玉米细菌性枯萎病菌PCR检测

 根据GenBank中玉米细菌性枯萎病菌及其近似种的16S序列差异,设计了一对玉米细菌性枯萎病菌特异性引物Ps2r/Ps3r,该引物能从供试的7株玉米细菌性枯萎病菌中特异性扩增出一条268 bp的预期条带,供试的32株近似种菌株都没有扩增产物;与国内外文献报道的其它5对特异性引物相比,除引物PSA/PSB外,引物DEP1/DEP2、ES16/ESIG2c、HRP1d/HRP3c和CPSL1/CPSR2c在不同程度上对部分近似种菌株出现了扩增。试验结果表明,引物Ps2r/Ps3r和PSA/PSB能特异性扩增玉米细菌性枯萎病菌,得到预期的扩增产物。对不同系列稀释度的DNA和玉米样品中病菌的检测结果表明,由引物Ps1/Ps4和Ps2r/Ps3r组合的巢式PCR方法的检测灵敏度高于引物ITSA/ITSB和PSA/PSB组合的巢式PCR方法,也高于Bio-PCR检测方法;前者可以检测到玉米种子中300 cfu/sample的目的细菌,该检测方法在进境玉米种子样品玉米细菌性枯萎病菌的检疫中具有比较理想的应有潜力和推广价值。

PCR detection of Pantoea stewartii subsp, stewartii

Seven strains of Pantoea stewartii subsp, stewartii (Pss) and 32 strains of related species were used to validate the specificity of the primer Ps2r/Ps3r designed from 16S sequence of Pss and other 5 pairs of primers specific for Pss. The primer Ps2r/Ps3r and PSA/PSB could amplify seven strains of Pss and get 268 bp product, but no band for other 32 strains of related species. To a certain extent, the primers DEP1/DEP2, ES16/ESIG2c,HRP1d/HRP3c and CPSL1/CPSR2c produced false positive amplification for the strains of genera Pantoea and Erwinia. The nested PCR combined with primers Ps1/Ps4 and Ps2r/Ps3r could detect as low as 300 cfu/sample bacteria of Pss in corn seed sample with better sensitivity than that with primers ITSA/ITSB or PSA/PSB and conventional Bio-PCR method. This more accurate and sensitive detection method for Pss exhibited potential role in the quarantine of imported corn seeds.