蛙源裂头蚴多靶标基因的PCR检测与分析

PCR detection and analysis of sparganum from frog based on multi-target genes

目的为食品来源的裂头蚴的分子生物学检测提供科学依据。方法PCR扩增裂头蚴线粒体nad5、rrnS、COX1基因并测序,利用Mega6.06软件中的NJ法构建系统发育树进行遗传分析。结果PCR扩增nad5、rrnS和COX1基因,分别得到531、328和155 bp的片段。经测序比对,本研究分离株的3个基因与GenBank数据库中猥迭宫绦虫对应序列的同源性分别为100%(nad5,Genbank No.KY114889.1、KU852381.1)、100%(rrnS,Genbank No.KY114889.1、KY114886.1、KU852381.1)和99.36%(COX1,Genbank No.MN432157.1、MN732156.1)。对COX1基因序列的系统发育树的分析结果表明,本实验的裂头蚴分离株与猥迭宫绦虫位于同一支上,确认本实验裂头蚴分离株为猥迭宫绦虫裂头蚴。结论基于PCR技术的裂头蚴分子生物学检测方法,可实现裂头蚴的快速监测,提高食源性寄生虫分子检测的效率和水平。

Objective To provide a scientific basis for the molecular biology detection of foodborne sparganum.Methods The mitochondrial NADH dehydrogenase subunit 5(nad5)gene,small subunit of ribosomal RNA(rrnS)gene and mitochondrial cytochrome oxidase subunit 1(COX1)gene were amplified by PCR and sequenced.The NJ method of the Mega6.06 program was used to construct a phylogenetic tree for genetic analysis.Results PCR amplification of nad5,rrnS,and COX1 genes resulted in fragments of 531,328 and 155 bp,respectively.After sequencing and comparison,the homology of the three genes isolated in this study between the corresponding sequences in the GenBank database of Spirometra erinaceieuropaei(S.erinaceieuropaei)were 100%(nad5,Genbank No.KY114889.1,KU852381.1),100%(rrnS,Genbank No.KY114889.1,KY114886.1,KU852381.1)and 99.36%(COX1,Genbank No.MN432157.1,MN732156.1),respectively.The results of the phylogenetic tree analysis of the COX1 gene sequence showed that the isolate in this study was located on the same branch with S.erinaceieuropaei,and it was confirmed that this isolate was S.erinaceieuropaei.Conclusion A PCR-based molecular detection method for sparganum was established,which could rapidly monitor of sparganum and improve the efficiency and level of molecular detection of food-borne parasites.