Design of a melting curve analysis (MCA) based on multiplex real-time PCR for detection of Aspergillus terreus and Aspergillus fumigatus in cereals and oilseeds samples

Abstract

Objective: In the early stages of food production, detection of Aspergillus species contamination is crucial to prevent potential harm to human health. The primary focus of this study was to evaluate the performance of Multiplex Real-Time PCR (MRT-PCR) and melting curve analysis (MCA) in identifying Aspergillus terreus and Aspergillus fumigatus in cereals and oilseeds. Results: The positive control data analysis indicates that the lovB gene displays melt peaks between 86.2 °C and 86.8 °C. The easL gene shows peaks from 89.2 °C to 89.7 °C. A high cycle threshold was observed in 100 ng of DNA using 5-fold DNA dilutions of A. fumigatus NCPF 7097 and A. terreus ATCC 20,542. Also, 36 A. fumigatus isolates (25.7%) and 21 A. terreus isolates (15%) were recognized among the 140 cereals and oilseeds samples through phenotypic analysis. Furthermore, the MRT-PCR technique detected 24 isolates (17.1%) of A. fumigatus and 11 (7.8%) of A. terreus. The detection of Aspergillus species in cereals and oilseed samples can be successfully achieved by utilizing DNA from pure culture with MRT-RCR assay and MCA.