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为建立苹果属冬生疫霉Phytophthora hibernali、丁香疫霉P.syringae和栗黑水疫霉P.cambivora 3种检疫性疫霉的同步分子检测方法,根据疫霉属18S rRNA,ITS,HSP90和Ypt1基因分别设计通用引物,冬生疫霉、丁香疫霉及栗黑水疫霉特异引物,建立了四重PCR检测方法,并进行了灵敏度和模拟带菌测试.建立了可同时检测苹果属上冬生疫霉、丁香疫霉和栗黑水疫霉的特异四重PCR检测体系:在20μL反应体系中,最佳引物浓度组合为10μmol/L的18SUF/18SUR,PHSF/PHSR,PCSF/PCSR和PSSF/PSSR分别为0.2,0.6,0.8,1.0μL;最佳退火温度和退火时间分别为63℃和20s.该体系扩增冬生疫霉出现884bp的18S rRNA条带和232bp的ITS基因特异带,丁香疫霉出现884bp的18S rRNA条带和683bp的HSP90基因特异带,扩增栗黑水疫霉出现884bp的18S rRNA条带和314bp的Ypt1基因特异带,对照菌只出现18S rRNA条带;四重PCR反应体系检测灵敏度低于单重PCR;模拟带菌试验可同时扩增出4个片段.表明该四重PCR检测方法能实现冬生疫霉、丁香疫霉和栗黑水疫霉的同步特异检测,实现苹果属类水果及种苗检疫性疫霉的快速检测.
In order to establish a synchronous molecular method for the determination of Phytophthora hibernali, P. syringae and P. cambivora, the phylogenetic tree was constructed based on the 18S rRNA, ITS, HSP90 and Ypt1 genes Four primers were designed, such as universal primers, Phytophthora infestans, Phytophthora capsici and Phytophthora nicotianae, respectively, and a quadruple PCR method was established for sensitivity and pathogenicity test. Phytophthora capsici, Phytophthora capsici and Phytophthora infestans: 18SUF / 18SUR, PHSF / PHSR, PCSF / PCSR and PSSF / PSSR at the optimum primer concentration of 10μmol / L in the 20μL reaction system 0.2, 0.6, 0.8 and 1.0μL respectively.The optimum annealing temperature and annealing time were 63 ℃ and 20s, respectively.The system amplified 884bp 18S rRNA and 232bp ITS specific bands, 884bp 18S rRNA band and 683bp HSP90 gene specific band appeared, 88Sbp 18S rRNA band and 314bp Ypt1 gene specific band were amplified, while only 18S rRNA band was observed in control bacterium. Quadruple PCR reaction System detection sensitivity is lower than single PCR; Four fragments were amplified at the same time, which indicated that the method of quadruple PCR could detect synchronous and specific detection of Phytophthora infestans, Phylidophthora and Pseudoperonospora cubensis and realize rapid detection of Phytophthora infestans in apple genus and fruit seedlings. .