含扩增内标的犬巴贝斯虫PCR检测方法的建立

Detection of canine babesiosis by PCR assay with an internal amplification control

为了提高犬巴贝斯虫的PCR检测方法准确性,采用复合引物重组方法构建两端含有检测巴贝斯虫18S r RN A基因的PCR检测引物的扩增内标质粒(含鸡特异性基因片段),通过优化扩增内标质粒的添加量,建立了一种新的检测犬巴贝斯虫PCR方法。结果显示,在25 L的PCR反应体系中加入1×106copies的扩增内标的可有效地检测犬巴贝斯虫基因组D NA,该PCR方法最低检测限为1×105copies。本试验建立的含扩增内标的PCR检测方法能快速、准确、简便的检测犬巴贝斯虫感染,同时能有效的避免假阴性现象的产生。

The aim of this study was to develop a real-time PCR method included an internal amplifi- cation control for rapid detection canine babesiosis. Primers were designed based on the 18S rRNA gene of canine Babesia for PCR. Internal amplification control （IAC）,which has the same primers with canine Babesia PCR amplification,was cloned from chicken genomic DNA. IAC was added into canine Babesia PCR reaction system. The results were shown that this method had a detection limit of 1×10^5 copies in 25 μL reaction systems. There was no effect on amplification of target gene in the presence of 1 × 10^Bcopies of IAC in 25 ~ L reaction system. The detection of canine Babesia by PCR included IAC developed in this study is certainly accurate and fast and can avoid the false positive results.