Paper-based nuclease protection assay with on-chip sample pretreatment for point-of-need nucleic acid detection

Abstract

Pathogen detection is crucial for human, animal, and environmental health; crop protection; and biosafety. Current culture-based methods have long turnaround times and lack sensitivity. Nucleic acid amplification tests offer high specificity and sensitivity. However, their cost and complexity remain a significant hurdle to their applications in resource-limited settings. Thus, point-of-need molecular diagnostic platforms that can be used by minimally trained personnel are needed. The nuclease protection assay (NPA) is a nucleic acid hybridization–based technique that does not rely on amplification, can be paired with other methods to improve specificity, and has the potential to be developed into a point-of-need device. In traditional NPAs, hybridization of an anti-sense probe to the target sequence is followed by single-strand nuclease digestion. The double-stranded target-probe hybrids are protected from nuclease digestion, precipitated, and visualized using autoradiography or other methods. We have developed a paper-based nuclease protection assay (PB-NPA) that can be implemented in field settings as the detection approach requires limited equipment and technical expertise. The PB-NPA uses a lateral flow format to capture the labeled target-probe hybrids onto a nitrocellulose membrane modified with an anti-label antibody. A colorimetric enzyme-substrate pair is used for signal visualization, producing a test line. The nuclease digestion of non-target and mismatched DNA provides high specificity while signal amplification with the reporter enzyme-substrate provides high sensitivity. We have also developed an on-chip sample pretreatment step utilizing chitosan-modified paper to eliminate possible interferents from the reaction and preconcentrate nucleic acids, thereby significantly reducing the need for auxiliary equipment.

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Acknowledgments

The authors would like to thank Dr. John Wydallis for technical assistance. We also would like to thank the members of the Henry and Geiss labs for the helpful comments and discussions.

Funding

This work was supported by Colorado State University to CSH, DSD, and BJG and the National Institutes of Health (R01 AI132668) to BJG.

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Correspondence to Brian J. Geiss or David S. Dandy or Charles S. Henry.

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Noviana, E., Jain, S., Hofstetter, J. et al. Paper-based nuclease protection assay with on-chip sample pretreatment for point-of-need nucleic acid detection. Anal Bioanal Chem 412, 3051–3061 (2020). https://doi.org/10.1007/s00216-020-02569-w

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Keywords

  • Nuclease protection assay
  • Nucleic acid detection
  • Paper-based device
  • Lateral flow assay