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Updated: Mar 24, 2026

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Phosphorothioate Modification-Regulated One-Pot CRISPR Assay for Arbovirus Detection.

Songkuan Zhuang1,2, Jie Li1, Zhoubin Fang1,3

  • 1Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China.

ACS Infectious Diseases
|March 23, 2026
PubMed
Summary
This summary is machine-generated.

A new psHOLMES method simplifies Chikungunya virus (CHIKV) diagnostics using CRISPR technology. This rapid, one-pot system offers high accuracy for detecting CHIKV RNA in clinical samples within 30 minutes.

Keywords:
CHIKVCRISPR diagnosticCas12aarthropod-borne virusesphosphorothioate modification

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Area of Science:

  • Molecular Biology
  • Virology
  • Biotechnology

Background:

  • Arthropod-borne viruses like Chikungunya virus (CHIKV) necessitate rapid and accurate diagnostic tools for effective epidemic control.
  • CRISPR diagnostic (CRISPR-Dx) technology presents a promising avenue, but optimizing one-pot systems requires balancing amplification and Cas cleavage.
  • Current methods often involve complex fine-tuning of Cas cis-cleavage activity.

Purpose of the Study:

  • To develop a simplified, one-pot, two-step CRISPR-Dx system for efficient Chikungunya virus RNA detection.
  • To introduce a novel method utilizing photocleavable partially phosphorothioate-modified DNA (ppPS-DNA) to regulate Cas12a activity.
  • To enable rapid development of sensitive and accurate diagnostic tools for clinical applications.

Main Methods:

  • Developed the psHOLMES method employing ppPS-DNA to control Cas12a activity in a one-pot, two-step process.
  • Cas12a activity was inhibited during isothermal amplification by ppPS-DNA binding.
  • Cas12a was reactivated post-amplification via UV-mediated photolysis of ppPS-DNA, initiating trans-cleavage detection.

Main Results:

  • psHOLMES achieved attomolar sensitivity for CHIKV RNA detection.
  • Demonstrated zero cross-reactivity with other related arboviruses.
  • Achieved 100% accuracy (50/50) in clinical samples, detecting CHIKV within 30 minutes.

Conclusions:

  • The psHOLMES method provides a simple and rapid approach for developing one-pot CRISPR-Dx systems.
  • Eliminates the need for traditional fine-tuning of Cas cis-cleavage activity, accelerating diagnostic development.
  • Offers a highly sensitive, accurate, and rapid diagnostic solution for Chikungunya virus, suitable for clinical settings.