湖北医药学院学报2016，Vol.35Issue(5)：439-444,6.DOI:10.13819/j.issn.1006-9674.2016.05.003
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
摘要
Abstract
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 (gDNA) was extracted by kit.The extracted gDNA 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.Tihe 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 gDNA 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 28S 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.Corclusion 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Key words
Trematode/Metacercaria/Polymerase chain reaction/Phylogenetic analysis引用本文复制引用
邵艳,李蓓,李健,孙彬,沈华飞,张轶静,尚荣华,杨树国,赵燕清,郭鄂平,杨靖..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[J].湖北医药学院学报,2016,35(5):439-444,6.基金项目
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) （JYB201448HBMU01）
