双重PCR检测板栗疫病菌的研究

A duplex PCR method for detection of Cryphonectria parasitica

由板栗疫病菌(Cyphonectria parasitica)引起的板栗疫病是板栗栽培区的主要病害,也是全国林业危险性有害生物。为建立C.parasitica的快速分子检测技术,根据C.parasitica与Gen Bank中同属其他物种的ITS序列差异设计了引物CP1/CP2,扩增了大小为285 bp的目的片段。进一步用RAPD技术从供试菌株中标记出C.parasitica的特异性片段,通过对RAPD特异片段克隆、测序后设计引物SC1/SC2,扩增的目的片段大小为389bp,实现了RAPD标记向SCAR标记的成功转化。采用引物对组合方式将引物CP1/CP2和SC1/SC2组成双重PCR,优化PCR反应条件并检测引物的特异性和灵敏度。双重PCR能从C.parasitica扩增出285 bp和389 bp的两条特异条带,而其他供试菌株及阴性对照均无条带,检测灵敏度达到300 fg/μL的基因组DNA。利用双重PCR能成功检测出自然条件下已发病板栗及处于潜伏期病害中的C.parasitica。

Chestnut blight caused by Cyphonectria parasitica is the main disease in Castanea mollissima cultivation area.According to the nucleotide sequence of internal transcribed spacer regions（ ITS） of the ribosomal gene of Cryphonectria in Gen Bank,a couple of primers CP1 / CP2 for C. parasitica were developed. By random amplification polymorphism technology,we identified a specific RAPD fragment of C. parasitica from all the strains tested. After recycling and purification,the specific fragment was cloned and sequenced. The sequence was used to design two specific primers SC1 /SC2 and converted RAPD marker to SCAR marker successfully. Combining the primers CP1 / CP2 and SC1 / SC2,a duplex PCR method for detecting C. parasitica had been established. The result indicated that the duplex PCR could amplify two unique fragments of 285 bp and 389 bp in size from C. parasitica,but other strains tested didn＇t present any fragments. Sensitivity testing showed that the detection limit was 300 fg / μL for genomic DNA. The duplex PCR method also successfully detected C. parasitica from infected chestnut tissues. Our results suggested that the duplex PCR method would have great significance for accurate identification and detection of C. parasitica.