青海省多房棘球绦虫分子种系发生研究

Study on the molecular phylogeny of Echinococcus multilocularis in Qinghai Province

目的对青海省多房棘球绦虫分离株Cox1、Nad1基因进行扩增和测序,并构建系统进化树及分子钟,为推测其进化关系及起源提供参考依据。方法收集2017年青海省人民医院收治的22份肝多房棘球蚴病患者术后标本,对多房棘球蚴线粒体DNA Cox1、Nad1基因进行PCR扩增和测序,并与Gen Bank数据库中的棘球属绦虫Cox1基因和Nad1基因序列进行比对,观察种内变异情况并构建进化树和分子钟。结果所有多房棘球绦虫标本与Gen Bank数据库中检索的多房棘球绦虫亚洲株Cox1、Nad1基因序列同源性均达99%以上,共获得6种不同基因型,其中有2个分离株在Gen Bank数据库中未找到同源性100%的基因序列。系统进化树显示,收集的多房棘球绦虫标本与多房棘球绦虫亚洲株聚类效果明显;分子钟推测青海地区多房棘球绦虫起源于9.4万年前。结论本研究发现青海省多房棘球绦虫存在6种不同基因型,其中首次发现2种基因型。青海地区多房棘球绦虫优势株为亚洲株,起源时间约在9.4万年前。

Objective To amplify and sequence Cox1 and Nad1 genes in Echinococcus multilocularis isolates from Qinghai Province,and to create phylogenetic trees and molecular clocks,so as to provide evidence for estimating the evolutionary relationships and origins of E.multilocularis in Qinghai Province.Methods Twenty-two post-surgical specimens of patients with hepatic alveolar echinococcosis were sampled from Qinghai Provincial People’s Hospital in 2017.The Cox1 and Nad1 genes were amplified from E.multilocularis samples and sequenced.Then,the gene sequences were aligned to the Cox1 and Nad1 genes of Echinococcus species in GenBank database.The intra-species variation was observed,and the phylogenetic tree and molecular clock were created.Results All E.multilocularis samples shared more than 99%genetic homology with the sequences of Cox1 and Nad1 genes from the E.multilocularis Asian strain in the GenBank database.A total of 6 genotypes were identified,including 2 isolates that had no 100%homology with the sequences of known genes in the GenBank database.Phylogenetic tree analysis revealed remarkable clustering of the E.multilocularis samples with the E.multilocularis Asian strain,and the E.multilocularis isolates from Qinghai Province were estimated to date back to 94000 years ago by the molecular clock.Conclusions The present study characterizes 6 E.multilocularis genotypes in Qinghai Province,including 2 novel genotypes.Asian strain is the predominant strain of E.multilocularis in Qinghai Province,and the E.multilocularis isolates from Qinghai Province date back to94000 years ago.