作　　者： ; ; ; ; ; ;

机构地区： 黄埔出入境检验检疫局

出　　处： 《植物检疫》 2016年第2期55-62,共8页

摘　　要： 苹果牛眼果腐病菌(Neofabraea malicorticis、N.perennans、N.alba和N.kienholzii)是我国检疫性植物病原真菌,为建立该病菌的实时荧光PCR检测法,根据病菌及其近缘种的翻译延伸因子(EF-1α)的保守序列设计了特异性探针,分别以苹果牛眼果腐病菌菌株和本研究构建的EF-1α重组质粒DNA为阳性标准品检验探针的特异性和灵敏度。结果显示探针MAL-P、PER-P、ALB-P和KIE-P分别对N.malicorticis、N.perennans、N.alba和N.kienholzii表现特异性阳性扩增,而与近缘种及其他常见的果腐病菌无交叉反应,单重探针和4种探针混合液的灵敏度分别达1 fg/μL和10 fg/μL DNA。总计从美国、智利、新西兰、法国进境截获的29批可疑病果的分离物中检测到PCR阳性荧光信号,包括20批N.perennans、8批N.alba和1批N.kienholzii。该方法在6 h内即可完成整个检测流程,其特异性强、灵敏度高,适用于实际样品中苹果牛眼果腐病菌的快速检测。 To establish a real-time fluorescence PCR assay for detecting the causal agent of bull＇s-eye rot on apple（Neofabraea malicorticis,N. perennans,N. alba and N. kienholzii）,which is presently subjected to phytosanitary legislation in China,specific probes were designed based on the conserved sequences of partial translation elongation factor 1-alpha（EF-1α） of the above four species and relative species.The specificity and sensitivity of the probes were evaluated by using DNA of bull＇s-eye rot strains and recombinant plasmids of EF-1α constructed as positive standard,respectively. The results showed the probes of MAL-P,PER-P,ALB-P and KIE-P display specificity to the species N. malicorticis,N. perennans,N. alba and N. kienholzii with positive amplification,respectively,while there were no crossing reaction with the other strains of relative species and common fruit rot pathogens. The sensitivity of single and the mixture of 4 probes reaches 1 fg/μL and 10 fg/μL DNA,respectively. In total,29 isolates from disease apple fruit samples which originated from USA,Chile,New Zealand and France were detected with a positive rate of 100%,including 20,8 and 1 isolate（s） of N. perennans,N. alba and N. kienholzii,respectively.The method developed in the present assay can be completed within 6 h and will be used to rapidly detect the causal pathogen of bull＇s-eye on apple in practical samples with strong specificity and high sensitivity.

关 键 词： 苹果牛眼果腐病菌 属 快速检测 探针 实时荧光

领　　域： [农业科学] [农业科学] [农业科学]