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由冬生疫霉(Phytophthora hibernalis)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,本文比较分析了冬生疫霉和其它疫霉的ITS序列,在此基础上设计了一对检测冬生疫霉的特异性引物751F/752R,该对引物从冬生疫霉中扩增得到一条616 bp的条带,而其它19种疫霉和其它真菌菌株均无扩增条带,表明该对引物对冬生疫霉具有特异性。在25μL PCR反应体系中,引物751F/752R检测灵敏度为10 fg基因组DNA;而以卵菌ITS区通用引物ITS1/ITS4和751F/752R进行套式PCR扩增,能够检测到10 ag的基因组DNA,使检测灵敏度提高了1 000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。结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术,在1个工作日内即可从人工接种发病的植物组织中特异性的检测到该病原菌。表明本研究建立的检测方法可用于冬生疫霉的快速分子检测。
The blight caused by Phytophthora hibernalis is a group of phytosanitary diseases. In order to establish a rapid detection technique of this pathogen, this paper analyzed the ITS sequences of Phytophthora and Phytophthora, and designed a pair of specific primers 751F / 752R for detecting P. infestans. Phytophthora thermophila amplified a 616 bp band, while the other 19 Phytophthora and other fungal strains were no amplification bands, indicating that the pair of primers specific for Phytophthora infestans. In the 25μL PCR reaction system, the detection sensitivity of primer 751F / 752R was 10 fg genomic DNA. The nested PCR amplification of ITS1 / ITS4 and 751F / 752R of ITS region of Oomycetes could detect genomic DNA of 10 μg, The detection sensitivity increased by 1 000 times. The detection system of zoospores in sterile water detection sensitivity of up to 0.5 zoospores. Combined with rapid alkaline lysis method to extract the DNA of the diseased tissue, the PCR detection technique can detect the pathogen specifically from the inoculated plant tissue in one working day. This indicates that the detection method established in this study can be used for the rapid molecular detection of Phytophthora infestans.