实时荧光PCR检测瓜类蔓枯病菌的方法研究  

Study on the Method of Detecting the Didymella Bryoniae by Real- Time PCR

作　　者：王翀[1] 张小菊[1] 张祥林[1] 张伟[1] 张瑜[1] 李亚伟[1] 孙燕飞[2] 

机构地区：[1]新疆出入境检验检疫局,乌鲁木齐830063 [2]石河子大学,新疆石河子832000

出　　处：《新疆农业科学》2015年第8期1472-1476,共5页Xinjiang Agricultural Sciences

基　　金：国家质量监督检验检疫总局科研计划项目(2013IK287;2011IK168);国家科技部质检公益性行业科研专项(201310091)

摘　　要：【目的】建立瓜蔓枯病菌(Didy mella bryoniae)的实时荧光PCR方法,为瓜蔓枯病菌快速分子检测奠定基础。【方法】通过Genebank网上已公布序列,选取瓜蔓枯病菌beta-tubulin(tub2)基因作为目的基因,设计了特异性引物和Taq Man探针,并对该引物和探针的反应条件进行优化。【结果】采用研究所设计的引物和探针对瓜上的其他菌株及近似菌株进行检测,该引物和探针可高度灵敏地检测到瓜蔓枯病菌。采用实时荧光PCR方法,25μL体系中只要有8.9 pg的核酸量就可以被检测到,检测灵敏度达到356 fg/μL,比普通PCR灵敏度高10倍。【结论】采用研究所设计的引物和探针对田间采集的病株和未知样品的检测,验证了引物和Taq Man探针的特异性。【Objective】Real time PCR was established in Didymella bryoniae. 【Method】According to published sequences in Genebank,the gene beta- tubulin（ tub2） was chosen as the target gene. The specific primer and Taq Man probe were designed according to beta- tubulin（ tub2） gene sequence in D. bryoniae. The reaction conditions were optimized. 【Result】The primers and probe were highly sensitive by detecting other strains in melon and similar strains. The sensitivity could reach to 356 fg / μL by real time PCR. The sensitivity was ten times than that of general PCR.【Conclusion】The primers and Taqman probe were specific by detecting infected plants and unknown samples.

关 键 词：实时荧光PCR 检测 瓜蔓枯病菌 

分 类 号：S41-30[农业科学—植物保护]