丁香疫霉病菌PCR和实时荧光PCR检测

Detection of Phytophthora syringae by PCR and real-time PCR

为了快速、准确地检测丁香疫霉病菌(Phytophthora syringae,PSY),根据Gene Bank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。

The primer Psyl/Psy2 and the probe P-Psy were designed from ITS sequence in the GenBank to de- tect 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 Psyl/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.