Molecular and feasibility assessment for local aflatoxin standard production in Nigeria

Aflatoxin contamination remains a major food safety and trade challenge in Nigeria. This study investigated the occurrence, molecular characteristics, and aflatoxigenic potential of Aspergillus flavus isolates from market samples to support development of locally derived reference materials. Thirty maize and groundnut samples collected from five markets in Ilorin, Nigeria, yielded 19 fungal isolates, of which four morphologically identified A. flavus strains were subcultured into fourteen monosporic isolates for molecular and phenotypic analyses. Species confirmation was performed using ITS1/ITS4 and FLA1/FLA2 primers, while aflatoxin biosynthetic genes (aflR, aflS, aflD, aflP, and aflQ) were assessed by PCR. Molecular analysis confirmed all isolates as A. flavus, with varying frequencies of aflatoxin biosynthetic genes: aflR (100%), aflS and aflQ (57% each), aflD (36%), and aflP (29%). Only one isolate (isolate 10) possessed the complete gene set. No significant associations were observed among gene distributions (p > 0.05), although aflS and aflP showed a near-significant relationship (p = 0.070). Under controlled laboratory conditions, isolate 10 produced 16.33 ppb total aflatoxins on maize, comprising AFB₁ (8.57 ppb, 52.5%), AFB₂ (4.57 ppb, 28.0%), and AFG₁ (3.18 ppb, 19.5%). HPLC-FLD showed excellent performance, including strong linearity (R² > 0.99), high recovery (97.8-99.5%), good precision (RSD < 3%), and high sensitivity (LOD 0.02-0.03 ppb), meeting AOAC SMPR 2020.013 criteria. These findings demonstrate the feasibility of producing and benchmarking locally derived aflatoxin fractions as a proof-of-concept toward local reference materials, pending further purification and validation before regulatory application.