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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 morphologically parasitic species of Acanthamoeba in sputum samples of patients with repeated cough. Methods The protozoa in sputum of patients were isolated and cultured in vitro. The protozoa trophozoites and cysts were observed under microscope. DNA of protozoa was extracted. Ami6F1 and Ami9R primers of Amoebae 18S rRNA, JDP1 JDP2, Acanthamoeba griffini S-7ATCC 18S rRNA full-length primer AacGF and AacGR for PCR amplification identification. The 18S rRNA gene of the sample was used as a molecular marker for homology analysis with each Acanthamoeba in GenBank. The sequences of related species were screened and the phylogenetic tree was constructed by using maximum likelihood method to analyze the genetic relationship. Results Microscopically, the trophozoites in the sputum of patients had acanthosis pseudopod protuberances characteristic of Acanthamoeba, with irregular amoebae. The cysts were two-layer membranous structures, and the intima had spinosad Pakistan characteristic asteroid. PCR results showed that the bands of 830, 479 and 1 954 bp were amplified by 3 sets of primers, which were consistent with the expected fragment size. After BLAST alignment, the similarity of the three amplified product sequences with the S. aurepian S-7 ATCC was 99%, 99%, 100% respectively. Phylogenetic tree analysis showed that the samples of Acanthamoebae and A. ketellanii, A.polyphaga, A.cullbertsoni ) And A. rhysodes were 91.4%, 99.6%, 94.5% and 91.8%, respectively. Conclusions The parasite in the sputum sample of this respiratory tract infection was identified as Acanthamoeba Gemma.