摘要
建立一种快速高效获取丝状真菌PCR反应模板的方法,提高丝状真菌PCR鉴定效率。通过单因素法对机械破壁联合微波法进行条件优化,利用优化后的方法获取13株不同种属丝状真菌的PCR反应模板,同时与Chelex-100法、机械破壁法作对比,以试剂盒抽提法作为阳性对照,进行ITS序列扩增,琼脂糖凝胶电泳检测扩增结果。机械破壁联合微波法获取丝状真菌PCR反应模板的最佳条件为40 Hz机械破壁1 min、微波700 W高温裂解3 min,采取该法与试剂盒抽提法获得的模板均成功扩增13株不同种属丝状真菌ITS序列,且PCR鉴定结果一致;Chelex-100法获得的模板成功扩增6株丝状真菌ITS序列;机械破壁法获得的模板虽成功扩增9株丝状真菌ITS序列,但扩增效果欠佳。机械破壁联合微波法能够有效获取丝状真菌PCR反应模板,与试剂盒抽提法相比具有操作简便、快速高效的优点,提高丝状真菌PCR鉴定效率。
A rapid and efficient method to obtain PCR template of filamentous fungi was established to improve PCR identification efficiency of filamentous fungi.Conditions of mechanical cell wall-breaking combined with microwave method were optimized by single factor method.The PCR reaction templates of 13 filamentous fungal of different genera were obtained by 4 methods such as mechanical cell wall-breaking,simultaneously compared with Chelex-100 method,and mechanical cell wall-breaking and kit as a positive control,the amplified results of different methods were detected through agarose gel electrophoresis.The results showed that PCR reaction template was obtained at optimum conditions:40 Hz of breaking frequency,for 1 min of breaking time,700 W of microwave power,for 3 min of microwave time.The results were consistent with mechanical cell wall-breaking combined with microwave method and kit extraction method.The amplified ITS sequences of 13 filamentous fungal genera were successfully.ITS sequence of six filamentous fungal strains was amplified successfully by Chelex-100 method.Nine filamentous fungal strains were amplified successfully by mechanical cell wall-breaking method yet with poor amplification results.Therefore,mechanical cell wall breaking combined with microwave method can effectively obtain PCR reaction template of filamentous fungi,having the advantages of simple operation,rapid and efficient as compared with kit extraction method.
作者
刘柳
张琨
邓百万
万一
LIU Liu;ZHANG Kun;DENG Bai-wan;WAN Yi(Schl.of Biol.Sci.&Engin.,Shaanxi Uni.of Technol.,Hanzhong 723000;Mol.Biol.Res.Ctr.,Shaanxi Inst,of Microbiol.,Xi'an 710043;Engin.Ctr of Mt.Qinling Nat.Prod.,Shaanxi Acad,of Sci.,Xi'an 710043)
出处
《微生物学杂志》
CAS
CSCD
2020年第3期98-103,共6页
Journal of Microbiology
基金
陕西省科学院一所一品专项(2018nk-01)
陕西省科技厅重点研发计划项目(2017NY-116)
陕西科技厅科技创新工程项目(2016HBGC-07)。
关键词
丝状真菌
细胞裂解
PCR
快速鉴定
filamentous fungi
cell lysis
PCR
rapid identification
