Rapid and sensitive detection of Acidovorax citrulli in cucurbit seeds by visual loop-mediated isothermal amplification assay

Abstract

A rapid, sensitive and visual loop-mediated isothermal amplification (LAMP) method for detecting Acidovorax citrulli in cucurbit seed was developed in this study. The LAMP primers were designed to recognize the non-ribosomal peptide synthetase (NRPS) gene (locus tag: Aave_4658) from A. citrulli. The LAMP assay was conducted at 64°C in 1 hr with calcein as an indicator. The sensitivity and specificity of the LAMP assay were further compared with those of a conventional polymerase chain reaction (PCR). The LAMP assay is highly specific to A. citrulli, and no cross-reaction was observed with other bacterial pathogen. The sensitivity of the LAMP assay was 100-fold higher than that of conventional PCR with a detection limit of 1 pg of genomic DNA. Using the LAMP assay, 7 of 12 cantaloupe seedlots collected from Xinjiang province were determined to be positive for A. citrulli. In contrast, only 2 of 12 seedlots showed positive for the pathogen with conventional PCR. Moreover, A. citrulli was detected in 100% of artificially infested seedlots with 0.01% infestation or greater. Our results demonstrated that the LAMP assay was simple, visual and sensitive for detecting A. citrulli, especially in seed health testing. Hence, this method has great potential application in routine detecting seed-borne pathogens and reducing the risk of epidemics.