Abstract
An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2− causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs.
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References
Gilbert N (2013) A hard look at gm crops. Nature 497:24–26
Kamle S, Ali S (2013) Genetically modified crops: detection strategies and biosafety issues. Gene 522:123–132
Ruttink T, Morisset D, Van Droogenbroeck B, Lavrač N, Van Den Eede G, Žel J, De Loose M (2010) Knowledge-technology-based discovery of unauthorized genetically modified organisms. Anal Biol Chem 396:1951–1959
Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl M, Shipley G, Vandesompele J, Wittwer C (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Fu W, Zhu P, Wang C, Huang K, Du Z, Tian W, Wang Q, Wang H, Xu W, Zhu S (2015) A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment. Sci Rep 5:12715–12725
Jiang X, Zhang H, Wu J, Yang X, Shao J, Qiu B, Lin Z, Chen G (2014) G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food. Talanta 128:445–449
Manzanares-Palenzuela C, Martín-Fernández B, López S, López-Ruiz B (2015) Electrochemical genosensors as innovative tools for detection of genetically modified organisms. Trac-trend Anal Chem 66:19–31
Passamano M, Pighini M (2006) QCM DNA-sensor for GMOs detection. Sensors Actuators B 118:177–181
Yoke-Kqueen C, Son R (2010) Surface plasmon resonance biosensor for real-time detection of genetically modified organisms. Food Res Int 17:477–483
Wu H, Liu Y, Wang H, Wu J, Zhu F, Zou P (2016) Label-free and enzyme-free colorimetric detection of microRNA by catalyzed hairpin assembly coupled with hybridization chain reaction. Biosens Bioelectron 81:303–308
Xu M, He Y, Gao Z, Chen G, Tang D (2015) Isothermal cycling and cascade signal amplification strategy for ultrasensitive colorimetric detection of nucleic acids. Microchim Acta 182:449–454
Lin Y, Zhang M, Yin B, Ye B (2012) Attomolar ultrasensitive microRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal amplification. Anal Chem 84:5165–5169
Liu X, Shuai H, Liu Y, Huang K (2016) An electrochemical biosensor for DNA detection based on tungsten disulfide/multi-walled carbon nanotube composites and hybridization chain reaction amplification. Sensors Actuators B 235:603–613
Song W, Zhang Q, Sun W (2015) Ultrasensitive detection of nucleic acids by template enhanced hybridization followed by rolling circle amplification and catalytic hairpin assembly. Chem Commun 51:2392–2395
Li D, Cheng W, Yan Y, Zhang Y, Yin Y, Ju H, Ding S (2016) A colorimetric biosensor for detection of attomolar microRNA with a functional nucleic acid-based amplification machine. Talanta 146:470–476
Cai Z, Chen Y, Lin C, Wu Y, Yang C, Wang Y, Chen X (2014) A dual-signal amplification method for the DNA detection based on exonuclease III. Biosens Bioelectron 61:370–373
Huang Y, Gao Z, Luo H, Li N (2017) Sensitive detection of HIV gene by coupling exonuclease III-assisted target recycling and guanine nanowire amplification. Sensors Actuators B 238:1017–1023
Xue Q, Lv Y, Cui H, Gu X, Zhang S, Liu J (2015) A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid. Anal Chim Acta 856:103–109
Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymeraseScience. Science 249:505–510
Ellington A, Szostak J (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Tang L, Liu Y, Ali M, Kang D, Zhao W, Li J (2012) Colorimetric and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme. Anal Chem 84:4711–4717
Yang L, Du F, Chen G, Yasmeen A, Tang Z (2014) A novel colorimetric PCR-based biosensor for detection and quantification of hepatitis B virus. Anal Chim Acta 840:75–81
Shimron S, Wang F, Orbach R, Willner I (2012) Amplified detection of DNA through the enzyme-free autonomous assembly of hemin/G-quadruplex DNAzyme nanowires. Anal Chem 84:1042–1048
Hang H, Kwak CH, Kim G, Kim SM, Huh YS, Jeon T-J (2016) Identification of genetically modified DNA found in Roundup Ready soybean using gold nanoparticles. Microchim Acta 183:2649–2654
Qiu B, Zhang Y, Lin Y, Lu Y, Lin Z, Wong K, Chen G (2013) A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter. Biosens Bioelectron 41:168–171
Tam P (2015) Genetically modified organism (GMO) detection by biosensor based on SWCNT material. Curr Appl Phys 15:397–401
Santago-Felipe S, Tortajada-Genaro LA, Puchades R, Maquieria A (2016) Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchim Acta 183:1195–1202
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This work was funded by the Project of Zhongshan Science and Technology Bureau (2015B1013).
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Zhang, D., Wang, W., Dong, Q. et al. Colorimetric detection of genetically modified organisms based on exonuclease III-assisted target recycling and hemin/G-quadruplex DNAzyme amplification. Microchim Acta 185, 75 (2018). https://doi.org/10.1007/s00604-017-2618-0
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DOI: https://doi.org/10.1007/s00604-017-2618-0