fig. 6d, lanes 4–5). Maize seeds have higher contents of polysaccharides than soybean seeds, and polysaccharides show strong inhibitory effects on guanidinium thiocyanate-based or phenol–chloroform extraction. As both RNeasy plant mini-kit and TRIzol contain guanidinium salts, CPBA-MNPs show better performance on RNA extraction from maize seeds than do the two commercial kits.
To assess the amount of gDNA in the extract, the nucleic acids extracted from genetically-modified maize seeds and soybean seeds using method B, Method C, RNeasy Plant mini-kit and TRIzol were used for PCR or real-time PCR. The nucleic acids extracted from genetically-modified maize seeds were used as templates to amplify the CaMV35S and NOS (Fig. 7). The mean Ct values of method B were 26.8 ± 0.26 (CaMV35S) and 26.7 ± 0.17 (NOS), and the mean Ct values of method C were 30.7 ± 0.29 (CaMV35S) and 30.4 ± 0.45 (NOS). The results indicated that the amount of gDNA in the extract of method C was ten times lower than that in the extract of method B. Although the amount of nucleic acids obtained from TRIzol and RNeasy Plant mini-kit was very low, gDNAs still existed in the extracts (Fig. 7). Furthermore, serial ten-fold dilutions of nucleic acids extracted from unmodified soybean seeds were amplified by PCR using the primers N-NS1 and C-18H (Bult
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b method B method C RNeasy Plant mini kit TRIzol
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Fig. 7 Amplification plots of CaMV35S (a) and NOS (b). The nucleic acids were extracted from genetically-modified maize seeds by method B, method C, TRIzol and RNeasy Plant mini-
Fig. 8 Agarose gel electrophoresis analysis of DNA fragments amplified by PCR. The nucleic acids were extracted from soybean seeds using method B (a), method C (b), TRIzol (c) and RNeasy Plant mini-kit (d), and were serially diluted ten-fold with RNase-free water. The dilutions were used as templates for PCR amplifications of 18S rDNA gene. Ten microliter of products was determined by 1.5 % agarose gel electrophoresis. Lane M, DL2000 DNA marker; lanes from 100 to 10-6 indicate 10-fold serial dilutions of nucleic acid extracts
et al. 1992). The details of reaction are shown in Supplementary Information. As shown in Fig. 8, the amount of gDNA in the extract of method C was at least 10 times lower than that in the extract of method
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kit, and subsequently used as templates for real-time PCR amplifications of CaMV35S and NOS. The experiments were done in triplex
B or two commercial kits (RNeasy Plant mini-kit and TRIzol). Therefore, method C can be effectively used for extracting RNA with relatively little gDNA contamination from seeds. Real-time RT-PCR was used to detect BPMV and TRSV carried by soybean seeds in order to examine the feasibility and applicability of RNA extracted by CPBA-MNPs for seed-borne virus detection. As shown in Table 2, the Ct values obtained from CPBA-MNPs were approximately equal to those obtained when using the commercial kits (RNeasy Plant mini-kit and TRIzol). The analysis of variance indicated that the differences among these three methods were not statistically significant (P = 0.831). The CPBA-MNPs-based RNA extraction method can be completed within 40 min, and its operation is relatively simple. Moreover, it does not involve any harmful reagents. Therefore, the method C shows good applicability for RNA extraction, and the extracted RNA is suitable for seed-borne virus detection. More importantly, RNA extracted by CPBAMNPs exhibits good integrity, which is potentially desirable for a variety of biological studies such as expression analysis or sequencing.
