Diagnosis of Sugarcane White Leaf Disease Using the Highly Sensitive DNA Based Voltammetric Electrochemical Determination

Porntip Wongkaew, Suta Poosittisak

Department of Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.
Plant Pathology Division, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand.

DOI: 10.4236/ajps.2014.515240                

Abstract

A simple and highly sensitive analysis by electrochemical voltammetry has been developed for diagnosis of the most destructive crop disease in Thailand known as sugarcane white leaf (SCWL). Determination of the corresponding DNA interaction has been obtained from the voltammetric signals of electroactive redox methylene blue (MB) by means of cyclic and differential pulse voltammetry. In this study, a chitosan-modified glassy carbon electrode (GCE) was created by self-assembly to produce electrostatic platform for effective immobilization of the DNA. Fabrication of SCWL-DNA hybridization detection system was performed by immobilizing the ssDNA probe as a specific sensor onto chitosan-modified GCE. Hybridization of complementary DNA from the real samples could then be detected by its respective MB signal. This fabricated DNA probe sensor was shown to be capable for discriminative identification among the DNAs from SCWL plants, mosaic virus infected sugarcane and healthy sugarcane plants. Relationship between the specific hybridization signal and DNA target concentration was also observed under optimal condition. The detection limit of 4.709 ng/μl with the regression coefficient (R²) of 0.998 and overall RSD of 2.44% were obtained by response curve fit analysis. The actual SCWL-ssDNA immobilization and hybridizing event were subsequently confirmed by an observation under atomic force microscope. Thus these experiments demonstrate the first successful and effective DNA based voltammetric electrochemical determination for a verification of the specific pathogenic infection within plants from the real epidemic field.

Keywords

Sugarcane White Leaf Disease, Chitosan-Modified Glassy Carbon Electrode, DNA Hybridization Sensor, Methylene Blue, Voltammetric Signal, Atomic Force Microscope

