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Colorimetric detection of nucleic acid sequences in plant pathogens based on CRISPR/Cas9 triggered signal amplification

Microchimica Acta volume 186, Article number: 243 (2019) Cite this article

Abstract

A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525 nm and the DNA concentration in the range from 0.2 pM to 20 nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA.

Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.

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References

  1. Fox A, Mumford RA (2017) Plant viruses and viroids in the United Kingdom: an analysis of first detections and novel discoveries from 1980 to 2014. Virus Res 241:10–18

    CAS  Article  Google Scholar 

  2. Khater M, de la Escosura-Muñiz A, Merkoçi A (2017) Biosensors for plant pathogen detection. Biosens Bioelectron 93:72–86

    CAS  Article  Google Scholar 

  3. Martinelli F, Scalenghe R, Davino S, Panno S, Scuderi G, Ruisi P, Villa P, Stroppiana D, Boschetti M, Goulart LR, Davis CE, Dandekar AM (2015) Advanced methods of plant disease detection. A review. Agron Sustain Dev 35(1):1–25

    Article  Google Scholar 

  4. Fang Y, Ramasamy RP (2015) Current and prospective methods for plant disease detection. Biosensors 4:537–561

    Article  Google Scholar 

  5. Martín S, Alioto D, Milne RG, Guerri J, Moreno P (2002) Detection of Citrus psorosis virus in field trees by direct tissue blot immunoassay in comparison with ELISA, symptomatology, biological indexing and cross-protection tests. Plant Pathol 51(2):134–141

    Article  Google Scholar 

  6. Shojaei TR, Salleh MAM, Sijam K, Rahim RA, Mohsenifar A, Safarnejad R, Tabatabaei M (2016) Fluorometric immunoassay for detecting the plant virus Citrus tristeza using carbon nanoparticles acting as quenchers and antibodies labeled with CdTe quantum dots. Microchim Acta 183:2277–2287

    CAS  Article  Google Scholar 

  7. Toh SY, Citartan M, Gopinath SCB, Tang TH (2015) Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. Biosens Bioelectron 64(15):392–403

    CAS  Article  Google Scholar 

  8. Babu BK, Sharma R (2015) TaqMan real-time PCR assay for the detection and quantification of Sclerospora graminicola, the causal agent of pearl millet downy mildew. Eur J Plant Pathol 142(1):149–158

    CAS  Article  Google Scholar 

  9. De Sousa MV, Machado J d C, Simmons HE, Munkvold GP (2015) Real-time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds. Plant Pathol 64(2):478–488

    Article  Google Scholar 

  10. Osman F, Hodzic E, Kwon SJ, Wang J, Vidalakis G (2015) Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus. J Virol Methods 220(3):64–75

    CAS  Article  Google Scholar 

  11. Yang L, Tao Y, Yue G, Li R, Qiu B, Guo L, Lin Z, Yang HH (2016) Highly selective and sensitive electrochemiluminescence biosensor for p53 DNA sequence based on nicking endonuclease assisted target recycling and hyperbranched rolling circle amplification. Anal Chem 88(10):5097–5103

    CAS  Article  Google Scholar 

  12. Kil EJ, Kim S, Lee YJ, Kang EH, Lee M, Cho SH, Kim MK, Lee KY, Heo NY, Choi HS, Kwon ST, Lee S (2015) Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination. J Virol Methods 213:68–74

    CAS  Article  Google Scholar 

  13. Kong C, Wang Y, Fodjo EK, Yang G, Han F, Shen X (2018) Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles. Microchim Acta 185(1):35

    Article  Google Scholar 

  14. Bentsink L, Leone G, Van Beckhoven JRCM, Van Schijndel HB, Van Gemen B, Van der Wolf JM (2002) Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato. J Appl Microbiol 93(4):647–655

    CAS  Article  Google Scholar 

  15. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278

    CAS  Article  Google Scholar 

  16. Zhang F, Wen Y, Guo X (2014) CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet 23(R1):R40–R46

    CAS  Article  Google Scholar 

  17. Liu W, Yu H, Zhou X, Xing D (2016) In vitro evaluation of CRISPR/Cas9 function by an electrochemiluminescent assay. Anal Chem 88(17):8369–8374

    CAS  Article  Google Scholar 

  18. Zhang K, Deng R, Li Y, Zhang L, Li J (2016) Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification. Chem Sci 7(8):4951–4957

    CAS  Article  Google Scholar 

  19. Huang M, Zhou X, Wang H, Xing D (2018) Clustered regularly interspaced short palindromic repeats/Cas9 triggered isothermal amplification for site-specific nucleic acid detection. Anal Chem 90(3):2193–2200

    CAS  Article  Google Scholar 

  20. Zhao Y, Chen F, Li Q, Wang L, Fan C (2015) Isothermal amplification of nucleic acids. Chem Rev 115(22):12491–12545

    CAS  Article  Google Scholar 

  21. Wang J, Zou B, Rui J, Song Q, Kajiyama T, Kambara H, Zhou G (2015) Exponential amplification of DNA with very low background using graphene oxide and single-stranded binding protein to suppress non-specific amplification. Microchim Acta 182:1095–1101

