利用Bio-real-time PCR技术检测玉米内州萎蔫病菌

Detection of Clavibacter michiganensis subsp. nebraskensis by Bio-real-time PCR

玉米内州萎蔫病是北美玉米生产上的严重病害之一,为预防其传入我国,利用人工注射接种的方法模拟自然感病的玉米籽粒,通过Clavibacter michiganensis不同亚种的转录间隔区序列,设计了特异性的TaqMan-MGB探针,将Clavibacter michiganensis subsp.nebraskensis(Cmn)的半选择性培养基sCNS和特异性的探针结合,建立了进出境玉米种子上Cmn的Bio-real-time PCR检测方法,该方法可以检测到105粒玉米种子中含有一粒感病籽粒(带菌量为1.2×106 CFU/粒)的种子样品,可以应用于实际检测。

Goss＇s Bacterial Wilt and Leaf Blight of Corn is a kind of severe disease occurring in the corn production in the North America. To avoid its entry into China, naturally infected com kernels were simulated by Cmn artificial injection. The intergenic transcribed spacer of Clavibacter michiganensis was analyzed to design the real-ttme PCR probe, then the Bio-real-time PCR, including semi-selective medium detection and molecular identification, was set up, which actually resulted in positive if there was a corn kernel of 1.2x10^6 CFU Cmn in one thousand seeds. After calculating, the method could reach the sensitivity when acom kernel containing the same amount of Crnn mixed in one hundred thousand healthy seeds. The system can be applied in quarantine oractice.