Conclusion CPBA-MNPs were used for the extraction of gDNA, total nucleic acids, and RNA from genetically-
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modified maize and unmodified soybean seeds infected with BPMV and TRSV. Genomic DNA extracted from the maize seeds was detected by realtime PCR, and RNA extracted from the soybean seeds was analyzed by real-time RT-PCR. Nucleic acids extracted by the CPBA-MNPs method show great potential for direct use as templates in PCR-based detection technologies. Acknowledgments This work was supported by a Special Fund from the General Administration of Quality Supervision (201110035 and 201310068) and the Chinese Ministry of Science and Technology (2011CB932803). Supporting information Reaction systems of real-time qPCR and RT-PCR PCR amplification of 18S rDNA from nucleic acid extracted from soybean seed Supplementary Table 1—Primers 1 and probes used in this study
References Bult C, Ka¨llersjo¨ M, Suh Y (1992) Amplification and sequencing of 16/18S rDNA from gel-purified total plant DNA. Plant Mol Biol Rep 10:273–284 C¸lc¸ek H (2005) Nucleotide isolation by boronic acid functionalized hydrogel beads. J Bioact Compat Pol 20:245–257 Demeke T, Jenkins GR (2010) Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 396:1977–1990 Deng C, Zhao X, Shuang L, Sun N, Chen J (2013) Bacterial magnetic particles real-time RT-PCR for detecting Bean pod mottle virus. Acta Phytophy Sin 40:91–92 Gomes AG, Azevedo AM, Aires-Barros MR, Prazeres DMF (2011) Studies on the adsorption of cell impurities from
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Biotechnol Lett (2015) 37:211–218 plasmid-containing lysates to phenyl boronic acid chromatographic beads. J Chromatogr A 1218:8629–8637 Li XY, Tang Y, Ge GL (2011) Preparation and f-potential characterization of highly dispersible phosphate-functionalized magnetite nanoparticles. Sci China-Phys Mech Astron 54:1766–1770 Liu M, Huang X, Ma Z, Chen H, Li M (2010) Detection of Tobacco ringspot virus by real-time RT-PCR assay. Lett Biotechnol 21:73–76 Nageswara-Rao M, Kwit C, Agarwal S, Patton MT, Skeen JA, Yuan JS, Manshardt RM, Stewart CN Jr (2013) Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.). BMC Biotechnol 13:1–11 ¨ zdemir A, Tuncel A (2000) Boronic acid-functionalized O HEMA-based gels for nucleotide adsorption. J Appl Pol Sci 78:268–277 Peano C, Samson MC, Palmieri L, Gulli M, Marmiroli N (2004) Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods. J Agr Food Chem 52:6962–6968 Singh N, Willson RC (1999) Boronate affinity adsorption of RNA: possible role of conformational changes. J Chromatogr A 840:205–213 Skakiba E, Chen P, Gergerich R, Li S, Shi A (2012) Reaction of mid-southern U.S. southern cultivars to Bean pod mottle virus and Tobacco ringspot virus. Crop Sci 52:1980–1989 Sun N, Deng CL, Zhao XL, Zhou Q, Ge GL, Liu Y, Yan WL, Xia Q (2014) Extraction of total nucleic acid based on silica-coated magnetic particles for RT-qPCR detection of plant RNA virus/viroid. J Virol Methods 197:204–211 Xu Y, Wu Z, Zhang L, Lu H, Yang P, Webley PA, Zhao D (2008) Highly specific enrichment of glycopeptides using boronic acid-functionalized mesoporous silica. Anal Chem 81:503–508 Yan J, Springsteen G, Deeter S, Wang B (2004) The relationship among pKa, pH, and binding constants in the interactions between boronic acids and diols—it is not as simple as it appears. Tetrahedron 60:11205–11209
ORIGINAL RESEARCH PAPER
Application of carboxyphenylboronic acid-functionalized magnetic nanoparticles for extracting nucleic acid from seeds Ning Sun • Congliang Deng • Guanglu Ge Qiang Xia
•
Received: 20 July 2014 / Accepted: 27 August 2014 / Published online: 12 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Magnetic iron oxide nanoparticles functionalized with 4-carboxyphenylboronic acid (CPBAMNPs) were developed for extracting genomic DNA, total RNA and nucleic acids from seeds. The seed samples were genetically-modified maize seeds and unmodified soybean seeds infected by bean pod mottle virus and tobacco ringspot virus. The total nucleic acids, genomic DNA, and RNA could be separately extracted from these seeds with high qualities using CPBA-MNPs under different conditions. Furthermore, the results of real-time quantitative qPCR and real-time reverse transcription (RT)-PCR indicated that the nucleic acids extracted from these seeds using CPBA-MNPs were suitable for the detection of genetically-modified seeds and seed-borne viruses.
