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

Abstract

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 96well 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.

Evaluation of the Impact of Skewness, Clustering, and Probe Sampling Plan on Aflatoxin Detection in Corn

“…To simulate the skewness in concentration, the aflatoxin level for uncontaminated kernels (20 ppb) and contaminated kernels (20 ppb) were assumed to follow different distributions. Based on our previous study (Cheng, Chavez, & Stasiewicz, 2020) where data of 432 individually‐collected aflatoxin‐contaminated kernels were used for distribution fitting, aflatoxin concentration in healthy kernels could be approximated by a modified PERT distribution (min = 0, mode = 0.7, max = 19.99, shape = 80) and concentration in contaminated kernels could be fitted by 20 + Gamma distribution (). Due to limited computation capacity, the aflatoxin concentration in contaminated kernels was simplified to a constant number of 40,000 ppb, which matched the most probable number from the aforementioned distribution.…”

“…As a result, the final aflatoxin distribution would be a mixture of both distributions with the weights proportional to the number of contaminated and healthy kernels. The validation of simulated aflatoxin distribution was described in detail in (Cheng et al., 2020 ).…”

“…Finally, measuring the concentration of aflatoxin is simply simulated by calculating the mean of the concentration values in the test sample vector. For this study, the limit of detection (LOD) was set at 1 ppb to align with the enzyme‐linked immunosorbent assay (ELISA) kit used to generate the single kernel data adopted to validate the simulated aflatoxin concentration in (Cheng et al., 2020 ). It is appropriate to also simulate ELISA method testing for bulk levels because many specific ELISA kits and testing methods are approved by the Federal Grain Inspection Service (FGIS) (USDA, 2018 ) for bulk toxin testing.…”

Developments in mycotoxin analysis: an update for 2020-2021

“…Research on sampling and sample processing published over the past year include characterisation of mycotoxin distributions among maize kernels (Chavez et al, 2022) and within fields (Yi et al, 2021), an enhanced analysis of existing data sets on mycotoxin spatial distribution in bulk volumes (Kerry et al, 2021), and descriptions of sampling techniques for blood (Vidal et al, 2021), grain dust (Limay-Rios and Schaafsma, 2021), and for pooling samples for mycotoxin screening (Cheng et al, 2020). A very welcome retrospective analysis was also published on the disappointing decrease in researchers' consideration of proper sampling and processing of maize (Kumphanda et al, 2021).…”

“…The low concentrations and detection frequencies observed precludes use of the model to predict concentrations in grain for the other mycotoxins included in the study. Cheng et al (2020) examined pooling strategies to reduce the cost of mycotoxin screening by 48-and 96well enzyme-linked immunosorbent assay (ELISA) tests using simulations incorporating the maize single kernel aflatoxin and fumonisin data from Chavez et al (2022). The authors modelled outcomes of combining extracts of multiple samples and analysing the composite extracts to quickly rule out negative samples and assessed if the reduction in numbers of analyses gained by pooling was offset by increased cost in supplies due to the complexity of pooling strategies.…”