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Microchimica Acta

, 186:243 | Cite as

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

  • Weidan Chang
  • Weipeng Liu
  • Ying Liu
  • Fangfang Zhan
  • Huifang Chen
  • Hongtao Lei
  • Yingju LiuEmail author
Original Paper

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.

Graphical abstract

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.

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 

Notes

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.

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2019_3348_MOESM1_ESM.docx (987 kb)
ESM 1 (DOCX 986 kb)

References

  1. 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 241:10–18CrossRefGoogle Scholar
  2. 2.
    Khater M, de la Escosura-Muñiz A, Merkoçi A (2017) Biosensors for plant pathogen detection. Biosens Bioelectron 93:72–86CrossRefGoogle Scholar
  3. 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):1–25CrossRefGoogle Scholar
  4. 4.
    Fang Y, Ramasamy RP (2015) Current and prospective methods for plant disease detection. Biosensors 4:537–561CrossRefGoogle Scholar
  5. 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):134–141CrossRefGoogle Scholar
  6. 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 183:2277–2287CrossRefGoogle Scholar
  7. 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):392–403CrossRefGoogle Scholar
  8. 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):149–158CrossRefGoogle Scholar
  9. 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):478–488CrossRefGoogle Scholar
  10. 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):64–75CrossRefGoogle Scholar
  11. 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):5097–5103CrossRefGoogle Scholar
  12. 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 213:68–74CrossRefGoogle Scholar
  13. 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):35CrossRefGoogle Scholar
  14. 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):647–655CrossRefGoogle Scholar
  15. 15.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278CrossRefGoogle Scholar
  16. 16.
    Zhang F, Wen Y, Guo X (2014) CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet 23(R1):R40–R46CrossRefGoogle Scholar
  17. 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):8369–8374CrossRefGoogle Scholar
  18. 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):4951–4957CrossRefGoogle Scholar
  19. 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):2193–2200CrossRefGoogle Scholar
  20. 20.
    Zhao Y, Chen F, Li Q, Wang L, Fan C (2015) Isothermal amplification of nucleic acids. Chem Rev 115(22):12491–12545CrossRefGoogle Scholar
  21. 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 182:1095–1101CrossRefGoogle Scholar
  22. 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):537CrossRefGoogle Scholar
  23. 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):33CrossRefGoogle Scholar
  24. 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):4819–4825CrossRefGoogle Scholar
  25. 25.
    Liu J (2012) Adsorption of DNA onto gold nanoparticles and graphene oxide: surface science and applications. Phys Chem Chem Phys 14(30):10485–10496CrossRefGoogle Scholar
  26. 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):490–495CrossRefGoogle Scholar
  27. 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 222:221–225CrossRefGoogle Scholar
  28. 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):4608–4618CrossRefGoogle Scholar
  29. 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):11773–11785CrossRefGoogle Scholar
  30. 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):1253–1258CrossRefGoogle Scholar
  31. 31.
    Sperling RA, Parak WJ (2010) Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles. Phil Trans R Soc A 368(1915):1333–1383CrossRefGoogle Scholar
  32. 32.
    Yoshitake k WS, Ueda H (2008) Dimerization-based homogeneous fluorosensor proteins for the detection of specific dsDNA. Biosens Bioelectron 23:1266–1271CrossRefGoogle Scholar
  33. 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 64:292–299CrossRefGoogle Scholar
  34. 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 61:28–37CrossRefGoogle Scholar
  35. 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):967–975CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Weidan Chang
    • 1
    • 2
  • Weipeng Liu
    • 1
  • Ying Liu
    • 1
  • Fangfang Zhan
    • 3
  • Huifang Chen
    • 1
  • Hongtao Lei
    • 2
  • Yingju Liu
    • 1
    Email author
  1. 1.College of Materials & EnergySouth China Agricultural UniversityGuangzhouChina
  2. 2.The Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural UniversityGuangzhouChina
  3. 3.Fujian Key Laboratory of Plant Virology, Institute of Plant VirologyFujian Agriculture and Forestry UniversityFuzhouChina

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