Development of multiplex PCR assay for detection of mycotoxigenic Fusarium species contamination in stored maize grains, India

Abstract

This investigation aimed to improve the detection of key mycotoxigenic Fusarium species to address agricultural biosecurity and food safety threats in stored maize grains in India. We developed a multiplex polymerase chain reaction (mPCR) assay for the selective detection of Fusarium species producing mycotoxin, fumonisin B₁ (FB₁), zearalenone (ZEA), and deoxynivalenol (DON) using their biosynthetic pathway genes viz., FUM1, FUM13, PKS4, PKS13 and TRI13, TRI7, respectively. The mPCR assay demonstrated a limit of detection (LOD) ranging from 1 × 10³ and 1 × 10² spores/g maize grain in both naturally contaminated and Fusarium spores spiked samples, respectively. Maize grain samples were also evaluated for the assessment of mycotoxigenic Fusarium species contamination by mPCR which resulted in A total of 53 maize grain samples (75 %) positive for mycotoxin chemotypes upon mPCR analysis. Among these, 40 sample tested positive for FB₁, 39 for ZEA and 25 for DON. The levels of contamination ranged from 4 to 6456 µg/kg for FB₁, 30–2192 µg/kg for ZEA and 5–1713 µg/kg for DON as determined by high-performance liquid chromatography (HPLC). Furthermore, the developed mPCR results exhibited a significant correlation (>95 %) with HPLC data confirming this assays reliability. This finding infers developed mPCR assay is an expeditious, cost-effective, sensitive and selective tool for detecting toxigenic Fusarium species. This method can play a crucial role in assessing food safety and public health, particularly in regions where maize contamination by mycotoxins is a significant concern.