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

Citrus Huanglongbing (HLB) is one of the most devastating diseases in the citrus industry and is caused primarily by ‘ Candidatus Liberibacter asiaticus’ ( C Las), a phloem-restricted gram-negative bacterium transmitted via citrus psyllids in the field. Precise C 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 C 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 C Las five-copy nrd 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 C 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 C Las in field conditions when coupled with simplified crude DNA extraction method, providing an alternative approach for on-site C Las detection in the context of HLB.