Ning Sun and Congliang Deng have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1658-3) contains supplementary material, which is available to authorized users. N. Sun Q. Xia (&) State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Sipailou No. 2, Nanjing 210096, China e-mail: [email protected] N. Sun Q. Xia Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou 215123, China
Keywords 4-Carboxyphenylboronic acid DNA extraction Magnetic nanoparticles Maize seeds RNA extraction Seed-borne virus Soybean seeds Introduction During the past two decades, genetically-modified organisms (GMOs) have been approved for cultivation in several countries. However, controversy continues to surround all aspects of GMOs, regardless of their rapidly growing planting areas and importance in trade. As more and more foods and feeds are produced from GMOs, the public has paid greater attention on GMOs than ever before. On the other hand, the qualities of maize and soybean seeds are of great importance to agricultural production, because the health and vigor of the seeds greatly affect crop growth. Seed-borne viruses may cause serious plant diseases which can result in enormous crop yield losses (Skakiba et al. 2012). Therefore, the detection of genetically-modified seeds and seed-borne viruses is necessary for the proper labeling of geneticallymodified seeds and foods and for protecting the security N. Sun C. Deng Beijing Entry-exit Inspection and Quarantine Bureau, Beijing 100026, China N. Sun G. Ge CAS Key Laboratory of Measurement and Standardization for Nanotechnology, National Center for Nanoscience and Technology, Beijing 100190, China
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of crop production. A number of PCR-based methods have been developed for the detection of GMOs (Nageswara-Rao et al. 2013; Peano et al. 2004) and plant viruses (Deng et al. 2013; Liu et al. 2010; Sun et al. 2014). The attainment of high-quality nucleic acids from seed samples is indispensable for the nucleic acid amplification-based detection method. However, the challenges in the conventional extraction methods remain unresolved because the abundant amounts of polysaccharides and polyphenols contained in seed samples strongly inhibit the extraction of nucleic acid (Demeke and Jenkins 2010). Therefore, the development of suitable nucleic acid extraction methods has been intensively pursued. Phenylboronic acid affinity separation has attracted increasing attention because of the reverse binding ability of phenylboronic acid to glycoproteins (Xu et al. 2008), RNA (Gomes et al. 2011; Singh and Willson 1999), ¨ zdemir and Tuncel 2000), and nucleotides (C¸lc¸ek 2005; O other compounds. The relationship between phenylboronic acid agarose and RNA has been investigated and the results suggest that the amount of RNA adsorption can be enhanced in the presence of divalent cations such as Mg2?, Ca2?, or Ba2? (Singh and Willson 1999). Both RNA and gDNA can be adsorbed on to phenylboronic acid for chromatography under alkaline lysis-derived Escherichia coli lysates (Gomes et al. 2011). Moreover, it is possible that nucleic acids can be isolated from biological samples using phenylboronic acid-functionalized magnetic nanoparticles, although such a study has not yet been reported. Therefore, it is necessary to investigate the feasibility and specificity of phenylboronic acid-functionalized magnetic nanoparticles for the extraction of nucleic acids. Herein, we developed a novel nucleic acid extraction method based on the use of 4-carboxyphenylboronic acid-functionalized magnetic nanoparticles (CPBA-MNPs) and applied the method for the detection of genetically-modified maize seeds and two types of seed-borne viruses, i.e., bean pod mottle virus and tobacco ringspot virus.
