一种改进的尖孢镰刀菌的实时荧光定量PCR检测方法

发布时间：2018-10-20 19:17

【摘要】：为实现荧光定量PCR对土壤病原真菌数量更为高效、灵敏的检测,本研究将液体培养的孢子悬浮液和长年耕作的水稻土制作成孢子浓度为4×10~1~4×10~6 spore/g的带菌土,采用MoBio PowerSoil@DNA Isolation Kit提取模拟带菌土壤总DNA,引入常规PCR预扩增包含qPCR目标序列的1 446bp片段,以预PCR产物为模板进行qPCR,构建荧光定量PCR标准曲线。研究结果表明:以试剂盒提取的带菌土壤总DNA为模板绘制的qPCR标准曲线相关系数r为0.985,检测下限为4×10~3 spore/g土;引入预PCR后,qPCR标准曲线相关系数r为0.974,检测下限为4×10~2 spore/g土,较未引入时提高了10倍,结合不同土壤病原真菌的特异性引物,该检测方法可为土壤病原真菌的有效定量检测提供技术支撑。
[Abstract]:In order to detect the number of soil pathogenic fungi more efficiently and sensitively by fluorescence quantitative PCR, spore suspension of liquid culture and paddy soil cultivated for years were used to produce spore carrying soil with a concentration of 4 脳 10 ~ (-1) and 4 脳 10 ~ (-6) spore/g. The total DNA, of simulated bacterial soil was extracted by MoBio PowerSoil@DNA Isolation Kit and the 1 446bp fragment containing the target sequence of qPCR was preamplified by conventional PCR. The fluorescence quantitative PCR standard curve was constructed by qPCR, using the pre-PCR product as template. The results showed that the correlation coefficient of qPCR standard curve drawn from the total DNA extracted by the kit was 0.985, the detection limit was 4 脳 10 ~ (-3) spore/g soil, the correlation coefficient of qPCR standard curve was 0.974 and the detection limit was 4 脳 10 ~ (2) spore/g soil after the introduction of pre-PCR, the correlation coefficient of qPCR standard curve was 0.974, and the detection limit was 4 脳 10 ~ (-2) spore/g soil. This method can provide technical support for the effective quantitative detection of soil pathogenic fungi by combining with the specific primers of different soil pathogenic fungi.
【作者单位】：中南大学研究生院隆平分院;湖南省西瓜甜瓜研究所;湖南省植物保护研究所;
【基金】：公益性行业(农业)科研专项(201503110-03)
【分类号】：S432.44

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