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为了快速准确检测进境玉米样品中的玉米内州萎蔫病菌Clavibacter michiganensis subsp.nebraskensis(Cmn),根据GenBank中Cmn的16S-23S序列设计引物CM1/CM4和引物PSM1/CM3。引物PSM1/CM3仅能从供试的4株Cmn菌株中扩增获得208 bp的预期产物,而其他36株对照菌株均不能扩增出预期条带。灵敏度测试结果表明引物CM1/CM4和PSM1/CM3组合的巢式PCR方法的检测灵敏度高于常规PCR,检测灵敏度可达40 fg DNA或6.8 CFU目标细菌。常规PCR和巢式PCR方法对进境美国玉米样品的阳性检出率分别为8%和24%,试验结果表明所建立的PCR方法可用于玉米样品中Cmn的快速检测。
In order to rapidly and accurately detect Clavibacter michiganensis subsp. Nebraskensis (Cmn), the primers CM1 / CM4 and PSM1 / CM3 were designed according to the 16S-23S sequence of Cmn in GenBank. Primer PSM1 / CM3 only obtained 208 bp of the expected product from the four tested Cmn strains, while the other 36 control strains did not amplify the expected band. Sensitivity test results showed that the detection sensitivity of the nested PCR method using the combination of primers CM1 / CM4 and PSM1 / CM3 is higher than that of the conventional PCR, and the detection sensitivity is up to 40 fg DNA or 6.8 CFU target bacteria. The positive detection rates of routine PCR and nested PCR were 8% and 24% respectively. The results showed that the established PCR method can be used for the rapid detection of Cmn in maize samples.