丁香疫霉病菌PCR和实时荧光PCR检测

 为了快速、准确地检测丁香疫霉病菌 (Phytophthora syringae， PSY)，根据GeneBank中PSY的ITS序列设计特异引物Psy1/Psy2和探针P-Psy，建立了常规PCR和实时荧光PCR检测方法。利用引物Psy1/Psy2扩增供试的26株PSY能得到585 bp的预期目标条带，但扩增其它61个非PSY供试菌株不能得到预期产物，检测灵敏度为12 pg菌丝DNA;探针P-Psy对供试26株PSY表现为阳性扩增，而对其它菌株和空白对照均表现为阴性扩增，检测灵敏度可达120 fg菌丝DNA，比常规PCR高100倍;引物Psy1/Psy2和探针P-Psy对5 g土壤中PSY卵孢子的检测灵敏度分别为20 000个和200个。样品检测试验表明两种PCR方法可用于口岸植物检疫中快速、准确和特异地检测丁香疫霉病菌。

Detection of Phytophthora syringae by PCR and real-time PCR

The primer Psy1/Psy2 and the probe P-Psy were designed from ITS sequence in the GenBank to detect Phytophthora syringae by conventional PCR and real time PCR. These two methods could be used to amplify 26 strains of P. syringae and yielded a 585-bp target band, but no expected target band or positive amplification appeared from other 61 strains of related species. The detection sensitivity of the probe P-Psy is 120 fg mycelial DNA, which is 100 times higher than that of the primer Psy1/Psy2. The conventional PCR and real time PCR can detect 20 000 and 200 oospores in 5 g soil sample, respectively. Our fruit-sample test demonstrate that the two PCR detection methods can be used to accurately and rapidly detect P. syringae from imported plant pro-ducts.