Single kernel aflatoxin and fumonisin contamination distribution and spectral classification in commercial corn


Aflatoxin and fumonisin contamination distribution in corn is non-homogeneous. Therefore, bulk sample testing may not accurately represent the levels of contamination. Single kernel analysis could provide a solution to these problems and lead to remediation strategies such as sorting. Our study uses extensive single kernel aflatoxin (AF) and fumonisin (FM) measurements to (i) demonstrate skewness, calculate weighted sums of toxin contamination for a sample, and compare those values to bulk measurements, and (ii) improve single kernel classification algorithm performance. Corn kernels with natural contamination of aflatoxin and fumonisin (n = 864, from 9 bulk samples) were scanned individually twice for reflectance between the ultraviolet–visible–near infrared spectrum (304 nm–1086 nm), then ground and measured for aflatoxin and fumonisin using ELISA. Single kernel contamination distribution was non-homogeneous with 1.0% (n = 7) of kernels with ≥20 ppb aflatoxin (range 0 – 4.2×10^5 ppb), and 5.0% (n = 45) kernels with ≥2 ppm fumonisin (range 0 – 7.0×10^2 ppm). A single kernel weighted sum was calculated and compared to bulk measurements. Average difference in mycotoxin levels (AF = 0.0 log(ppb), FM = 0.0 log(ppm), weighted sum – measured bulk levels) calculated no systematic bias between the two methods, though with considerable range of −1.4 to 0.7 log(ppb) for AF and −0.6 to 0.8 log(ppm) for FM. Algorithms were trained on 70% of the kernels to classify aflatoxin (≥20ppb) and fumonisin (≥2ppm), while the remaining 30% of kernels were used for testing. For aflatoxin, the best performing algorithm was stochastic gradient boosting model with an accuracy of 0.83 (Sensitivity (Sn) = 0.75, Specificity (Sp) = 0.83), for both training and testing set. For fumonisin, the penalized discriminant analysis outperformed the rest of the algorithms, with a training accuracy of 0.89 (Sn = 0.87, Sp = 0.88), and testing accuracy of 0.86 (Sn = 0.78, Sp = 0.87). The present study improves the foundations for single kernel classification of aflatoxin and fumonisin in corn, and can be applied to high throughput screening. This study demonstrates the heterogeneous distribution of aflatoxin and fumonisin contamination at single kernel level, comparing bulk levels calculated from those data to traditional bulk tests, and utilizing a UV–Vis–NIR spectroscopy system to classify single corn kernels by aflatoxin and fumonisin level.


When to use one-dimensional, two-dimensional, and Shifted Transversal Design pooling in mycotoxin screening


While complex sample pooling strategies have been developed for large-scale experiments with robotic liquid handling, many medium-scale experiments like mycotoxin screening by Enzyme-Linked Immunosorbent Assay (ELISA) are still conducted manually in 48- and 96-well plates. At this scale, the opportunity to save on reagent costs is offset by the increased costs of labor, materials, and risk-of-error caused by increasingly complex pooling strategies. This paper compares one-dimensional (1D), two-dimensional (2D), and Shifted Transversal Design (STD) pooling to study whether pooling affects assay accuracy and experimental cost and to provide guidance for when a human experimentalist might benefit from pooling. We approximated mycotoxin contamination in single corn kernels by fitting statistical distributions to experimental data (432 kernels for aflatoxin and 528 kernels for fumonisin) and used experimentally-validated Monte-Carlo simulation (10,000 iterations) to evaluate assay sensitivity, specificity, reagent cost, and pipetting cost. Based on the validated simulation results, assay sensitivity remains 100% for all four pooling strategies while specificity decreases as prevalence level rises. Reagent cost could be reduced by 70% and 80% in 48- and 96-well plates, with 1D and STD pooling being most reagent-saving respectively. Such a reagent-saving effect is only valid when prevalence level is < 21% for 48-well plates and < 13%-21% for 96-well plates. Pipetting cost will rise by 1.3–3.3 fold for 48-well plates and 1.2–4.3 fold for 96-well plates, with 1D pooling by row requiring the least pipetting. Thus, it is advisable to employ pooling when the expected prevalence level is below 21% and when the likely savings of up to 80% on reagent cost outweighs the increased materials and labor costs of up to 4 fold increases in pipetting.