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<p>Citrus Huanglongbing (HLB) is one of the most devastating diseases in the citrus industry and is caused primarily by ‘<i>Candidatus</i> Liberibacter asiaticus’ (<i>C</i>Las), a phloem-restricted gram-negative bacterium transmitted via citrus psyllids in the field. Precise <i>C</i>Las detection is crucial for HLB control, particularly during extensive surveys in new emerging regions. Unfortunately, the lack of a practical on-site detection method for <i>C</i>Las due to the limited specificity of immunostrips and the costive non-user friendliness approaches prevent the effective disease control. In this study, a probe-based recombinase polymerase amplification (RPA) targeting the <i>C</i>Las five-copy <i>nrd</i>B gene (β-subunit of ribonucleotide reductase, RNR) was developed and evaluated for its specificity, sensitivity, and reliability. To enhance user-friendliness and facilitate widely use, an All-in-one kit was premade as freeze-dried pellets in individual tubes, containing all necessary components except DNA template. The applicability of the All-in-one kit for <i>C</i>Las detection was confirmed using plant or psyllid crude DNA extracts. Collectively, the All-in-one kit offers a specific, sensitive, rapid, cost-effective, and practical alternative for diagnosing <i>C</i>Las in field conditions when coupled with simplified crude DNA extraction method, providing an alternative approach for on-site <i>C</i>Las detection in the context of HLB.</p>

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Field applicable all-in-one kit developed for rapid detection of ‘Candidatus Liberibacter asiaticus’ in plants and psyllids

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月31日

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  • Shimin Fu,
  • Hongcen Wan,
  • Qiuyuan Duan,
  • Xuefeng Wang,
  • Binghai Lou,
  • Ziqin Bai,
  • Changyong Zhou

摘要

Citrus Huanglongbing (HLB) is one of the most devastating diseases in the citrus industry and is caused primarily by ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-restricted gram-negative bacterium transmitted via citrus psyllids in the field. Precise CLas detection is crucial for HLB control, particularly during extensive surveys in new emerging regions. Unfortunately, the lack of a practical on-site detection method for CLas due to the limited specificity of immunostrips and the costive non-user friendliness approaches prevent the effective disease control. In this study, a probe-based recombinase polymerase amplification (RPA) targeting the CLas five-copy nrdB gene (β-subunit of ribonucleotide reductase, RNR) was developed and evaluated for its specificity, sensitivity, and reliability. To enhance user-friendliness and facilitate widely use, an All-in-one kit was premade as freeze-dried pellets in individual tubes, containing all necessary components except DNA template. The applicability of the All-in-one kit for CLas detection was confirmed using plant or psyllid crude DNA extracts. Collectively, the All-in-one kit offers a specific, sensitive, rapid, cost-effective, and practical alternative for diagnosing CLas in field conditions when coupled with simplified crude DNA extraction method, providing an alternative approach for on-site CLas detection in the context of HLB.