Materials and methods Chemicals and reagents Iron(III) acetylacetonate [Fe(Acac)3] and 4-carboxyphenylboronic acid (CPBA) were purchased from Sigma-Aldrich. Plant Genomic DNA kit was
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purchased from Tiangen Biotech Co., Ltd. RNeasy Plant mini-kit was obtained from Qiagen GmbH. RNase A1, One-step PrimeScript RT-PCR kit (Perfect Real-time), Premix Ex Taq (Probe qPCR), k-HindIII digest DNA Marker, DL2000 DNA marker, DL500 DNA marker, and 100 bp DNA ladder were purchased from TaKaRa Biotech. Co. Ltd. The geneticallymodified maize seeds and non-genetically-modified soybean seeds infected with bean pod mottle virus (BPMV) and tobacco ringspot virus (TRSV) were kindly provided by Beijing Entry–Exit Inspection and Quarantine Bureau. Preparation and characterization of MNPs Fe3O4 nanoparticles were synthesized by following the protocol of Li et al. (2011). The particles were washed three times with ethanol and lyophilized using a vacuum freeze-drying apparatus for 48 h. Fe3O4 nanoparticles, 10 lg, were added to 2 ml ethanol containing 30 mg CPBA. The mixture was sonicated for 45 min until it became transparent and uniform, and was then left overnight at room temperature. After the addition of 2 ml saturated NaCl solution into the mixture to destroy the stability of the solution, the particles were collected with an external magnet and washed several times with ethanol and deionized water. The nanoparticles modified with CPBA (CPBA-MNPs) were then dispersed in the deionized water at 5 mg/ml. Afterward, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy and dynamic light scattering (DLS; Malvern Zetasizer NanoZS) analyses were performed to characterize the particles. Extraction of nucleic acid The procedures for the extraction of gDNA (method A), total nucleic acid (method B) and RNA (method C) from seeds are shown in Fig. 1. In method A, 900 ll CTAB lysis buffer (2 % CTAB and 1.4 M NaCl, pH 8.0) was added into a tube containing 50 mg seeds, held at 65 °C for 15 min and then cooled to room temperature. After the addition of 2 ll RNase A1 (50 mg/ll), the tube was incubated at 37 °C for 15 min. After the addition of 100 ll potassium acetate (5 M, pH 5.2), the tube was placed on ice for 5 min and centrifuged at 12,0009g for 2 min. Subsequently, 200 ll supernatant (more than 800 ll in total) was
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Fe3O4 nanoparticles
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CTAB lysis + RNase CTAB lysis
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method A. Finally, 10 ll extracted nucleic acid was analyzed by 1.5 % agarose gel electrophoresis. In addition, three commercial kits, Plant Genomic DNA kit, TRIzol and RNeasy Plant mini-kit, were used for the extraction of gDNA or RNA for comparing the efficiencies of different extraction methods. To produce an accurate comparison, the nucleic acid was extracted from 50 mg of the seeds using these three commercial kits, and finally, 200 ll elution buffer was used to dissolve the nucleic acid in these experiments. Ten microlitre extracted nucleic acid was analyzed by agarose gel electrophoresis, and the concentrations were measured at 260 nm. Real-time PCR and reverse transcription (RT)PCR
Total nucleic acid method A
method B
method C
Fig. 1 Schematic procedure for the extraction of gDNA (method A), total nucleic acid (method B) or RNA (method C) from seed using CPBA-MNPs
transferred into a new tube containing 1 mg CPBAMNPs and 200 ll ethanol was added then vortexed for 15 s and incubated for 3 min at room temperature. The particles were collected by DynaMag-2 Magnet and the remaining supernatant was discarded. The collected particles were washed twice with 70 % (v/v) ethanol and dried at room temperature. Fifty microlitre of TE buffer (50 mM Tris/HCl and 5 mM EDTA, pH 8.8) were added into the tube and held at room temperature for 3 min. The supernatant was transferred into a new tube after the CPBA-MNPs were collected. Ten microlitre extracted nucleic acid was analyzed by 1 % agarose gel electrophoresis. In the extraction of total nucleic acids (method B), the step of using RNase A to digest RNA was omitted from method A. In method C, 1 ml of SDS lysis buffer (2 % SDS, pH 8.0) was added into a tube containing 50 mg seeds. The tube was incubated at 65 °C for 15 min and placed on ice for 5 min, followed by centrifugation at 12,0009g for 2 min. [If the supernatant was not clear, one vol phenyl/chloroform (1:1, v/v) was added to 200 ll supernatant, followed by centrifugation at 12,0009g for 5 min]. Then 200 ll supernatant was transferred into a new tube containing 1 mg CPBA-MNPs, 200 ll ethanol was added followed by the steps similar to those described in
Real-time PCR was used for detecting the geneticallymodified maize seeds, while real-time RT-PCR was performed for the detection of BPMV and TRSV. Both detection processes were performed on an ABI 7900HT real-time PCR system (Applied Biosystems). In the detection of genetically-modified maize, the cauliflower mosaic virus 35S (CaMV35S) promoter region and the nopaline synthase (NOS) terminator region were amplified by real-time PCR according to the instructions of entry-exit inspection and quarantine standards of the People’s Republic of China (SN/T 1024–2003. Protocol of real-time PCR for detecting genetically-modified plants and their products, China, 2003). The detection of BPMV and TRSV by real-time RT-PCR was conducted by referring to the procedures described in previous studies (Deng et al. 2013; Liu et al. 2010). The primers and probes used in this study are given in Supplementary Table 1. Mg2? can be used to improve the activity of enzymes due to the complexation of EDTA in the elution buffer.
