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

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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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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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