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<p>Rapid, sensitive, and field-deployable diagnostic tools are crucial for effective outbreak management. To address this need, we developed a one-pot, contamination-free assay integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR-Cas12a) for the rapid and precise detection of the measles virus (MeV). RT-LAMP primers were designed to target conserved regions of the MeV genome. Reaction conditions—including temperature, MgSO<sub>4,</sub> deoxynucleotide triphosphates (dNTPs), and Bst polymerase concentrations—were systematically optimized to achieve robust amplification. CRISPR RNA (crRNA) was designed to target these regions. CRISPR-Cas12a reagents were preloaded into the tube lid. Following RT-LAMP amplification, CRISPR reagents were added to the tubes and mixed by manual shaking. CRISPR-Cas12a demonstrated highly specific target recognition and collateral reporter cleavage activities. Fluorescence detection in laboratory settings and lateral-flow readouts in point-of-care applications were evaluated. Representative sensitivity experiments detected MeV templates down to 10 copies per reaction, while Probit regression analysis estimated LoD<sub>95</sub> of 32.25 copies per reaction, compared with 97.32 copies per reaction RT-LAMP alone. The assay demonstrated high analytical specificity, with no cross-reactivity observed against other common respiratory viruses. The MeV-CRISPR assay was completed within 45–50&#xa0;min. Overall, this platform provided rapid, sensitive, and specific detection of MeV, showing strong potential for field applications in outbreak surveillance.</p>

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CRISPR-based dual-mode detection of measles virus for laboratory and point-of-care applications

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月5日

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  • Wahedul Karim Ansari,
  • Seung-Hyun Jung,
  • Yeun-Jun Chung

摘要

Rapid, sensitive, and field-deployable diagnostic tools are crucial for effective outbreak management. To address this need, we developed a one-pot, contamination-free assay integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR-Cas12a) for the rapid and precise detection of the measles virus (MeV). RT-LAMP primers were designed to target conserved regions of the MeV genome. Reaction conditions—including temperature, MgSO4, deoxynucleotide triphosphates (dNTPs), and Bst polymerase concentrations—were systematically optimized to achieve robust amplification. CRISPR RNA (crRNA) was designed to target these regions. CRISPR-Cas12a reagents were preloaded into the tube lid. Following RT-LAMP amplification, CRISPR reagents were added to the tubes and mixed by manual shaking. CRISPR-Cas12a demonstrated highly specific target recognition and collateral reporter cleavage activities. Fluorescence detection in laboratory settings and lateral-flow readouts in point-of-care applications were evaluated. Representative sensitivity experiments detected MeV templates down to 10 copies per reaction, while Probit regression analysis estimated LoD95 of 32.25 copies per reaction, compared with 97.32 copies per reaction RT-LAMP alone. The assay demonstrated high analytical specificity, with no cross-reactivity observed against other common respiratory viruses. The MeV-CRISPR assay was completed within 45–50 min. Overall, this platform provided rapid, sensitive, and specific detection of MeV, showing strong potential for field applications in outbreak surveillance.