A Loop-Mediated Isothermal Amplification (LAMP) Assay for Fumonisin Gene Detection in Corn Fusarium Ear Rot

This study reports on the development of a LAMP assay for the rapid detection of the fumonisin gene in corn kernels with Fusarium ear rot (FER) caused by Fusarium verticillioides. Fumonisin is a mycotoxin that can be found on FER-infected grains or even in symptomless kernels, and can cause fatal diseases in animals and even in humans. FER can lead to varying disadvantages in the field, depending on the severity of infestation and timeliness of the control measures. In this study, the LAMP assay was targeted to rapidly identify the presence of fumonisin gene, for the visual detection of the gene in corn samples. LAMP permits DNA amplification through a relatively convenient set of requirements compared to the more common gene amplification technique, polymerase chain reaction (PCR). These advantages include single cycling temperature, short incubation time, ocular visual determination of results, and high specificity of gene products. Initially, six LAMP primers were designed from a fumonisin 1 gene homolog that was isolated and sequenced from FER-infected corn kernels. These six primers were used in LAMP reactions for the amplification of fumonisin gene in DNA extracted from FER-infected corn. Results showed that the optimal LAMP isothermal condition for fumonisin gene detection was at 65 ??C for 60 min. Further, it was determined that in the presence of the hydroxynaphthol blue dye, LAMP products from DNA of FER-infected corn developed a sky-blue color, while the negative controls showed dark blue to dark purple color. The LAMP products, when resolved by agarose gel electrophoresis, formed ladder-like series of bands that was typical of amplification products of LAMP reactions. Moreover, the specificity for the fumonisin gene of the LAMP protocol formulated in this study was comparable to a commercially available Fumonisin ELISA kit. The developed LAMP kit is recommended for faster, easier, and more cost-effective fumonisin gene detection in the field and in postharvest facilities compared to PCR-based and serological assays available.