犬源A型贾第虫real-time PCR检测方法的建立

摘要

To establish a detection method for zoonotic Giardia larnblia, a real-time PCR assay based on SYBR Green I was developed with a pair of primers designed according to the 16S Rrna gene of G. Lamblia from dogs and a recombinant plasmid containing the target gene was constructed as a standard control. Tm value of the amplified product was confirmed to be 94.20 ℃. This technique was highly sensitive with a detection limit of 3.39 copy/Μl of positive recombinant plasmid without any cross-reaction with Cryptosporidium, coccidium and Toxoplasma gondii. The correlation coefficient of the standard curve was 0.99, and had a coefficient of variations less than 3% for both intra- and inter-assay. The developed real-time PCR using SYBR Green I was specific, highly sensitive, and could be further used in clinic detection and epidemic investigation of giardiosis.%为建立贾第虫定性定量的检测方法,本研究针对犬源贾第虫16S rRNA基因片段设计一对引物,将构建的重组质粒作为阳性对照,建立了贾第虫DNA的SYBR Green Ⅰ real-time PCR检测方法.结果显示,特异性产物Tm值为94.20℃,最低可检测到3.39 copy/μL的阳性质粒,标准曲线的相关系数为0.99.与其他常见原虫隐孢子虫、球虫、弓形虫均不发生交叉反应,重复性变异系数小于3％.该检测方法具有较好的特异性和敏感性,为贾第虫病的临床检测和流行病学调查提供了新的技术手段.