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Background <p>Chronic hepatitis B (CHB) represents a leading driver of hepatocellular carcinoma and end-stage liver disease. Serum hepatitis B virus pregenomic RNA (HBV-pgRNA) has emerged as a new bioindicator strongly related to the efficacy and prognosis of CHB treatment. Seeking ultrasensitive, rapid, highly specific, and straightforward HBV-pgRNA detection, we constructed an innovative CRISPR-HBV-pgRNA platform through the integration of a CRISPR/Cas12b system with loop-mediated isothermal amplification (LAMP). Then, we interpreted the detection results via either real-time fluorescence (RTF) or a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB).</p> Methods <p>Herein, the AuNPs-based biosensor used was manufactured following our design. The unique LAMP primers and guide RNA (gRNA) were designed against the HBV-pgRNA gene, ensuring optimized diagnostic conditions: Reaction temperature and time. Both assay sensitivity and specificity were validated, and the feasibility was validated via clinical specimens from patients having chronic HBV infection.</p> Results <p>The developed AuNPs-based biosensor was successfully fabricated. Primers LAMP and gRNA were specifically designed to target the HBV pgRNA sequence. The integrated assay protocol, comprising RNA extraction (45&#xa0;min), RT-LAMP amplification (30&#xa0;min), CRISPR/Cas12b cleavage (5&#xa0;min), and visual readout (2&#xa0;min), was completed in 85&#xa0;min without reliance on costly instrumentation. The method achieved a detection limit of 10 copies/reaction and demonstrated no cross-reactivity with other tested pathogens.</p> Conclusions <p>The CRISPR-HBV-pgRNA assay is a powerful diagnostic tool and exhibits considerable potential for POC testing for the evaluation of chronic HBV infection status and antiviral drug efficacy, especially for resource-limited regions.</p>

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Nanoparticle-based biosensor integrated with CRISPR/Cas12b platform for sensitive and visual identification of hepatitis B virus pregenomic RNA in chronic hepatitis B patients

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月24日

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  • Xu Chen,
  • Yanyan Qin,
  • Shilei Dong,
  • Cencen Jia,
  • Ya Li,
  • Yalan Liu,
  • Qi Zhao,
  • Qingxue Zhou

摘要

Background

Chronic hepatitis B (CHB) represents a leading driver of hepatocellular carcinoma and end-stage liver disease. Serum hepatitis B virus pregenomic RNA (HBV-pgRNA) has emerged as a new bioindicator strongly related to the efficacy and prognosis of CHB treatment. Seeking ultrasensitive, rapid, highly specific, and straightforward HBV-pgRNA detection, we constructed an innovative CRISPR-HBV-pgRNA platform through the integration of a CRISPR/Cas12b system with loop-mediated isothermal amplification (LAMP). Then, we interpreted the detection results via either real-time fluorescence (RTF) or a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB).

Methods

Herein, the AuNPs-based biosensor used was manufactured following our design. The unique LAMP primers and guide RNA (gRNA) were designed against the HBV-pgRNA gene, ensuring optimized diagnostic conditions: Reaction temperature and time. Both assay sensitivity and specificity were validated, and the feasibility was validated via clinical specimens from patients having chronic HBV infection.

Results

The developed AuNPs-based biosensor was successfully fabricated. Primers LAMP and gRNA were specifically designed to target the HBV pgRNA sequence. The integrated assay protocol, comprising RNA extraction (45 min), RT-LAMP amplification (30 min), CRISPR/Cas12b cleavage (5 min), and visual readout (2 min), was completed in 85 min without reliance on costly instrumentation. The method achieved a detection limit of 10 copies/reaction and demonstrated no cross-reactivity with other tested pathogens.

Conclusions

The CRISPR-HBV-pgRNA assay is a powerful diagnostic tool and exhibits considerable potential for POC testing for the evaluation of chronic HBV infection status and antiviral drug efficacy, especially for resource-limited regions.