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Kusalwong, A. (1980) Sugarcane Disease. In: Suriyapan, P., Ed., Sugarcane, Monograph Number 1, Department of Agriculture, Ministry of Agriculture and Cooperatives, Bangkok, 131-144.
[2]	Wongkaew, P. (2012) Sugarcane White Leaf Disease Characterization, Diagnosis Development, and Control Strategies. Functional Plant Science Biotechnology, 6, 73-84.
[3]	Wongkaew, P., Hanboonsong, Y., Sirithorn, P., Choosai, C., Boonkrong, S., Tinnangwattana, T., Kitchareonpanya, R., and Damak, S. (1997) Differentiation of Phytoplasmas Associated with Sugarcane and Gramineous Weed White Leaf Disease and Sugarcane Grassy Shoot Disease by RFLP and Sequencing. Theoretical Applied Genetics, 95, 660-663. http://dx.doi.org/10.1007/s001220050609
[4]	Wongkaew, P. (2013) Plant Diseases Caused by Phytoplasma. Biosensor Technology for Sustainable Development Research Group, Khon Kaen University Publishing, Khon Kaen.
[5]	Leu, L.S. (1983) White Leaf Disease. In: Agnihotri, V.P., Ed., Disease of Sugarcane, Oxford and IBH Publishing Co., Delhi, 250-267.
[6]	Kumarrasinghe, N.C. and Jones, P. (2001) Identification of white Leaf Disease of Sugarcane in Sri Lanka. Sugar Technology, 3, 55-58. http://dx.doi.org/10.1007/BF02945532
[7]	Win, N.K.K and Jung, H.Y. (2012) The Distribution of Phytoplasmas in Myanmar. Journal of Phytopathology, 160, 139-145. http://dx.doi.org/10.1111/j.1439-0434.2011.01875.x
[8]	Wongkaew, P. and Fletcher, J. (2004) Sugarcane White Leaf Phytoplasma in Tissue Culture: Long-Term Maintenance, Transmission, and Oxytetracycline Remission. Plant Cell Reports, 23, 426-434. http://dx.doi.org/10.1007/s00299-004-0847-2
[9]	Wongkaew, P., Sirithorn, P., Chaleeprom, W., Nakashima, K., Hayashi, T. and Koizumi, M. (1995) Detection of Sugarcane White Leaf Mycoplasma-Like Organism in Field Plants and Tissue Cultures by DNA Probes. In: Matangkasombat, P. and Yoshida, T., Eds., Microbial Utilization of Renewable Resources, ICCR Biotechnology, 9, 406-419.
[10]	Sarindu, N. and Clark, M.F. (1993) Antibody Production and Identify of MLOs Associated with Sugarcane White Leaf Disease and Bermuda Grass White Leaf Disease from Thailand. Plant Pathology, 42, 396-402. http://dx.doi.org/10.1111/j.1365-3059.1993.tb01517.x
[11]	Wongkaew, P. (1999) Sugarcane White Leaf Disease Management. Pimpatana Press, Khon Kaen.
[12]	Nakashima, K., Chaleeprom, W., Wongkaew, P. and Sirithorn, P. (1994) Detection of Mycoplasmalike Organism Associated with White Leaf Disease of Sugarcane in Thailand Using DNA Probes. JIRCAS Journal, 1, 57-67.
[13]	Palecek, E., Fojta, M. and Jelen, F. (2002) New Approaches in the Development of DNA Sensors: Hybridization and Electrochemical Detection of DNA and RNA at Two Different Surfaces. Bioelectrochemistry, 55, 85-90.
[14]	Yin, B-C., Guan, Y-M. and Ye, B-C. (2012) An Ultrasensitive Electrochemical DNA Sensor Based on the ssDNA-Assisted Cascade of Hybridization Reaction. Chemical Communications, 48, 4208-4210. http://dx.doi.org/10.1039/c2cc30997a
[15]	Wei, F., Lillehoj, P.B. and Ho, C-M. (2010) DNA Diagnostics: Nanotechnology-Enhanced Electrochemical Detection of Nucleic Acids. Pediatric Research, 67, 458-468. http://dx.doi.org/10.1203/PDR.0b013e3181d361c3
[16]	Oliveira-Brett, A.M., Piedade, J.A.P., Silva, L.A. and Diculescu, V.C. (2004) Voltammetric Determination of All DNA Nucleotides. Analytical Biochemistry, 332, 321-329. http://dx.doi.org/10.1016/j.ab.2004.06.021
[17]	Pedano, M.L. and Rivas, G.A. (2005) Immobilization of DNA at Glassy Carbon Electrodes: A Critical Study of Adsorbed Layer. Sensors, 5, 424-447. http://dx.doi.org/10.3390/s5060424
[18]	Erdem, A., Kerman, K., Meric, B., Akarca, U.S. and Ozsoz, M. (1999) DNA Electrochemical Biosensor for the Detection of Short DNA Sequences Related to the Hepatitis B Virus. Electroanalysis, 11, 586-587. http://dx.doi.org/10.1002/(SICI)1521-4109(199906)11:8<586::AID-ELAN586>3.0.CO;2-J
