致人体呼吸道感染的似棘阿米巴形态学和分子生物学鉴定  

Morphological and Molecular Identification of Acanthamoeba sp.,A New Pathogen for Human Respiratory Tract Infection

作　　者：乔岩[1,2] 彭恒[2] 朱淮民[2] 严继舟[1] 

机构地区：[1]上海海洋大学海洋生物和神经科学研究所,上海201306 [2]第二军医大学病原生物学教研室,上海200032

出　　处：《中国寄生虫学与寄生虫病杂志》2016年第2期113-117,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：卫生行业科研专项经费资助项目(No.201202019);国家自然科学基金(No.81141085)~~

摘　　要：目的鉴定某反复咳嗽患者痰液样本中形态学似棘阿米巴的寄生虫种属。方法分离患者痰液中原虫进行体外培养,显微镜观察原虫滋养体和包囊形态,并提取原虫DNA,采用阿米巴科18S rRNA通用引物Ami6F1和Ami9R、棘阿米巴属18S rRNA通用引物JDP1和JDP2、葛氏棘阿米巴(Acanthamoeba griffini)S-7ATCC 18S rRNA全长序列引物AacGF和AacGR进行PCR扩增鉴定。以样本的18S rRNA基因为分子标志,与GenBank中各棘阿米巴序列进行同源性分析,筛选相关物种序列,采用最大似然法构建系统进化树,分析亲缘关系。结果镜下可见,患者痰样中的滋养体具有棘阿米巴特征性的棘状伪足凸起,并呈无规则的变形虫状;包囊为两层膜结构,内膜具有棘阿米巴特征性的星状突起。PCR检测结果显示,采用3组引物分别扩增出830、479和1 954 bp的条带,与预期片段大小一致。经BLAST比对后,3个扩增产物序列与棘阿米巴S-7 ATCC相似度分别达99%、99%、100%。系统进化树结果显示,样本棘阿米巴与引起角膜炎的卡氏棘阿米巴(A.castellanii)、多噬棘阿米巴(A.polyphaga)、柯氏棘阿米巴(A.cullbertsoni)和条脊棘阿米巴(A.rhysodes)同源性较高,分别为91.4%、99.6%、94.5%和91.8%。结论该呼吸道感染患者痰液样本中的寄生虫鉴定为葛氏棘阿米巴。Objective To identify the species of a morphologically Acanthamoeba-like pathogen in sputum from a patient with repeated cough. Methods Protozoa were isolated from the sputum and cultured for morphological observation of the trophozoites and cysts. DNA was extracted from the cultivated sample, and PCR was performed using primers as follows： 18S universal primers for amoeba family（Ami6F1 and Ami9R） and for amoeba genus（JDP1 and JDP2）, and primers for 18S full-length sequence of S-7 ATCC reference strain of Acanthamoeba griffini （AacGF and AscGR）. The 18S rRNA was sequenced, followed by homology analysis. The maximum likelihood method was used to construct phylogenetic tree. Results Microscopic examination showed that the trophozites had spine and irregular-shape pseudopodia bulge. The cysts were encapsulated by double membrane layer with the inner membrane having star-like processes. As expected, PCR amplification resulted in bands of 830, 479 and 1 957 bp, respectively, which were blasted to be 99%, 99% and 100% homologous to those of A. griffini（U07412.1）. Phylogenetic tree indicated that this acanthamobe in the patient’s sample was 91.4%, 99.6%, 94.5% and 91.8% homologous to keratitis-associated A. castellanii, A. polyphage, A. cullbertsoni and A. rhysodes. Conclusion The parasite in sputum of the patient with respiratory tract infection is Acanthamoeba griffini.

关 键 词：葛氏棘阿米巴 呼吸道 形态鉴定 分子鉴定 

分 类 号：R372[医药卫生—病原生物学]