土壤中棉花黄萎病菌SYBR Green Ⅰ荧光RT-PCR定量检测技术研究  

SYBR Green Ⅰ real time RT-PCR assay for quantitatively detecting the occurrence of Verticillium dahliae of cotton in naturally infested soil

作　　者：肖蕊[1] 余真真 Elsharawy AA 魏锋 杨家荣[1] 

机构地区：[1]西北农林科技大学植物保护学院陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《菌物学报》2011年第4期598-603,共6页Mycosystema

基　　金：国家公益性行业(农业)科研专项(3-19);高等学校学科创新引智计划项目(No.B07049)

摘　　要：为实现田间土壤棉花黄萎病菌的早期检测,建立了土壤中棉花黄萎病菌的SYBR Green I荧光定量PCR检测方法。以含342bp PCR扩增产物的阳性质粒为参考,构建了标准曲线,并对该曲线的特异性、敏感性、可重复性进行了评价。结果表明,该方法具有快速、特异性强、敏感度高等特点。检测范围在3.8×103-3.8×108copies/μL之间有良好的线性关系,相关系数R2为0.996,扩增效率为101.5%,灵敏度比常规PCR方法高102倍。In order to explore early detection the occurrence of Verticillium dahliae of cotton in naturally infested soil,a SYBR Green I based Real time RT-PCR assay was developed.The positive plasmid included a 342bp PCR product was used as the reference and the specification curve was created on that basis.The specificity,sensitivity and reproducibility of the curve were evaluated and compared with conventional PCR techniques.The result showed that the Real time RT-PCR assay was rapid,highly sensitive and specific.A linear relationship was observed between the amount of input plasmid DNA and cycle threshold （Ct） values over a range of 3.8×103copies/μL to 3.8×108copies/μL,and correlation coefficient was 0.996.PCR amplification efficiency was 101.5%,which was 102 more sensitive than that of the conventional PCR.

关 键 词：大丽轮枝菌 SYBR Green I 荧光实时定量 

分 类 号：S435.621[农业科学—农业昆虫与害虫防治]