一种改进的尖孢镰刀菌的实时荧光定量PCR检测方法

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。

An improved real time fluorescence quantitative PCR method for detecting Fusarium

The aim of this study is to develop a more efficient and sensitive method for quantitative detection of soil pathogens by real time PCR. The soil samples containing 4×101-4×106 spore/g were prepared by mixing the cultured spore suspension and long term cultivated paddy soil. The total DNA was extracted from the prepared soil samples using MoBio PowerSoil@ DNA Isolation Kit. A 1 446 bp DNA fragment containing the qPCR target sequence was amplified from soil DNA by conventional PCR, then the PCR products were used as templates to construct the standard curve of the fluorescence quantitative PCR. The results indicated that the correlation coefficient of qPCR standard curve was 0.985, and the lower limit of detection was 4×103 spore/g when the soil DNA was used as PCR templates. When the pre amplified PCR products were used as template, the correlation coefficient of qPCR standard curve was 0.974, and the lower limit of detection was 4×102 spore/g, that is the sensitivity of the method increased by 10 fold. These results showed that when combined with the specific primers of different soil pathogenic fungi, this method can provide technical support for effective quantitative detection of soil pathogens.