摘要
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 GreenⅠreal-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。
To establish a detection method for zoonotic Giardia lamblia, 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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2011年第12期961-964,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(30972179)
