吉林地区华支睾吸虫虫卵的鉴定及PCR检测方法的建立  

Identification of Clonorchis sinensis Eggs in Jilin and Establishment of PCR Detection

作　　者：唐艺芝[1] 郝玉花[2] 孙青松[1] 彭鹏[1] 吴秀萍[1] 王学林[1] 刘明远[1] 

机构地区：[1]吉林大学人兽共患病研究所/教育部重点实验室,长春130062 [2]吉林大学第一临床学院,长春130062

出　　处：《湖北农业科学》2013年第7期1601-1603,1606,共4页Hubei Agricultural Sciences

基　　金：国家自然科学基金资助项目(NSFC30700600);吉林省科技厅基金资助项目(20060417)

摘　　要：对华支睾吸虫病流行地区人粪样进行华支睾吸虫(Clonorchis sinensis)虫卵的鉴定、收集及DNA的提取,然后根据华支睾吸虫基因设计两对特异性引物,以粪便中提取的虫卵DNA为模板,采用聚合酶链式反应(PCR)方法扩增目的基因片段,对PCR方法的各种反应条件进行优化。结果两对引物中以上游引物(5′-TGGCCTGACTGGCTGGCCGG-3′)、下游引物(5′-CGGCACCCCACACACATACA-3′)为最适引物,用PCR方法可扩增到分子量大小为255 bp的特异性条带,测序结果经BLAST工具进行分析,与中国沈阳分离株(AF217099)和朝鲜分离株(AF217094)的同源性为100%;50μL PCR反应体系的最优化反应条件为:dNTP 2.6μL;10×PCR Buffer(含Mg2+)4.0μL;上下游引物各0.5μL;DNA模板5.5μL;Taq DNA聚合酶0.4μL。扩增程序为:94℃预变性5 min,94℃变性30 s,56℃退火1 min,72℃延伸30 s,30个循环,72℃延伸5 min,4℃保存。试验成功对吉林地区人粪样中华支睾吸虫虫卵进行了鉴定,并建立了华支睾吸虫虫卵PCR检测方法,为从分子生物学角度检测华支睾吸虫病提供了理论依据。The Clonorchis sinensis eggs were collected from the human fecal samples which infected with C.sinensis and then used to extract DNA for PCR detection.With the extracted DNA as template,three specific primers were designed according to the gene sequences of C.sinensis for PCR amplification.The reaction systems of PCR among different react conditions were also optimized simultaneously.The results showed that the upstream primer 5′-TGGCCTGACTGGCTGGCCGG-3′ and downstream primer 5′-CGGCACCCCACACACATACA-3′ were considered as the most optimum primer,the obtained specific band by amplification sized 255 bp.The gene sequence analysis showed that the homology was 100% compared with the genome of C.sinensis(AF217099) and(AF217094)by BLAST.The optimal reaction conditions of PCR system were as followed:10×PCR Buffer(Containing Mg2+)4.0 μL,dNTP 2.6 μL,upstream and downstream 0.5 μL respectively,template DNA 5.5 μL and Taq DNA polymerase 0.4 μL in total 50 μL reaction volume.The amplification program were: after 5 min at 94 ℃,the reactions were cycled through 30 s denaturation at 94 ℃,1 min annealing at 56 ℃,30 s extension at 72 ℃ for 30 cycles,followed for 5 min extension at 72 ℃ and reservation at 4 ℃.Through this experiment the PCR detection method was successfully established to identify the C.sinensis eggs from the human fecal samples which provided scientific experimental basis on the molecular level for detecting C.sinensis.

关 键 词：华支睾吸虫(Clonorchis sinensis) 虫卵DNA 鉴定 PCR 

分 类 号：S[农业科学]