[19]	Erdem, A., Kerman, K., Meric, B., Akarca, U.S. and Ozsoz, M. (2000) Novel Hybridization Indicator Methylene Blue for the Electrochemical Detection of Short DNA Sequences Related to the Hepatitis B Virus. Analytica Chimica Acta, 422, 139-149. http://dx.doi.org/10.1016/S0003-2670(00)01058-8
[20]	Ye, Y.K. and Ju, H.X. (2003) DNA Electrochemical Behaviors, Recognition and Sensing by Combining with PCR Technique. Sensors, 3, 128-145. http://dx.doi.org/10.3390/s30600128
[21]	Guo, M.D., Li, Y.Q., Guo, H.X., Wu, X.Q. and Fan, L.F. (2007) Electrochemical Detection of Short Sequences Related to the Hepatitis B Virus Using MB on Chitosan-Modified CPE. Bioelectrochemistry, 70, 245-249. http://dx.doi.org/10.1016/j.bioelechem.2006.09.002
[22]	Erdem, A., Kerman, K., Meric, B., Ozkan, D., Kara, P. and Ozsoz, M. (2002) DNA Biosensor for Microcystis spp. Sequence Detection by Using Methylene Blue and Ruthenium Complex as Electrochemical Hybridization Label. Turkish Journal of Chemistry, 26, 851-862.
[23]	Zhang, D., Peng, Y., Qi, H., Gao, Q. and Zhang, C.X. (2010) Label-Free Electrochemical DNA Biosensor Array for Simultaneous Detection of the HIV-1 and HIV-2 Oligonucleotides Incorporating Different Hairpin-DNA Probes and Redox Indicator. Biosensors and Bioelectronics, 25, 1088-1094. http://dx.doi.org/10.1016/j.bios.2009.09.032
[24]	Sun, W., Qin, P., Gao, H., Li, G. and Jiao, K. (2010) Electrochemical DNA Biosensor Based on Chitosan/Nano-V2O5/MWCNTs Composite Film Modified Carbon Ionic Liquid Electrode and Its Application to the LAMP Product of Yersinia enterocolitica Gene Sequence. Biosensors and Bioelectronics, 25, 1264-1270. http://dx.doi.org/10.1016/j.bios.2009.10.011
[25]	Rai, V., Deng, J. and Toh, C.S. (2012) Electrochemical Nanoporous Alumina Membrane-Based Label-Free DNA Biosensor for the Detection of Legionella sp. Talanta, 98, 112-117. http://dx.doi.org/10.1016/j.talanta.2012.06.055
[26]	Rai, V., Hapuarachchi, H.C., Ng, L.C., Soh, S.H., Leo, Y.S. and Toh, C.S. (2012) Ultrasensitive cDNA Detection of Dengue Virus RNA Using Electrochemical Nanoporous Membrane-Based Biosensor. PLoS ONE, 8, Article ID: e42346. http://dx.doi.org/10.1371/journal.pone.0042346
[27]	Jiang, D., Liu, F., Liu, C., Liu, L. and Pu, X. (2013) An Electrochemical Sensor Based on Allosteric Molecular Beacons for DNA Detection of Escherichia coli O157:H7. International Journal of Electrochemical Science, 8, 9390-9398.
[28]	Li, J., Liu, Q, Liu, Y., Liu, S. and Yao, S.Z. (2005) DNA Biosensor Based on Chitosan Film Doped with Carbon Nanotubes. Analytical Biochemistry, 346, 107-114. http://dx.doi.org/10.1016/j.ab.2005.07.037
[29]	Jin, Y., Yao, X., Liu, Q. and Li, J.H. (2007) Hairpin DNA Probe Based Electrochemical Biosensor Using Methylene Blue as Hybridization Indicator. Biosensors and Bioelectronics, 22, 1126-1130. http://dx.doi.org/10.1016/j.bios.2006.04.011
[30]	Pänke, O., Kirbs, A. and Lisdat, F. (2007) Voltammetric Detection of Single Base-Pair Mismatches and Quantification of Label-Free Target ssDNA Using a Competitive Binding Assay. Biosensors and Bioelectronics, 22, 2656-2662. http://dx.doi.org/10.1016/j.bios.2006.10.033
[31]	Sun, W., Qin, P., Gao, H., Li, G. and Jiao, K. (2010) Electrochemical DNA Biosensor Based on Chitosan/Nano-V2O5/MWCNTs Composite Film Modified Carbon Ionic Liquid Electrode and Its Application to the LAMP Product of Yersinia enterocolitica Gene Sequence. Biosensors and Bioelectronics, 25, 1264-1270. http://dx.doi.org/10.1016/j.bios.2009.10.011
[32]	Motomura, T. and Aoyama, Y. (1992) Multiple Electrostatic Interaction of Polycationic Chitosan Oligomers with Nucleic Acids: Polyamine Promotes Stabilization of the A-Form of RNA and DNA in Aqueous Solutions. Bulletin of the Chemical Society of Japan, 65, 1755-1760. http://dx.doi.org/10.1246/bcsj.65.1755
[33]	Xu, C., Cai, H., He, P. and Fang, Y. (2001) Electrochemical Detection of Sequence-Specific DNA Using a DNA Probe Labeled with Aminoferrocene and Chitosan Modified Electrode Immobilized with ssDNA. Analyst, 126, 62-65. http://dx.doi.org/10.1039/b005847p