Results and discussion Characterization The morphologies of Fe3O4 nanoparticles with or without CPBA modification were characterized by TEM after the samples were dried using ethanol (Fig. 2). The Fe3O4 nanoparticles without any modifications are not dispersible in either water or hexane (Li et al. 2011) and can maintain their stability in
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Fig. 3 FT-IR spectra of Fe3O4 nanoparticles (a), CPBA-MNPs (b), and CPBA (c). The absorption peaks at *1,345 cm-1 and at*1,710 cm-1 are assigned to the carboxyl group, demonstrating that CPBA was successfully immobilized onto the iron oxide nanoparticles
Fig. 2 TEM images of Fe3O4 nanoparticles (a) and CPBAMNPs (b) dried through ethanol
ethanol for 10 min precipitating due to aggregation. However, CPBA-MNPs after surface functionalization can maintain their stability in ethanol for a prolonged time. This indicates that Fe3O4 nanoparticles can be functionalized with CPBA, which can significantly improve the dispersibility of the functionalized nanoparticles (Fig. 2b). FT-IR spectra of Fe3O4 nanoparticles, CPBAMNPs, and CPBA are shown in Fig. 3. As compared with the spectrum of Fe3O4 nanoparticles, a
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characteristic band (1,340 cm-1), which is ascribed to the B–O bond, can be observed from the spectrum of CPBA-MNPs (Xu et al. 2008). As compared with CPBA, CBPA-MNPs do not exhibit the characteristic band of COOH at 1,710 cm-1. These results provide strong evidence for the attachment of the carboxyl group of CPBA to Fe3O4 nanoparticles and indicate the success of using CPBA for particle modification. In our previous study, Fe3O4 nanoparticles dispersed in deionized water immediately aggregated together or attached to the sidewalls of the tube (Li et al. 2011), indicating the hydrophobicity of the particles. The hydrodynamic size and f-potential of CPBA-MNPs were characterized by using the Malvern Zetasizer NanoZS system. The characterization results revealed an average particle size of approximately 11 nm (Fig. 4a). CPBA-MNPs were positively charged below pH 9, above which the particles were slightly negatively-charged (Fig. 4b), and only a slight difference was observed in the pKa value from that reported for CPBA (Yan et al. 2004). The reason might be that the immobilization of CPBA on the surface of Fe3O4 nanoparticles affected the particle ionization. Extraction of gDNA and detection of geneticallymodified seeds To confirm the efficiency of the method, CPBA-MNPs were used to extract gDNAs from geneticallymodified maize seeds and non-genetically-modified
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Fig. 5 Agarose gel electrophoresis analysis of nucleic acids extracted from genetically-modified maize seeds and nongenetically-modified soybean seeds by method A and a commercial kit (Plant Genomic DNA kit). Lane 1 k-Hind IIIdigested DNA marker; lanes 2–3 and lanes 6–7 nucleic acids extracted from genetically-modified maize seeds and soybean seeds, respectively, using method A; lanes 4–5 and lanes 8–9: nucleic acids extracted from genetically-modified maize seeds and soybean seeds, respectively, using the commercial kit. All of the nucleic acids which were analyzed by agarose gel electrophoresis were extracted from independent samples
result indicates that gDNAs extracted by CPBAMNPs are suitable for PCR-based detection methods with demonstrated good performance. Total nucleic acid and RNA extracted from maize and soybean seeds Fig. 4 Hydrodynamic size distribution (a) and f-potential (b) of CPBA-MNPs in water measured by the Malvern Zetasizer NanoZS system