    CAS  Article  Google Scholar 

  22. Piao J, Zhou X, Wu X (2018) Colorimetric human papillomavirus DNA assay based on the retardation of avidin-induced aggregation of gold nanoparticles. Microchim Acta 185(12):537

    Article  Google Scholar 

  23. Ma X, Guo Z, Mao Z, Tang Y, Miao P (2018) Colorimetric theophylline aggregation assay using an RNA aptamer and non-crosslinking gold nanoparticles. Microchim Acta 185(1):33

    Article  Google Scholar 

  24. Hu B, Guo J, Xu Y, Wei H, Zhao G, Guan Y (2017) A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology. Anal Bioanal Chem 409(20):4819–4825

    CAS  Article  Google Scholar 

  25. Liu J (2012) Adsorption of DNA onto gold nanoparticles and graphene oxide: surface science and applications. Phys Chem Chem Phys 14(30):10485–10496

    CAS  Article  Google Scholar 

  26. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529(7587):490–495

    CAS  Article  Google Scholar 

  27. Shi C, Liu Q, Zhou M, Zhao H, Yang T, Ma C (2016) Nicking endonuclease-mediated isothermal exponential amplification for double-stranded DNA detection. Sensors Actuators B Chem 222:221–225

    CAS  Article  Google Scholar 

  28. Xu SY, Zhu Z, Zhang P, Chan SY, Samuelson JC, Xiao J, Ingalls D, Wilson GG (2007) Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI. Nucleic Acids Res 35(14):4608–4618

    CAS  Article  Google Scholar 

  29. Dharanivasan G, Riyaz SUM, Jesse DMI, Muthuramalingam TR, Rajendran G, Kathiravan K (2016) DNA templated self-assembly of gold nanoparticle clusters in the colorimetric detection of plant viral DNA using a gold nanoparticle conjugated bifunctional oligonucleotide probe. RSC Adv 6(14):11773–11785

    CAS  Article  Google Scholar 

  30. Deng H, Xu Y, Liu Y, Che Z, Guo H, Shan S, Sun Y, Liu X, Huang K, Ma X, Wu Y, Liang XJ (2012) Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence. Anal Chem 84(3):1253–1258

    CAS  Article  Google Scholar 

  31. Sperling RA, Parak WJ (2010) Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles. Phil Trans R Soc A 368(1915):1333–1383

    CAS  Article  Google Scholar 

  32. Yoshitake k WS, Ueda H (2008) Dimerization-based homogeneous fluorosensor proteins for the detection of specific dsDNA. Biosens Bioelectron 23:1266–1271

    Article  Google Scholar 

  33. Qiu L, Shen Z, Wu ZS, Shen GL, Yu R (2015) Discovery of the unique self-assembly behavior of terminal suckerscontained dsDNA onto GNP and novel “light-up” colorimetric assay of nucleic acids. Biosens Bioelectron 64:292–299

    CAS  Article  Google Scholar 

  34. Ermini ML, Mariani S, Scarano S, Minunni M (2014) Bioanalytical approaches for the detection of single nucleotide polymorphisms by surface plasmon resonance biosensors. Biosens Bioelectron 61:28–37

    CAS  Article  Google Scholar 

  35. Li S, Liu H, Jia Y, Deng Y, Zhang L, Lu Z, He N (2012) A novel SNPs detection method based on gold magnetic nanoparticles array and single base extension. Theranostics 2(10):967–975

    CAS  Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Scientific Foundation of China (21705051, 21874048), the Scientific Foundation of Guangdong Province (2017A030313077), the Science and Technology Planning Project of Guangdong Province (2016B030303010), National Key Research and Development Program of China (SQ2017YFC160089), the Program for the Top Young Innovative Talents of Guangdong Province (2016TQ03N305), and the Foundation for High-level Talents in South China Agricultural University.

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Author notes

  1. Weidan Chang and Weipeng Liu contributed equally to this work.

Authors and Affiliations

  1. College of Materials & Energy, South China Agricultural University, Guangzhou, 510642, China

    Weidan Chang, Weipeng Liu, Ying Liu, Huifang Chen & Yingju Liu

  2. The Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou, 510642, China

    Weidan Chang & Hongtao Lei

  3. Fujian Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Fangfang Zhan

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  1. Weidan Chang
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Correspondence to Yingju Liu.

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Chang, W., Liu, W., Liu, Y. et al. Colorimetric detection of nucleic acid sequences in plant pathogens based on CRISPR/Cas9 triggered signal amplification. Microchim Acta 186, 243 (2019). https://doi.org/10.1007/s00604-019-3348-2

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  • DOI: https://doi.org/10.1007/s00604-019-3348-2

Keywords

  • Isothermal amplification
  • Gold nanoparticles
  • AuNP probes
  • Triggered aggregation
  • Single-base mismatch
  • Phytophthora infestans
  • Cas9/sgRNA complex
  • Localized surface plasmon resonance
  • Rolling circle amplification
  • Double-strand break