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<p>Huanglongbing, associated with <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las), is a devastating citrus disease that necessitates early and accurate detection for effective management. While PCR-based diagnostics offer high sensitivity, their reliance on laboratory infrastructure limits field applicability. This study aimed to develop and validate a rapid, field-deployable recombinase polymerase amplification (RPA) assay combined with lateral flow assay (LFA) detection for <i>C</i>Las, targeting both single and dual gene regions. Initially, an RPA-LFA assay targeting the multi-copy <i>nrdB</i> gene was optimized and demonstrated a detection limit of 1 pg, equivalent to real-time PCR and superior to conventional PCR. To further enhance diagnostic accuracy, a dual-target RPA-LFA assay combining 16S rRNA and <i>nrdB</i> targets was developed to improve detection in low-titer samples and outperform prior single-gene RPA assays. Both assays exhibited 100% sensitivity and specificity compared to qPCR in validation using 61 field samples, with perfect diagnostic agreement (κ = 1.00), and no cross-reactivity with other citrus pathogens. The assays yielded rapid, visually interpretable results within 30&#xa0;min and were effective even with crude DNA extracts. These findings establish the dual-target RPA-LFA as a robust, sensitive, and scalable tool for on-site <i>C</i>Las detection, supporting its integration into citrus certification programs and early HLB surveillance strategies.</p>

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Enhanced RPA-LFA assays targeting the nrdB and 16S rRNA genes for the detection of Candidatus Liberibacter asiaticus

  • 期刊论文

  • 发表日期：

    2025年11月16日

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  • Mrugendra G. Gubyad,
  • Gurudayal Ram Guru,
  • Rushikesh D. Bharsakale,
  • Sunil B. Kokane,
  • Amol D. Kokane,
  • Siddrame Gowda,
  • Dilip Kumar Ghosh

摘要

Huanglongbing, associated with Candidatus Liberibacter asiaticus (CLas), is a devastating citrus disease that necessitates early and accurate detection for effective management. While PCR-based diagnostics offer high sensitivity, their reliance on laboratory infrastructure limits field applicability. This study aimed to develop and validate a rapid, field-deployable recombinase polymerase amplification (RPA) assay combined with lateral flow assay (LFA) detection for CLas, targeting both single and dual gene regions. Initially, an RPA-LFA assay targeting the multi-copy nrdB gene was optimized and demonstrated a detection limit of 1 pg, equivalent to real-time PCR and superior to conventional PCR. To further enhance diagnostic accuracy, a dual-target RPA-LFA assay combining 16S rRNA and nrdB targets was developed to improve detection in low-titer samples and outperform prior single-gene RPA assays. Both assays exhibited 100% sensitivity and specificity compared to qPCR in validation using 61 field samples, with perfect diagnostic agreement (κ = 1.00), and no cross-reactivity with other citrus pathogens. The assays yielded rapid, visually interpretable results within 30 min and were effective even with crude DNA extracts. These findings establish the dual-target RPA-LFA as a robust, sensitive, and scalable tool for on-site CLas detection, supporting its integration into citrus certification programs and early HLB surveillance strategies.