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

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

关键词:  黄曲霉素  产毒真菌  多重PCR  优化体系

Key words:  aflatoxin  toxigenic fungi  multiplex PCR  optimization system

摘要:

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。

Abstract:

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.