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<p>Piper yellow mottle virus (PYMoV), the causal agent of stunt disease in black pepper, poses a major threat to cultivation worldwide, leading to substantial yield losses and deterioration of product quality. Timely and reliable detection of PYMoV is therefore essential for effective disease management. However, conventional diagnostic methods are often labor-intensive, time-consuming, and require sophisticated laboratory equipment. In this study, we developed a rapid and highly sensitive RPA–CRISPR–LFA assay (recombinase polymerase amplification coupled with CRISPR-Cas12a and lateral flow assay) for the specific detection of PYMoV in black pepper. In this assay, the RPA-amplified product is specifically recognized and cleaved by the Cas12a–crRNA complex, producing a visual signal detectable on a lateral flow strip. Optimal reaction conditions were established using 500 nM LF reporter, 75 nM Cas12a, and 125 nM crRNA. Distinct test lines were observed within 30&#xa0;min, with maximum signal intensity recorded at 1&#xa0;h. The assay exhibited high specificity, showing no cross-reaction with other viruses infecting black pepper, and demonstrated a detection sensitivity comparable to that of qPCR. Validation with field samples confirmed its reliability and suitability for on-site detection of PYMoV in black pepper.</p>

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CRISPR-Cas12a coupled RPA-LFA for the specific detection of piper yellow mottle virus in crude plant extracts

  • 期刊论文

  • 发表日期：

    2026年8月5日

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  • Malayathodi Greeshma,
  • Alangar Ishwara Bhat

摘要

Piper yellow mottle virus (PYMoV), the causal agent of stunt disease in black pepper, poses a major threat to cultivation worldwide, leading to substantial yield losses and deterioration of product quality. Timely and reliable detection of PYMoV is therefore essential for effective disease management. However, conventional diagnostic methods are often labor-intensive, time-consuming, and require sophisticated laboratory equipment. In this study, we developed a rapid and highly sensitive RPA–CRISPR–LFA assay (recombinase polymerase amplification coupled with CRISPR-Cas12a and lateral flow assay) for the specific detection of PYMoV in black pepper. In this assay, the RPA-amplified product is specifically recognized and cleaved by the Cas12a–crRNA complex, producing a visual signal detectable on a lateral flow strip. Optimal reaction conditions were established using 500 nM LF reporter, 75 nM Cas12a, and 125 nM crRNA. Distinct test lines were observed within 30 min, with maximum signal intensity recorded at 1 h. The assay exhibited high specificity, showing no cross-reaction with other viruses infecting black pepper, and demonstrated a detection sensitivity comparable to that of qPCR. Validation with field samples confirmed its reliability and suitability for on-site detection of PYMoV in black pepper.