摘要
为建立一种可以同时检测猪囊尾蚴和华支睾吸虫的液相基因芯片方法,本研究以猪囊尾蚴ITS基因和华支睾吸虫ITS基因为靶序列,设计并合成特异性探针和引物,通过PCR方法扩增目的片段,构建阳性重组质粒标准品并对其进行测序,建立一种基于双重PCR的液相基因芯片检测方法。结果显示,扩增目的片段长度分别约为150 bp和170 bp,测序结果与Gen Bank登录的猪囊尾蚴和华支睾吸虫相关基因一致性分别为99.35%和98.27%;液相基因芯片对单重PCR产物和双重PCR产物的检测结果一致,特异性和重复性良好。应用该方法检测猪囊尾蚴和华支睾吸虫的灵敏度分别为5.13×104拷贝/μL和2.03×104拷贝/μL,比琼脂糖凝胶电泳灵敏高约8倍;应用该方法检测猪囊尾蚴和华支睾吸虫的变异系数均在8%以内,模拟污染样品的检验试验准确率达98%。本研究初步建立了可以同时高效、灵敏和特异检测猪囊尾蚴和华支睾吸虫的液相基因芯片方法,为人畜共患寄生虫的检测和监控提供了新方法。
To develop a sensitive protocol for simultaneous detecting Cysticercus cellulose and Clonorchis sinensis, a liquid gene chip assay based on duplex PCR was established with two pairs of primers and probes designed according to ITS sequences of the two parasites, which amplified the DNA ffagrnents of 150 bp for C.eeHulose and 170 bp for C.sinensis, respectively. In addition, the test results showed that the assay was specific to detect C.cellulose and C.sinensis, but no cross-react was found to other pathogens. The minimum limit of detectability for the liquid gene chip technique was 5.13 × 104 copies/μL and 2.03 × 104 copies/μL for C.cellulose and C.sinensis, respectively, which was 8-fold more sensitive than that of PCR assay. The repeatability test showed that the coefficient of variation of liquid gene chip for each was less than 8%. The results showed that liquid gene chip assay could be valuable for sensitive, rapid and simultaneous detection of the C.cellulose and C.sinensis.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2015年第5期387-391,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省教育厅科学技术研究(重点)项目(1153Lz04)
关键词
液相基因芯片
猪囊尾蚴
华支睾吸虫
liquid gene chip
Cysticercus cellulose
Clonorchis sinensis
