黄曲霉素产毒真菌ver-1、verB及ITS基因序列多重PCR体系的优化

目的 对黄曲霉素产毒真菌的多重PCR体系进行优化，确定最佳PCR体系。方法 以黄曲霉素产生过程中的主要调控基因ver-1、verB及通用基因片段ITS序列为目的片段，设计多重PCR引物，采用单因素和正交优化试验，对DNA模板、Mg²⁺浓度、引物用量、dNTPs用量、退火温度等因素进行考察。结果 最佳多重PCR体系为：50 µL体系中含有引物(5 µmol·L^-1)3 mL，dNTPs(2.5 mmol·L^-1)5 µL，Mg²⁺(2.5 mmol·L^-1)4 µL，DNA浓度10 ng·µL^-1，退火温度56 ℃。结论 建立的体系可用于黄曲霉素产毒真菌的多重PCR鉴定，对黄曲霉产毒菌的源头鉴定具有一定的意义。

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.