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<p>The Asian citrus psyllid, <i>Diaphorina citri</i> Kuwayama, is the vector of huanglongbing (HLB) citrus disease caused by the bacterium <i>Candidatus</i> Liberibacter asiaticus. The disease has rapidly spread throughout Pakistan, necessitating early detection for effective quarantine management. To address the urgent need for a&#xa0;rapid and cost-effective field diagnostic tool, we optimized a&#xa0;real-time loop-mediated isothermal amplification (LAMP) assay using crude DNA extracts from citrus leaf midribs and psyllid samples. This method demonstrated approximately 100-fold higher sensitivity than quantitative polymerase chain reaction (qPCR) and successfully detected diverse <i>Ca</i>. L.&#xa0;asiaticus isolates. Primers were designed to target highly conserved genomic regions, including phage-related sequences and the 16S rDNA, for specificity. The real-time LAMP assay enabled detection within 20 min using crude DNA from between five and&#xa0;10 psyllids, and citrus leaves mid-ribs. Amplification was monitored through a&#xa0;Smart-DART device paired with Android-based software for real-time fluorescence detection. Post-amplification, LAMP products were validated using gel electrophoresis and conventional PCR. While the assay confirmed <i>Ca</i>. L.&#xa0;asiaticus in both plant and psyllid samples, plant-derived crude DNA produced faint electrophoretic bands as compared to distinct patterns from psyllid DNA and purified plant DNA. Field application of this real-time LAMP protocol can enhance epidemiological studies by enabling large-scale screening of psyllid populations, thereby improving our understanding of <i>Ca</i>. L.&#xa0;asiaticus transmission dynamics and outbreak patterns. This approach offers a&#xa0;practical solution for early HLB detection, critical for mitigating crop losses in Pakistan’s citrus industry.</p>

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High-Sensitivity RT-LAMP Assay Enables Rapid, Field-Deployable Detection of Candidatus Liberibacter Asiaticus in Crude DNA Extracts from Citrus Phyllosphere and Psyllid Vectors

  • 期刊论文

  • 发表日期：

    2025年6月24日

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  • Ummad ud Din Umar,
  • Syed Muhammad Zaka,
  • Muhammad Nauman Tahir,
  • Muhammad Kashif,
  • Ateeq Ur Rehman,
  • Sagheer Atta,
  • Syed Atif Hasan Naqvi

摘要

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the vector of huanglongbing (HLB) citrus disease caused by the bacterium Candidatus Liberibacter asiaticus. The disease has rapidly spread throughout Pakistan, necessitating early detection for effective quarantine management. To address the urgent need for a rapid and cost-effective field diagnostic tool, we optimized a real-time loop-mediated isothermal amplification (LAMP) assay using crude DNA extracts from citrus leaf midribs and psyllid samples. This method demonstrated approximately 100-fold higher sensitivity than quantitative polymerase chain reaction (qPCR) and successfully detected diverse Ca. L. asiaticus isolates. Primers were designed to target highly conserved genomic regions, including phage-related sequences and the 16S rDNA, for specificity. The real-time LAMP assay enabled detection within 20 min using crude DNA from between five and 10 psyllids, and citrus leaves mid-ribs. Amplification was monitored through a Smart-DART device paired with Android-based software for real-time fluorescence detection. Post-amplification, LAMP products were validated using gel electrophoresis and conventional PCR. While the assay confirmed Ca. L. asiaticus in both plant and psyllid samples, plant-derived crude DNA produced faint electrophoretic bands as compared to distinct patterns from psyllid DNA and purified plant DNA. Field application of this real-time LAMP protocol can enhance epidemiological studies by enabling large-scale screening of psyllid populations, thereby improving our understanding of Ca. L. asiaticus transmission dynamics and outbreak patterns. This approach offers a practical solution for early HLB detection, critical for mitigating crop losses in Pakistan’s citrus industry.