PCR技术鉴定南水北调中线水源地淡水虾囊蚴并行系统进化分析  

Molecular Identification and Phylogenetic Analysis of Metacercariae from Infected Shrimp in the Water Source of Middle Route Project of South-to-North Water Diversion

作　　者：邵艳[1] 孙彬[2] 沈华飞[2] 张轶静[2] 尚荣华[2] 杨树国[2] 赵燕清[2] 郭鄂平[2] 杨靖[2] 李蓓[2] 李健[1,2] 

机构地区：[1]十堰市太和医院·湖北医药学院附属医院门诊部,湖北十堰442000 [2]湖北医药学院基础医学研究所,湖北十堰442000

出　　处：《湖北医药学院学报》2016年第5期439-444,共6页Journal of Hubei University of Medicine

基　　金：教育部留学回国人员启动金(JYB201448HBMU01);湖北医药学院研究生启动金(2013QDJZR04);十堰市科学技术研究与开发项目(14Y05);校级重点学科建设项目(2014XKJSXJ04)

摘　　要：目的:基于PCR结合测序分析建立一种准确判断淡水虾类感染吸虫囊蚴的分子鉴定方法,并对其系统进化进行分析,追溯其起源。方法:采用肌肉压片直接挑取法取出囊蚴,试剂盒提取基因组DNA。用吸虫通用引物对所提DNA进行PCR扩增,琼脂糖凝胶电泳鉴定。PCR阳性样本采用各类吸虫特异性引物分别进行扩增,PCR产物电泳鉴定,阳性样本送公司进行双向测序。使用DNAstar软件拼接序列,离线软件MEGA5比对测序结果并建立系统进化树并分析其起源。结果:制作虾肌肉压片20张共挑取囊蚴32个,获取了囊蚴基因组DNA。三对通用引物均扩出480 bp左右的单一条带。吸虫特异性引物扩增时,仅有针对于吸虫异形科的ITS2和28S rRNA序列分别扩增出了约480 bp和1 300 bp的特异性条带。软件分别拼接出了419 bp和770 bp的截断序列。系统进化树分析显示,虾类所感染吸虫囊蚴有两种,分别与Neochoanostoma spp和Dimerosaccus oncorhynchi高度同源。结论:应用PCR结合测序分析在十堰地区首次建立了淡水虾类囊蚴的分子鉴定方法,为我国吸虫系统分类和南水北调中线水源地食源性吸虫病的研究奠定了基础。Objective To establish a molecular method based on PCR with sequencing analysis for identification of metacercariae from infected shrimp in Shiyan and analyse the constructed polygenetic tree for retrospecting its origin. Methods The metacercariaes were obtained from the muscle compression under microscopy. The genomic DNA（ g DNA） was extracted by kit. The extracted g DNA was amplified by PCR with universal trematode primer and the PCR product was identified through electrophoresis. For the positive sample,it was amplified by using specific trematode primers. The positive PCR products were screened by electrophoresis and sent to company for bi-directional sequencing. The sequences were assembled by DNA star software.Using the offline software MEGA5,the assembled sequences were analyzed and polygenetic tree was established. Results Totally,20 slides containing 32 metacercarias were prepared,and the g DNA was extracted. The single band（ 480 bp） was amplified respectively by three pairs of universal primers. For amplification with trematode specificity primer,only products from ITS2（ 480 bp） and 28 S rRNA（ 1 300 bp） sequences were successfully obtained. The truncated sequences（ 419 bp and 770 bp） were assembled with the software. The polygenetic tree results showed two kinds of trematode metacercariae were infected the local shrimp and they had highly homology with Neochoanostoma spp and Dimerosaccus oncorhynchi. Conclusion A molecular method for freshwater shrimp infected with metacercarias was primary established in Shiyan by using PCR coupled sequencing analysis. It establishes the foundation for the trematode phylogenetic systematics in China and the study of food-borne trematodiasis in the water source of the Middle Route Project for South-to-North Water Diversion.

关 键 词：吸虫 囊蚴 PCR 系统进化树 

分 类 号：S945.4[农业科学—水产养殖]