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Background and Aims <p>: Persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, is the leading cause of cervical cancer. While molecular diagnostics offer high sensitivity, their deployment in decentralized settings remains limited. This study presents a proof-of-concept CRISPR/dCas9-based membrane-assisted detection platform for HR-HPV genotyping.</p> Methods <p>A membrane-based assay integrating recombinase polymerase amplification (RPA) with CRISPR/dCas9-mediated sequence-specific recognition was developed. FAM-labeled amplicons were captured by immobilized dCas9-sgRNA ribonucleoprotein complexes and detected via antibody-mediated colorimetric readout. Results: The assay enabled specific detection of HPV16 and HPV18 using genotype-specific sgRNAs, producing visually interpretable signals on a nitrocellulose membrane. No signal was observed in negative controls, demonstrating high analytical specificity. Semi-quantitative signal assessment confirmed clear differentiation between positive and negative samples.</p> Conclusion <p>This study demonstrates the feasibility of a CRISPR/dCas9-based membrane-assisted detection system for HR-HPV genotyping. While not yet configured as a fully integrated lateral flow device, the platform provides a foundation for future development of simplified, point-of-care molecular diagnostics.</p>

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Development of a CRISPR/dCas9-based membrane-assisted colorimetric assay for detection of high-risk HPV16 and HPV18: a proof-of-concept study

  • 期刊论文

  • 发表日期：

    2026年7月4日

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  • Vaibhav Kumar Tamrakar,
  • Kuldeep Sharma,
  • Pushpendra Singh,
  • Anudita Bhargava,
  • Pushpawati Thakur,
  • Sanjay Singh Negi

摘要

Background and Aims

: Persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, is the leading cause of cervical cancer. While molecular diagnostics offer high sensitivity, their deployment in decentralized settings remains limited. This study presents a proof-of-concept CRISPR/dCas9-based membrane-assisted detection platform for HR-HPV genotyping.

Methods

A membrane-based assay integrating recombinase polymerase amplification (RPA) with CRISPR/dCas9-mediated sequence-specific recognition was developed. FAM-labeled amplicons were captured by immobilized dCas9-sgRNA ribonucleoprotein complexes and detected via antibody-mediated colorimetric readout. Results: The assay enabled specific detection of HPV16 and HPV18 using genotype-specific sgRNAs, producing visually interpretable signals on a nitrocellulose membrane. No signal was observed in negative controls, demonstrating high analytical specificity. Semi-quantitative signal assessment confirmed clear differentiation between positive and negative samples.

Conclusion

This study demonstrates the feasibility of a CRISPR/dCas9-based membrane-assisted detection system for HR-HPV genotyping. While not yet configured as a fully integrated lateral flow device, the platform provides a foundation for future development of simplified, point-of-care molecular diagnostics.