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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。
A pair of Ps2r / Ps3r primers was designed based on the 16S sequences of the bacterial wilt pathogen and their approximate species in GenBank. The primers were designed to detect the specific resistance of seven strains of Fusarium oxysporum f. A predicted 268 bp band was amplified and no amplified products were obtained from the 32 strains tested. Compared with the other 5 pairs of specific primers reported in domestic and foreign literature, in addition to the primers PSA / PSB, the primers DEP1 / DEP2, ES16 / ESIG2c, HRP1d / HRP3c and CPSL1 / CPSR2c amplified to some extent strains of some similar species. The results showed that the primers Ps2r / Ps3r and PSA / PSB could specifically amplify Fusarium oxysporum coronatum and obtain the expected amplification products. The detection results of the bacteria in different serial dilutions of DNA and corn samples showed that the detection sensitivity of the nested PCR method combined with the primers Ps1 / Ps4 and Ps2r / Ps3r is higher than that of the primers ITSA / ITSB and PSA / PSB Method, which is also higher than the Bio-PCR detection method. The former can detect 300 cfu / sample of the target bacteria in the corn seed, and the detection method has ideal potential in the quarantine of the corn bacterial wilt pathogen entering the corn seed sample And promote the value.