冬生疫霉(Phytophthora hibernalis)的快速分子检测

Rapid molecular detection of Phytophthora hibernalis by PCR

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 diseases caused by Phytophthora hibernalis were risky and quarantinable, Here a species-specific PCR assay for rapid and accurate detection of the pathogenic oomycete P. hibernalis in diseased plant tissues was developed in the study. Based on differences in internal transcribed spacer （ITS） sequences of P. hi- bernalis and other Phytophthora spp. , a pair of species-specific primers, 751F/752R, was synthesized. More than 19 species of Phytophthora and 6 other species of pathogens were used to test the specificity of the primers. 751F/752R amplified only a unique 616 bp band from P. hibernalis. The detection sensitivity with 751F/752R was 10 fg of genomic DNA in 25 μL recation solution. A nested PCR procedure using ITS1/ITS4 as the first-round primers and followed with 751F/752R increased detection sensitivity 1 000-fold to 10 ag. The detection sensitivity for the zoospores in distilled water was 0.5 zoospore. To detect the pathogen from the diseased plant tissues by PCR could be completed within 24 h. The results suggested that the assay detected the pathogen more rapidly and accurately than the standard isolation methods. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.