松材线虫PCR标准化阳性对照构建及检测体系的建立

Establishment of Standard Positive Control and PCR Detection System for Bursaphelenchus xylophilus

建立了松材线虫PCR检测标准化阳性对照及特异性强的PCR检测体系.根据松材线虫rDNA-ITS区和BxPe12基因的特异基因序列设计引物,并从中筛选出一对特异引物cqubs01/cquba01,该引物能从松材线虫特异性扩增出196bp片段,而不能对其他种类线虫进行扩增.基于优化PCR反应体系和反应程序,稳定的检测体系得以建立,检测灵敏度为100pg/μL.以松材线虫基因组DNA为模板,以上述特异引物进行PCR扩增,将纯化后的PCR产物与PMD18-T载体连接之后转入大肠杆菌中,筛选出阳性克隆进行测序验证,最终获得了松材线虫的无害化阳性对照,建立了松材线虫标准化阳性对照的PCR检测体系.对来自于不同地区的12批次近100个样品进行了实际检测验证,其结果与实际发生情况一致,说明本检测体系稳定可靠.

A pair of primer sets cqubs01/cquba01 was selected from the primers which were designed based on the specific gene sequences rDNA-ITS and BxPel2 of the pine wood nematode （Bursaphelenchus xylophilus）. After optimization, a stable, sensitive （The detection limition was 100 pg/μL with the genome DNA of B. xylophilus as template） and specific PCR detection method was established. Using the specific primers, the 196 bp amplified fragments, which were not homologous with any nematodes but B. xylophilus, were obtained and purified. The product was then ligated with the pMD18-T vector and transformed into Escherichia coli JM109. The selected positive clones were sequenced and those validated were used as a harmless positive control. Nearly 100 samples of 12 batches collected from different areas were detected with this PCR system and the anticipated results were obtained. Fig 3, Tab 2, Ref 8