[34]	Xu, C., Cai, H., Xu, Q., He, P. and Fang, Y. (2001) Characterization of Single Stranded DNA on Chitosan-Modified Electrode and Its Application to the Sequence-Specific DNA Detection. Fresenius’ Journal of Analytical Chemistry, 369, 428-432. http://dx.doi.org/10.1007/s002160000673
[35]	Wongkaew, P. and Poosttisak, S. (2008) Direct Electrochemical DNA Sensor for Sugarcane White Leaf Disease Diagnosis Using Label Free DNA Probes and Oligochitosan Self Assembled Monolayer-Modified Glassy Carbon Electrodes. Proceeding of the 2nd International Conference on Technology and Innovation for Sustainable Development, Khon Kaen, 28-29 January 2008, 504-507.
[36]	Wongkaew, P. and Poosittisak, S. (2012) Electro-Affinity of SCWL-dsDNA on Different High Deacetylation Degree Chitosans Deposited Glassy Carbon Electrode. Proceeding of the International Conference on Technological Challenges in Developing Affordable in Vitro Molecular Diagnostics, Mumbai, 11-14 March 2012, 249-258.
[37]	Poosittisak, S. and Wongkaew, P. (2012) Electrochemical Characterization of the Hexacyanides of a Bare Screen Printed Carbon Electrode and Chitosan Thin Film Modified-Screen Printed Carbon Electrode. Proceeding of the International Conference on Technological Challenges in Developing Affordable in Vitro Molecular Diagnostics, Mumbai, 11-14 March 2012, 259-266.
[38]	Venkateswaran, N. and Venkateswaran, K.S. (1991) Nucleic Acid Probes in Microbiology. Defence Science Journal, 41, 335-356.
[39]	Kollar, A., Seemuller, E., Bonnet, F., Saillard, C. and Bove, J.M. (1990) Isolation of the DNA of Various Plant Pathogenic Mycoplasmalike Organisms from Infected Plants. Phytopathology, 80, 233-237. http://dx.doi.org/10.1094/Phyto-80-233
[40]	Kara, P., Kerman, K., Ozkan, D., Meric, B., Erdem, A. and Ozsoz, M. (2002) Electrochemical Genosensor for the Detection of Interaction between Methylene Blue and DNA. Electrochemistry Communications, 4, 705-709. http://dx.doi.org/10.1016/S1388-2481(02)00428-9
[41]	Henry, O.Y.F., Gutierrez Perez, J., Acero Sanchez, J.L. and O’Sullivan, C.K. (2010) Electrochemical Characterization and Hybridization Efficiency of Co-Assembled Monolayers of PEGylated ssDNA and Mercaptohexanol on Planar Gold Electrodes. Biosensor and Bioelectronics, 25, 978-983. http://dx.doi.org/10.1016/j.bios.2009.09.007
[42]	Patel, M.K., Solanki, P.R., Kumara, A., Khare, S., Gupta, S. and Malhotra, B.D. (2010) Electrochemical DNA Sensor for Neisseria meningitidis Detection. Biosensors and Bioelectronics, 25, 2586-2591. http://dx.doi.org/10.1016/j.bios.2010.04.025
[43]	Patel, M.K., Ali, A., Zafaryab, M., Agrawal, V.V., Rizvi, M.M.A., Ansari, Z.A., Ansari, S.G. and Malhotra, B.D. (2013) Biocompatible Nanostructured Magnesium Oxide-Chitosan Platform for Genosensing Application. Biosensors and Bioelectronics, 45, 181-188. http://dx.doi.org/10.1016/j.bios.2012.12.055
[44]	Peterson, A.W., Heaton, R.J. and Georgiadis, R.M. (2001) The Effect of Surface Probe Density on DNA Hybridization. Nucleic Acids Research, 29, 5163-5168. http://dx.doi.org/10.1093/nar/29.24.5163
[45]	Petrovynkn, D.Y., Kimura-Suda, H., Whitman, L.J. and Tarlov, M.J. (2003) Quantitative Analysis and Characterization of DNA Immobilized on Gold. Journal of American Chemistry Society, 125, 5219-5226. http://dx.doi.org/10.1021/ja029450c
[46]	Steel, A.B., Herne, T.M. and Tarlov, M.J. (1998) Electrochemical Quantitation of DNA Immobilized on Gold. Analytical Chemistry, 70, 4670-4677. http://dx.doi.org/10.1021/ac980037q
[47]	Ricci, F., Lai, R.Y., Heeger, A.J., Plaxco, K.W. and Sumner, J.J. (2007) Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor. Langmuir, 23, 6827-6834. http://dx.doi.org/10.1021/la700328r
[48]	Mirmomtaz, E., Castronovo, M., Grunwald, C., Bano, F., Scaini, D., Ensafi, A.A., Scoles, G. and Casalis, L. (2008) Quantitative Study of the Effect of Coverage on the Hybridization Efficiency of Surface-Bound DNA Nanostructures. Nano Letters, 12, 4134-4139. http://dx.doi.org/10.1021/nl802722k