soybean seeds. The obtained results were compared with those of the commercial extraction kit (Plant Genomic DNA kit; Tiangen). Genomic DNAs were analyzed by 1 % agarose gel electrophoresis (Fig. 5). The concentration was determined from the absorbance at 260 nm, and the A260/280 ratio was between 1.68 and 1.82. Approximately 11 lg gDNA was extracted from 50 mg seeds using CPBA-MNPs, whereas only 7.5 lg gDNA was obtained using the commercial kit, indicating a higher efficiency of the CPBA-MNPs-based method. The nucleic acids extracted from genetically-modified maize seeds were detected by real-time PCR. As shown in Table 1, the threshold cycle (Ct) values obtained from CPBA-MNPs were slightly lower than those from Plant Genomic DNA kit, although the difference between the two methods was not statistically significant (Student’s t test, P = 0.208). This
Figure 6a and b show the electrophoresis results of the nucleic acids extracted from the maize and soybean seeds, respectively. Total nucleic acids were successfully extracted from the seeds with potassium acetate (KA) and gave an enhanced quality with an average A260/280 ratio of 1.697, as compared with the extraction without KA. Although the extraction of gDNA or total nucleic acids from seed samples has been successfully demonstrated, the extraction of RNA remains as a challenging task. In magnetic particlebased nucleic acid extraction, gDNA and RNA are usually co-extracted because of their chemical similarities (Sun et al. 2014). In the present study, 2 % SDS, pH 8.0, was used as the lysis buffer to extract nucleic acids from the independent samples. The extracted nucleic acids were determined by 1.5 % agarose gel electrophoresis. As shown in Fig. 6a and b, the total RNA could be successfully extracted from the samples without visible gDNA bands (Fig. 6a, lanes 5–7; Fig. 6b, lanes 3–4). Then, the performance of CPBA-MNPs for RNA extraction from maize or soybean seeds was compared with that of the
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Fig. 6 Gel agarose electrophoresis analysis of nucleic acids extracted using CPBA-MNPs, TRIzol, and RNeasy Plant minikit. a Nucleic acids extracted from soybean seeds by method B (lanes 2–4) and method C (lanes 5–7), and k-Hind III-digested DNA marker (lane 1); b nucleic acids extracted from maize seeds by method B (lanes 1–2) and method C (lanes 3–4); c nucleic acids extracted from maize seeds (lanes 2–3) and
soybean seeds (lanes 4–5) using TRIzol, and 100-bp DNA ladder (lane 1); d nucleic acids extracted from maize seeds (lanes 2–3) and soybean seeds (lanes 4–5) using RNeasy Plant mini-kit, and 100-bp DNA ladder (lane 1). All of the nucleic acids which were analyzed by agarose gel electrophoresis were extracted from independent samples
commercial kits (TRIzol and RNeasy Plant minikit) (Fig. 6c and d). Only a minute amount of RNA was extracted from the maize seeds by both commercial kits (Fig. 6c, lanes 1–2; Fig. 6d, lanes 1–2), and the resulting RNA integrity was inferior to that obtained from the CPBA-MNPs method (Fig. 6a). In addition, a large amount of degraded RNA was obtained from the soybean seeds using TRIzol (Fig. 6c, lanes 4–5), and high-quality RNA containing much gDNA contamination was extracted by using RNeasy Plant mini-kit (Fig. 6d, lanes 4–5). Maize seeds have higher contents of polysaccharides than soybean seeds, and polysaccharides show strong inhibitory effects on guanidinium thiocyanate-based or phenol–chloroform extraction. As both RNeasy plant mini-kit and TRIzol contain guanidinium salts, CPBA-MNPs show better performance on RNA extraction from maize seeds than do the two commercial kits.
