CAJ | 학술논문

为建立花生青枯病菌的快速检测技术，本研究利用细菌16S‐23S rDNA内源转录间隔区（ITS）通用引物L1／L2扩增花生青枯病菌基因组DNA并对其扩增序列进行克隆测序，通过与其同属近缘种做比较，设计1对特异性引物W1／W2，利用该对引物与细菌通用引物L1／L2建立了花生青枯病菌的巢式PCR检测方法。结果显示，引物W1／W2只能从花生青枯病菌中扩增出374 bp的特异片段；巢式PCR检测灵敏度可达10 fg ·μL －1基因组DNA ，较常规PCR提高1000倍；该技术可用于花生青枯感病期或者发病潜伏期时的病害检测。
위건립화생청고병균적쾌속검측기술，본연구이용세균16S‐23S rDNA내원전록간격구（ITS）통용인물L1／L2확증화생청고병균기인조DNA병대기확증서렬진행극륭측서，통과여기동속근연충주비교，설계1대특이성인물W1／W2，이용해대인물여세균통용인물L1／L2건립료화생청고병균적소식PCR검측방법。결과현시，인물W1／W2지능종화생청고병균중확증출374 bp적특이편단；소식PCR검측령민도가체10 fg ·μL －1기인조DNA ，교상규PCR제고1000배；해기술가용우화생청고감병기혹자발병잠복기시적병해검측。
To develop a technique for the quick detection of Ralstonia solanacearum of Peanut ,a nested PCR was established. Bacterial universal primer pair L1 /L2 ,based on the intergenic transcribed spacer (ITS) region of 16S-23S ribosomal DNA of bacteria ,was used as the first round primers and the PCR products were cloned and sequenced. A pair of special PCR primers W1/W2 was designed as the second round primers. The prime pair could amplify a single 374 bp band from genomic DNA of Ralstonia solanacearum in peanut ,but not from other bacteria. The sensibility of nested PCR reached 10 fg ·μL-1 of DNA ,1 000 times higher than that of a sample PCR. Using nested PCR assay ,the pathogen could be specifically detected from infected peanut plants with or without disease symptom.