Validation of loop-mediated isothermal amplification (LAMP) assay for identifying Fusarium verticilloides infection in fast extracted corn DNA
2018
Santos, M.M. | Rancho, K. | Tumolva, J.A. | Pascual, C.B. | Ocampo, E.T.M.
One common problem in postharvest handling and storage of corn is the infection of Fusarium verticilloides, which greatly decreases yield quality and quantity by mycotoxin, is coded for by the FUM1 gene, which can be used as a marker in detecting early stages of Fusarium infection. The authors developed a loop-mediated isothermal amplification (LAMP) assay for FUM1 that provides a fast, easy and sensitive approach in identifying infection by providing products observable by the naked eye. To test for the possibility of using the developed LAMP protocol, a fast DNA extraction protocol for corn kernels was also developed. Kernels were ground in liquid nitrogen in extraction buffer composed of EDTA, Tris-HCI and KCI. After centrifugation, the supernatant was mixed with isopropanol for DNA precipitation, and resuspended in TE-RNAse solution. The amplification of FUM1 gene was done by mixing designed internal, outer and loop primer pairs, and Bst polymerase in a single LAMP cocktail, followed by incubation at 65 deg C. LAMP products were visualized by changes in solution color made possibly by the dyes hydroxynaphtol blue and SYBR Green 1 that were added before and after incubation respectively. The LAMP assay showed positive amplification with a detection level as low as 1.526 ng/muL DNA. These results show that LAMP can be utilized in developing and marketing an easy-to-use detection kit for fumonisin production corn kernels.
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