Rapid and quantitative detection of Aspergillus nigerVan Tieghem using loop-mediated isothermal amplification assay

Peanut (Arachis hypogaeaL.) crown rot and root rot are common diseases caused by Aspergillus nigerVan Tieghem. Early and accurate detection of A. nigeris key to disease management. In this study, the design of two to five sets of loop-mediated isothermal amplification (LAMP) primers was based on the EglA, GOD, Tub, NRPS, Tan, CbhA, and CbhBgenes of A. niger. Of these, primer set GOD-91 was selected for optimization of the three-factor LAMP system: the BstDNA polymerase concentration, the concentration ratio of the inner and outer primers, and the concentration of Mg2+. In addition, the optimized LAMP reaction system for A. nigerdetection was validated for specificity, sensitivity, and on-site feasibility. The specificity test showed that A. nigercould be specifically detected with the proposed method without cross-amplification of other pathogenic fungi DNA. Moreover, based on the sensitivity test, the lowest detection limit of this reaction system was 5.1 × 10−7ng/µL pAN01 plasmid DNA, after which a standard curve was generated for the quantitative detection of A. niger.The LAMP method was further applied for field sample assessment before and after A. nigerinfection, successfully detecting A. nigerpresence in the samples collected in the field. This study yielded a sensitive, specific, and reproducible LAMP system that can be used to assess on-site samples within 45 min. It is an effective approach for the rapid and quantitative detection of A. niger.