利什曼原虫实时荧光定量PCR方法的建立

以利什曼原虫的小环状动基体DNA(Leishmaniak DNA)为靶基因,建立实时荧光定量PCR检测利什曼原虫的方法。根据GenBank报道的利什曼原虫小环状动基体DNA保守序列设计合成特异性引物,经PCR扩增后与pMD18-T载体连接,转化入大肠杆菌DH5α感受态细胞。挑选阳性克隆重组质粒经鉴定正确后,作为模板建立SYBR-GreenⅠ荧光定量PCR标准曲线和熔解曲线。结果构建的标准曲线线性关系良好,相关系数为0.996,而且特异性强,重复性好。本研究成功建立了实时荧光定量PCR检测利什曼原虫的方法,该方法可用于利氏曼原虫病的诊断、流行病学监测和科学研究。

Development of the Method for Detection and Quantitation of Leishmania with Real-time Fluorescent Quantitative PCR

To establish a SYBR-Green I fluorescent quantitative PCR method to detect the minicircles of the kinetoplast DNA of Leishmania，special primers were designed and synthesized to this conserved sequence. The special fragment was cloned into pMD18-T vector after PCR amplification，and then was transformed into E.coli DH5α. After the positive recombinant plasmid was identified，it was used as quantitative template to generate standard curve and melt curve. Results showed that the standard curve had a good linear relationship between cycle threshold(Ct) and template concentration，and the correlation coefficient was 0.996. We have successfully established a SYBR-Green I fluorescent quantitative PCR method to detect Leishmania，which can be used in the diagnosis and epidemiological surveillance of Leishmaniasis.