Optimization of Multiplex PCR System for Aflatoxin producing fungi based on ver-1, verB and ITS genes

OBJECTIVE To optimize the multiplex PCR system of aflatoxin producing fungi and to determine the optimal PCR system. METHODS Multiple PCR primers were designed based on the sequences of ver-1, verB and ITS, which were the main regulatory genes in aflatoxin production, DNA template, Mg²⁺concentration, primer dosage, dNTPs dosage, annealing temperature and other factors were investigated by single factor and orthogonal test. RESULTS The optimal multiplex PCR system was as follows:50 mL system contained primer(5 µmol·L^-1) 3 µL, dNTPs(2.5 mmol·L^-1) 5 µL, Mg²⁺(2.5 mmol·L^-1) 4 µL, DNA concentration 10 ng·µL^-1, annealing temperature 56℃. CONCLUSION The established system can be used for the identification of aflatoxin producing fungi by multiplex PCR and has certain significance for the source identification of aflatoxin producing fungi.