Establishment and application of the Recombinase-Aided Amplification-Lateral Flow Dipstick detection method for Pantoea ananatis on rice

Aiying, Wang; Ju, Luo; Cilin, Wang; Yuxuan, Hou; Baojun, Yang; Jian, Tang; Shuhua, Liu

doi:10.1007/s13313-023-00918-8

Cited by 2 publications

(2 citation statements)

References 24 publications

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“…First introduced in 2006 [23], RPA has lately become the molecular diagnostic tool of choice for rapid (results within 5-20 min), simple (requiring minimally processed test material, few and easy hands-on steps), specific (single-target copy number and singlenucleotide polymorphisms detection) and cost-effective (approximately 4.3 USD per test) identification of pathogens [24]. RPA-based diagnostic tools for several economically important phytopathogens have been developed in recent years [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. However, production and mass availability of affordably priced RPA assays that carry the possibility of multiple detections through the analysis of a single sample remains the single most formidable challenge for phytopathogen diagnostics at the consumer level [43].…”

Section: Discussionmentioning

confidence: 99%

“…RPA-based molecular toolkits have been developed for several agronomically important phytopathogens including DNA [31,32] and RNA [30,33,34] viruses, nematodes [35], bacterial [26,36,37], and fungal [27,29,38,39] species. To date, field diagnosis of rice leaf blight pathogen P. ananatis-using an integrated RPA-lateral flow dipstick assay [40]-remains the only diagnostic tool for characterizing any Pantoea species in a non-laboratory setting. The objectives of the current research were two-fold: (a) design and validate PCR primers for precise molecular identification and differentiation of P. stewartii subsp.…”

Section: Introductionmentioning

confidence: 99%

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Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Cai,

Tian,

Meng

et al. 2023

Agriculture

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Stewart’s vascular wilt and leaf blight of sweet corn is caused by the Gram-negative enteric bacterium Pantoea stewartii subsp. stewartii. Stewart’s wilt results in substantial yield losses worldwide warranting rapid and accurate disease diagnosis. Recombinase polymerase amplification (RPA) is an isothermal technique that is tolerant to host plant-derived inhibitors and is, therefore, ideally suited for rapid in-field detection vis-à-vis traditional polymerase chain reaction-based molecular assays. An RPA assay coupled with a Lateral Flow Device (LFD) was developed for rapid, accurate, and sensitive real-time detection of P. stewartii subsp. stewartii directly from the infected host offering in-field pathogen detection, timely disease management, and satisfying quarantine and phytosanitary requirements. Twelve novel primer sets were designed against conserved genomic regions of P. stewartii subsp. Stewartii; however, only the primers for amplification of the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB were discernibly unique and adequate for unambiguous identification of P. stewartii subsp. stewartii. The P. stewartii subsp. stewartii-specific primers were further validated in a simplex RPA assay for specificity against twenty-six bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii subsp. stewartii with an empirical detection limit of 0.0005 pg μL−1 bacterial DNA and 1 × 102 CFU mL−1 (app. two bacterial cells used per RPA reaction) in minimally processed samples for accurate, low-cost, and point-of-need diagnosis of the quarantine pathogen P. stewartii subsp. stewartii.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Cai,

Tian,

Meng

et al. 2023

Agriculture

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Rapid and sensitive detection of Pseudomonas syringae pv. actinidiae causing bacterial canker of kiwifruit using recombinase‐aided amplification with lateral flow dipstick assay

Liu,

Chen,

Peng

et al. 2024

Plant Pathology

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Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), is a highly destructive disease that results in significant economic losses. Timely detection of Psa is essential for implementing effective disease control measures. In this study, a recombinase‐aided amplification with lateral flow dipstick (RAA‐LFD) assay targeting the type III effector avirulence D1 (AvrD1) gene was developed. The assay was optimized for reaction conditions to ensure a swift and accurate detection protocol suitable for field application. In tandem, conventional PCR was also selected for comparison with the RAA‐LFD assay. The RAA‐LFD assay demonstrated a detection limit of 1 pg/μL for genomic DNA and 104 cfu/mL cell suspension. Specificity testing confirmed that the assay exclusively targeted Psa without cross‐reactivity with other endophytic bacteria present in kiwifruit. To validate the RAA‐LFD assay, it was compared with the conventional PCR method using 56 field samples. The results from both methods were found to be in complete agreement, validating the RAA‐LFD as a reliable diagnostic tool. Therefore, the established Psa RAA‐LFD assay provided technical support for the early and accurate diagnosis of bacterial canker in kiwifruit, which is crucial for disease management and prevention strategies.

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Establishment and application of the Recombinase-Aided Amplification-Lateral Flow Dipstick detection method for Pantoea ananatis on rice

Cited by 2 publications

References 24 publications

Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Rapid and sensitive detection of Pseudomonas syringae pv. actinidiae causing bacterial canker of kiwifruit using recombinase‐aided amplification with lateral flow dipstick assay

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