Detection of Aflatoxin B1 Producing Aspergillus flavus Genes from Maize Feed using Loop-mediated Isothermal Amplification (LAMP) Technique
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Abstract
Aflatoxin contamination in maize feed, one of several agriculture crops grown for livestock feeding, is still a problem throughout the world mainly under hot and humid weather conditions like Thailand. In this study Aspergillus flavus (A. flavus), the key fungus for aflatoxin production especially aflatoxin B1 (AFB1), isolated from naturally infected maize were identified and characterized according to colony morphology and PCR detected on ITS, β-tubulin and calmodulin genes. The isolates were analysed for the presence of four aflatoxigenic biosynthesis genes in relation to their capability to produce AFB1, Ver1, Omt1, Nor1, and aflR. AFB1 production of the isolates was then confirmed using high performance liquid chromatography (HPLC) technique. A loop-mediated isothermal amplification (LAMP) was applied as an innovative technique for rapid detection of target nucleic acid. The reaction condition was optimized at 65°C for 60 min and calcein fluorescent reagent was added before amplification. The LAMP results showed clear differences between positive and negative reactions in end point analysis under daylight and UV light by the naked eye. In daylight, the samples with AFB1 producing A. flavus genes developed a yellow color, but those without the genes retained the orange color. When excited with UV light, the positive samples become visible by bright green fluorescence. LAMP reactions were positive after addition of purified target DNA until dilutions of 10-6 (89 pg/µl) whereas PCR reactions were positive after addition of purified target DNA until dilutions of 10-5 (0.0089 ng/µl). The reaction products were then confirmed and visualized with 1% agarose gel electrophoresis. To test the LAMP utility, 50 maize samples were collected from livestock farms and tested for the presence of four aflatoxigenic biosynthesis genes. The results were positive in 18 samples (36%) but negative in 32 samples (64%). All of the samples were rechecked by multiplex PCR and the results were the same as LAMP, indicating 100% specificity. Additionally, when assessing LAMP results with the HPLC aflatoxin analysis results using correlation coefficient, there was a significant correlation between the two methods (r = 0.83, P<0.05) which suggested that positive maize samples were likely to be a high- risk feed. In conclusion, the LAMP technique in this study can provide a simple and rapid approach for detecting AFB1 producing A. flavus genes from maize and appeared to be a promising tool for the prediction of potential aflatoxigenic risk in livestock feedings.
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