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<p>Tilapia lake virus (<i>Tilapinevirus tilapiae</i>; TiLV) remains a significant threat to global tilapia aquaculture, underscoring the urgent need for rapid, sensitive, and field-ready diagnostic approaches. In this study, we developed a CRISPR-Cas12a assisted reverse transcriptase-recombinase polymerase amplification assay (Cr-TiLV) targeting a conserved region of segment 6 of the TiLV genome. The integration of RT-RPA with Cas12a significantly enhanced analytical sensitivity, enabling the detection of viral RNA at levels as low as 10 copies. Diagnostic assessment using 40 clinical samples demonstrated that the Cr-TiLV assay achieved sensitivities of 86.67% and 82.29%, and accuracies of 90% and 85%, respectively, relative to semi-nested PCR and RT-qPCR, with specificity approaching 99.9% for both benchmark methods. Although less sensitive than RT-qPCR for samples with low viral loads, the assay consistently detected low to moderate viral concentrations and showed substantial concordance with semi-nested PCR. Operating isothermally at 39&#xa0;°C and requiring minimal laboratory resources, the Cr-TiLV assay offers a rapid and field-deployable diagnostic solution with strong potential for routine TiLV surveillance in resource-limited aquaculture settings.</p>

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Comparative Evaluation and Optimization of a CRISPR/Cas-Based Assay for Rapid Detection of Tilapinevirus tilapiae (TiLV) in Indian Isolates, Validated Against Conventional Nucleic acid Detection Methods

  • 期刊论文

  • 发表日期：

    2026年3月22日

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  • Kaliyamoorthy Thangadurai,
  • Megha Kadam Bedekar,
  • Chandraprakasham Manojkumar,
  • Meshram Supradhnya Namdeo,
  • Maharshi Limbola,
  • Kezhedath Jeena,
  • Kiran Dashrath Rasal,
  • Kooloth Valappil Rajendran,
  • Kundan Kumar

摘要

Tilapia lake virus (Tilapinevirus tilapiae; TiLV) remains a significant threat to global tilapia aquaculture, underscoring the urgent need for rapid, sensitive, and field-ready diagnostic approaches. In this study, we developed a CRISPR-Cas12a assisted reverse transcriptase-recombinase polymerase amplification assay (Cr-TiLV) targeting a conserved region of segment 6 of the TiLV genome. The integration of RT-RPA with Cas12a significantly enhanced analytical sensitivity, enabling the detection of viral RNA at levels as low as 10 copies. Diagnostic assessment using 40 clinical samples demonstrated that the Cr-TiLV assay achieved sensitivities of 86.67% and 82.29%, and accuracies of 90% and 85%, respectively, relative to semi-nested PCR and RT-qPCR, with specificity approaching 99.9% for both benchmark methods. Although less sensitive than RT-qPCR for samples with low viral loads, the assay consistently detected low to moderate viral concentrations and showed substantial concordance with semi-nested PCR. Operating isothermally at 39 °C and requiring minimal laboratory resources, the Cr-TiLV assay offers a rapid and field-deployable diagnostic solution with strong potential for routine TiLV surveillance in resource-limited aquaculture settings.