| 引用本文: | 潘锋君,叶垚敏,张晓芹.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[J].中国现代应用药学,2022,39(7):912-917. |
| PAN Fengjun,YE Yaomin,ZHANG Xiaoqin.Optimization of Multiplex PCR System for Aflatoxin producing fungi based on ver-1, verB and ITS genes[J].Chin J Mod Appl Pharm(中国现代应用药学),2022,39(7):912-917. |
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| 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。 |
| 关键词: 黄曲霉素 产毒真菌 多重PCR 优化体系 |
| DOI：10.13748/j.cnki.issn1007-7693.2022.07.008 |
| 分类号:R927 |
| 基金项目:浙江省基础公益研究计划项目（LGF19H280002） |
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| Optimization of Multiplex PCR System for Aflatoxin producing fungi based on ver-1, verB and ITS genes |
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PAN Fengjun1, YE Yaomin2, ZHANG Xiaoqin2
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1.Lishui Municipal Central Hospital, Lishui 323000, China;2.Lishui Hospital of Traditional Chinese Medicine, Lishui 323000, China
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| 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. |
| Key words: aflatoxin toxigenic fungi multiplex PCR optimization system |