To assess the amount of gDNA in the extract, the nucleic acids extracted from genetically-modified maize seeds and soybean seeds using method B, Method C, RNeasy Plant mini-kit and TRIzol were used for PCR or real-time PCR. The nucleic acids extracted from genetically-modified maize seeds were used as templates to amplify the CaMV35S and NOS (Fig. 7). The mean Ct values of method B were 26.8 ± 0.26 (CaMV35S) and 26.7 ± 0.17 (NOS), and the mean Ct values of method C were 30.7 ± 0.29 (CaMV35S) and 30.4 ± 0.45 (NOS). The results indicated that the amount of gDNA in the extract of method C was ten times lower than that in the extract of method B. Although the amount of nucleic acids obtained from TRIzol and RNeasy Plant mini-kit was very low, gDNAs still existed in the extracts (Fig. 7). Furthermore, serial ten-fold dilutions of nucleic acids extracted from unmodified soybean seeds were amplified by PCR using the primers N-NS1 and C-18H (Bult
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Fig. 7 Amplification plots of CaMV35S (a) and NOS (b). The nucleic acids were extracted from genetically-modified maize seeds by method B, method C, TRIzol and RNeasy Plant mini-
Fig. 8 Agarose gel electrophoresis analysis of DNA fragments amplified by PCR. The nucleic acids were extracted from soybean seeds using method B (a), method C (b), TRIzol (c) and RNeasy Plant mini-kit (d), and were serially diluted ten-fold with RNase-free water. The dilutions were used as templates for PCR amplifications of 18S rDNA gene. Ten microliter of products was determined by 1.5 % agarose gel electrophoresis. Lane M, DL2000 DNA marker; lanes from 100 to 10-6 indicate 10-fold serial dilutions of nucleic acid extracts
et al. 1992). The details of reaction are shown in Supplementary Information. As shown in Fig. 8, the amount of gDNA in the extract of method C was at least 10 times lower than that in the extract of method
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kit, and subsequently used as templates for real-time PCR amplifications of CaMV35S and NOS. The experiments were done in triplex
B or two commercial kits (RNeasy Plant mini-kit and TRIzol). Therefore, method C can be effectively used for extracting RNA with relatively little gDNA contamination from seeds. Real-time RT-PCR was used to detect BPMV and TRSV carried by soybean seeds in order to examine the feasibility and applicability of RNA extracted by CPBA-MNPs for seed-borne virus detection. As shown in Table 2, the Ct values obtained from CPBA-MNPs were approximately equal to those obtained when using the commercial kits (RNeasy Plant mini-kit and TRIzol). The analysis of variance indicated that the differences among these three methods were not statistically significant (P = 0.831). The CPBA-MNPs-based RNA extraction method can be completed within 40 min, and its operation is relatively simple. Moreover, it does not involve any harmful reagents. Therefore, the method C shows good applicability for RNA extraction, and the extracted RNA is suitable for seed-borne virus detection. More importantly, RNA extracted by CPBAMNPs exhibits good integrity, which is potentially desirable for a variety of biological studies such as expression analysis or sequencing.
Conclusion CPBA-MNPs were used for the extraction of gDNA, total nucleic acids, and RNA from genetically-
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modified maize and unmodified soybean seeds infected with BPMV and TRSV. Genomic DNA extracted from the maize seeds was detected by realtime PCR, and RNA extracted from the soybean seeds was analyzed by real-time RT-PCR. Nucleic acids extracted by the CPBA-MNPs method show great potential for direct use as templates in PCR-based detection technologies. Acknowledgments This work was supported by a Special Fund from the General Administration of Quality Supervision (201110035 and 201310068) and the Chinese Ministry of Science and Technology (2011CB932803). Supporting information Reaction systems of real-time qPCR and RT-PCR PCR amplification of 18S rDNA from nucleic acid extracted from soybean seed Supplementary Table 1—Primers 1 and probes used in this study
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