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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 identification method for accurate judgment of freshwater shrimp infestation metacercariae based on PCR and sequencing analysis, and to analyze its phylogeny and trace its origin. Methods: The musculoskelet was taken out directly by muscle compression and the kit was used to extract genomic DNA. The common DNA primers were used to amplify the DNA and identified by agarose gel electrophoresis. PCR positive samples were all kinds of fluke-specific primers were amplified, PCR product electrophoresis identification, positive samples sent to the company for bidirectional sequencing. Using DNAstar software to splicing sequence, offline software MEGA5 compared the sequencing results and established phylogenetic tree and analyzed its origin. Results: Twenty pieces of shrimp muscle tablets were prepared and 32 metacercarids were picked up to obtain the metacercaria genomic DNA. Three pairs of universal primers all expanded a single band of about 480 bp. When the flukes specific primers were amplified, only the specific bands of about 480 bp and 1 300 bp were amplified for ITS2 and 28S rRNA sequences respectively. The software spliced 419 bp and 770 bp truncated sequences respectively. Phylogenetic analysis showed that there are two kinds of metacercariae in shrimp, which are highly homologous to Neochoanostoma spp and Dimerosaccus oncorhynchi, respectively. Conclusion: The molecular identification method of freshwater shrimp metacercariae was established for the first time in Shiyan by PCR and sequencing analysis, which lays the foundation for the study on the classification of the trematodes in China and the study on the foodborne trematodes in the midline of South-to-North Water Diversion Project.