【摘要】 由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。更多还原

【Abstract】 The diseases caused by Phytophthora lateralis were risky and quarantinable. A species-specific PCR assay for rapid and accurate detection of the pathogenic oomycete P. lateralis in diseased plant tissues was developed in the study. Based on differences in tRNA sequences of P. lateralis and other Phytophthora spp.,a pair of species-specific primers T1 / T2 was synthesized. More than 15 species of Phytophthora and other species of pathogens were used to test the specificity of the primers; T1 / T2 amplified only a unique 192 bp band from P. lateralis. The detection sensitivity with T1 / T2 was 10 pg of genomic DNA in 25 μL PCR reaction solution. A nested PCR procedure using T3 / T4 as the first-round primers and followed with T1 / T2 increased detection sensitivity 10 000-fold to 1 fg. The detection sensitivity for the zoospores in distilled water was 0. 5 zoospore. The duplex PCR method combining primers(T1 / T2 and T3 / T4) was used to detect the pathogen in the plant tissues inoculated with pathogens. Furthermore,Real-time fluorescent quantitative PCR assays were developed to detect and monitor the P. lateralis from the diseased plant tissues.更多还原