龟源肺炎克雷伯菌SYBR-GreenⅠ荧光定量PCR检测方法的建立

The Establishment of the SYBR-Green Ⅰ Fluorescence Quantitative PCR Detection Method of Turtle Source Klebsiella pneumoniae

本研究在黄喉拟水龟中分离的肺炎克雷伯菌基础上,根据肺炎克雷伯菌Ⅲ型菌毛的Mrk D基因序列设计引物,通过对SYBR-GreenⅠ荧光定量PCR反应条件、特异性、灵敏性实验进行调节优化,建立了肺炎克雷伯菌的SYBR-GreenⅠ荧光定量PCR检测方法。研究结果表明,建立的肺炎克雷伯菌荧光定量PCR方法,检测时间短,用时73 min;特异性强,对非肺炎克雷伯菌无交叉反应;灵敏度高,检测肺炎克雷伯菌DNA的最低检测量为2.78 fg。该方法的建立,为肺炎克雷伯菌的辅助诊断、流行病学调查、毒力基因的分析提供科学借鉴。

The research based on isolation of Klebsiella pneumoniae from Mauremys mutica, primers were designed from Mrk D gene of type 3 fimbriae of Klebsiella pneumoniae for fluorescent quantitative PCR. After adjusted and optimized fluorescent quantitative PCR reaction, specificity and sensitivity experiment, a SYBRGreen Ⅰ fluorescent quantitative PCR detection method of Klebsiella pneumoniae was established. The result showed that the established method was a high specific, high sensitivity and time-saved method, which reaction time was 73 min, no cross reaction to non-klebsiella pneumoniae, and the lowest detection DNA mount to 2.78 fg.The method provides scientific reference to auxiliary diagnosis, epidemiological investigation and virulence gene analysis of Klebsiella pneumoniae.