葡萄苦腐病菌的分子检测方法

Molecular detection for Greeneria uvicola.

葡萄苦腐病菌(Greeneria uvicola)是我国检疫性有害生物。本研究根据G.uvicola的ITS基因序列,设计了PCR引物GUA-1和GUA-2以及实时荧光PCR引物GUA-P1和GUA-P2及荧光探针GUA-Probe-117。利用引物GUA-1和GUA-2进行PCR扩增,G.uvicola和感染G.uvicola的红提葡萄可产生354 bp的特异性片段;实时荧光PCR扩增,G.uvicola和感染G.uvicola的红提葡萄均出现强荧光信号(△Rn)增加。而葡萄上其他常见真菌样品以及健康的红提葡萄既无特异性扩增也无荧光信号增加。灵敏度测试中,普通PCR可检测到含G.uvicolaDNA浓度0.05ng/μL以上的样品,实时荧光PCR可检测到含G.uvicolaDNA浓度0.0005 ng/μL以上的样品。由此确立了G.uvicola的两种稳定可靠、灵敏的分子检测方法。

Greeneria uvicola is one of quarantine pests of China. One pair of PCR primers GUA - 1 and GUA - 2 and one pair of real - time PCR primers GUA - P1, GUA - P2 and GUA - Probe - 117 for G . uvicola were de- signed basing on the sequence of ITS genes. A specific band of 354 bp was amplified by PCR using GUA - 1 and GUA -2 from G. uvicola and the grape infected by G. uvicola. The results of real - time PCR showed that the PCR products from G. uvicola and the grape infected by G. uvicola had strong fluorescent signals. Other patho- genic fungus of grape and the healthy grape had none. The sensitivity test indicated that more than 0.05ng/μL DNA of G. uvicola could be detected by PCR and more than 0. 0005 ng/μL DNA of G. uvicola could be detected by real -time PCR. Two stable and sensitive molecular detection methods for Greeneria uvicola were established. Key words Greeneria uvicola, detection, PCR, real time PCR