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选择玉米内州萎蔫病菌基因组DNA高度特异保守区域设计环介导等温扩增技术(LAMP)引物,通过对其反应条件优化,建立玉米内州萎蔫病菌的LAMP检测体系。实验得出内引物与外引物最佳比例为6∶1,反应最佳温度为64℃。运用玉米内州萎蔫病菌克隆质粒梯度稀释液对LAMP检测体系的灵敏度进行了验证。LAMP检测体系的检测限达到100 fg,与常规PCR相比,该方法的检测限提高了100倍。以多种参比菌DNA为模板对LAMP检测体系的特异性进行了验证,结果表明LAMP检测体系仅对玉米内州萎蔫病菌有扩增,对非靶标菌没有扩增。为玉米内州萎蔫病菌快速高效检测提供了一种新型的检测方法。
LAMP primers were designed based on the highly conserved regions of the genomic DNA of wilt Neisseria gonorrhoeae, and the LAMP detection system of Neisseria gonorrhoeae was established by optimizing the reaction conditions. The optimal ratio of inner primer to outer primer was 6:1, and the optimal reaction temperature was 64 ℃. The sensitivity of LAMP detection system was validated by using a gradient gradient of cloning plasmids of wilt in Maize. The detection limit of LAMP detection system reached 100 fg. Compared with the conventional PCR, the detection limit of this method increased by 100 times. The specificity of LAMP detection system was validated by using a variety of reference bacteria DNA as template. The results showed that the LAMP detection system only amplified the wilt of Maize wilt and did not amplify the non-target bacteria. It provides a new detection method for rapid and efficient detection of wilt pathogens in maize.