Total Aflatoxin Colloidal Gold Test Cassette

As a pathogenic fungus, Aspergillus flavus can produce carcinogenic aflatoxins (AFs), which poses a great threat to crops and animals. Msb2, the signalling mucin protein, is a part of mitogen-activated protein kinase (MAPK) pathway which contributes to a range of physiological processes. In this study, the roles of membrane mucin Msb2 were explored in A. flavus by the application of gene disruption. The deletion of msb2 gene (Delta msb2) caused defects in vegetative growth, sporulation and sclerotia formation when compared to WT and complement strain (Delta msb2) in A. flavus. Using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis, it was found that deletion of msb2 down-regulated aflatoxin B-1 (AFB(1)) synthesis and decreased the infection capacity of A. flavus. Consistently, Msb2 responds to cell wall stress and osmotic stress by positively regulating the phosphorylation of MAP kinase. Notably, Delta msb2 mutant exhibited cell wall defect, and it was more sensitive to inhibitor caspofungin when compared to WT and Delta msb2. Taking together, these results revealed that Msb2 plays key roles in morphological development process, stresses adaptation, secondary metabolism and pathogenicity in fungus A. flavus.

In this work, a green enzyme-linked immunosorbent assay (ELISA) based on the single-stranded binding protein (SSB)-assisted aptamer was designed for biosensing applications. Combined with the biotin-streptavidin (SA) system and the high catalytic activity of horseradish peroxidase (HRP), this SSB-assisted aptamer sensor was applied for the detection of aflatoxin B1, ochratoxin A, and zearalenone. In this novel ELISA, mycotoxin-protein conjugations were replaced by SSB to avoid the hazard of mycotoxin, whereas antibodies were replaced by aptamer to avoid the complex and tedious preparation of antibodies. In the absence of target mycotoxins, SSB can bind the aptamer-biotin specifically. Detection was performed using the strong combination of biotin and SA after adding SA-HRP and substrate/chromogen solution, thereby resulting in a strong yellow color signal. In the presence of target mycotoxins, the aptamerbiotin cannot bind to the SSB, thereby leading to a weak yellow color signal. Under optimal conditions, the designed method was successfully applied for the determination of real sample and exhibited high specificity and low limits of detection in corn (112 ng L-1 for aflatoxin B1, 319 ng L-1 for ochratoxin A, and 377 ng L-1 for zearalenone). The green ELISA may also be extended to the detection of other biohazardous targets by changing the aptamer.