论文部分内容阅读
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。
Apple (Neofabraea malicorticis, N. perennans, N.alba and N.kienholzii) is a quarantine plant pathogenic fungus in China. In order to establish this real-time fluorescence PCR detection method, according to the translation of germs and their related species A specific probe was designed based on the conserved sequence of the elongation factor (EF-1α), and the specificity and sensitivity of the probe were tested using positive strains of P. corniculatus and EF-1α recombinant plasmid DNA constructed in this study. The results showed that the probes MAL-P, PER-P, ALB-P and KIE-P were specifically positive for N.malicorticis, N.perennans, N.alba and N.kienholzii, respectively, but closely related to related species and other Common fruit rot bacteria did not cross-react, single probe and 4 probe mixture sensitivity of 1 fg / μL and 10 fg / μL DNA. PCR-positive fluorescent signals were detected in the isolates of 29 batches of suspected fruitings intercepted from the United States, Chile, New Zealand and France, including 20 batches of N. perennans, eight batches of N. alba and one batch of N. kienholzii. The method can complete the entire detection process within 6 h, and has the advantages of high specificity and high sensitivity, and is suitable for the rapid detection of pathogenic bacteria of apple eye fruit in the actual sample.