宿根矮化病菌的竞争定量PCR检测方法建立

利用宿根矮化病菌的特异检测引物,构建竞争定量PCR检测的非同源模板,建立宿根矮化病菌的竞争定量PCR检测方法.实验中非同源模板(竞争模板)构建是采用PCR方法对大肠杆菌基因组进行低严谨度扩增,回收合适的DNA片段并将其构建到T-esay载体上,通过测序和序列比对,除两端引物序列完全相似外,其余部分没有同源性,因此可以作为定量检测的非同源模板.通过计算非同源模板的拷贝数,进行竞争定量PCR检测宿根矮化病,建立宿根矮化病菌的标准竞争曲线和直线回归方程.本研究建立的竞争定量PCR检测方法操作简单、特异性和敏感性高,适合于宿根矮化病菌的检测,并可为其它病菌竞争定量PCR检测方法的建立提供参考.

Establishment of a Quantitative Competitive PCR Assay for Detection of Clavibacter xyli subsp.xyli

To develop a specific and sensitive detection method for Clavibacter xyli subsp.xyli in sugarcane by using quantitative competitive polymerase chain reaction(QC-PCR), heterologous template had been built following a pair of specific detection primers of Clavibacter xyli subsp.xyli. The E. coli genomic DNA was low stringently amplified to construct the heterologous template, and then the amplified fragments were purified and ligated into pGEM-T Easy Vector. Through sequencing and sequence alignment, no other homologous sequences were found with the exception of primer sequences at both ends of the heterologous template, and these amplified fragments could be used for quantitative detection of non -homologous template. By calculating the copy number of non-homologous template for QC-PCR detection of ratoon stunning disease(RSD), the standard RSD competition curve and linear regression equation were established. This QC-PCR detection method is easy to operate with high specificity and sensitivity and is good for detection of ratoon stunning disease. It can also be a reference for construction of competitive template for QC-PCR assay of other microbes.