Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of <i>Acidovorax citrulli</i> (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants

Abstract

Acidovorax citrulli (Ac) is an important pathogenic bacterium causing bacterial fruit blotch (BFB) in Cucurbitaceae plants and is an important quarantine pest in China. This study was conducted to establish a rapid, convenient, and accurate visual method for detecting A. citrulli. A. citrulli-specific primers and a prober were designed based on the conserved region of the YD-repeat protein gene. Loop-mediated isothermal amplification combined with lateral flow dipstick (LAMP-LFD) was used to establish an assay for the rapid visual detection of A. citrulli by optimizing the reaction temperature and time. The specificity, sensitivity, and performance of the optimized LAMP-LFD assay were evaluated using the genomic DNA of the tested isolates, A. citrulli pure culture, infested seeds, commercial seeds, and leaf samples. The optimal assay temperature and time were 64 °C and 60 min, respectively. The assay specifically detected A. citrulli, and no cross-reactions were observed with the genomic DNA of other closely related species. The detection sensitivity of the LAMP-LFD for detecting pure genomic DNA, the bacterial suspension, bacterial amount on seeds (colony-forming units (CFU)·g−1), and infection rate of seeds (%) were 1 fg·μL−1, 8 CFU·mL−1, 5 CFU·g−1, and 0.05% infestation per reaction, respectively. The positive detection rate of the LAMP-LFD assay was 20–100% in seed samples (n = 1000 seeds) with 0.05–0.1% infestation. The LAMP-LFD assay rapidly and accurately detected A. citrulli in seeds and leaf tissues carrying pathogens. This assay thus offers the advantages of easy operation, rapidity, high specificity and sensitivity, low cost (no need for complex and expensive precision instruments), visualization of detection results, good stability, and strong applicability, which can be used for epidemiological studies and disease management.

Keywords

• bacterial fruit blotch of <i>Cucurbitaceae</i> plants
• <i>Acidovorax citrulli</i>
• loop-mediated isothermal amplification
• lateral flow dipstick
• YD-repeat protein gene
• visual rapid detection