云南大理片形吸虫扫描电镜观察和分子生物学鉴定

发布时间：2018-10-12 11:44

【摘要】：[目的]观察云南大理片形吸虫头锥部分(口吸盘、腹吸盘、生殖孔、体棘)的超微结构,用分子生物学方法对该地区片形吸虫的虫种进行鉴定,为云南大理片形吸虫的生物学特性及遗传变异提供基础信息。[方法]1.云南大理片形吸虫部分结构的扫描电镜观察:从大理泰兴农贸市场自然感染片形吸虫的4头黄牛肝胆管中获得了片形吸虫各10条,用生理盐水冲洗去血迹后各自分装到装满生理盐水的玻璃瓶中,依次标记为1、2、3、4号。从4号瓶中选取两条较完整的虫体编号为A、B号,从3号瓶选取了两条较完整的虫体编号为C、D号。剪取四条虫体头锥部分经冷藏过夜处理后按照制作扫描电镜标本的步骤:取材、固定、脱水、包埋、切片、染色后放在电子显微镜下进行观察。2.提取片形吸虫成虫基因组DNA:1号瓶中获得较完整虫体6条,编号为1-6号;2号瓶中获得虫体2条,编号7-8号,取这些虫体前端1/3部分,生理盐水冲洗,再用超纯水冲洗4到5遍,装于已消毒的1.5ml离心管。用手持式研磨仪捣碎虫体,将所得组织碎片参照TaKaRa MiniBEST Universal Genomic DNA Extraction KitVer.5.0试剂盒说明书上的方法提取片形吸虫成虫DNA,用核酸定量仪检测8个样品的DNA浓度。3.PCR扩增和测序:扩增片形吸虫的核糖体ITS-2基因片段、线粒体cox1部分基因片段及线粒体nad5部分基因片段,将所得扩增产物送至大连宝生物公司测序。4.序列分析:用DNAStar7.0软件将所得序列与之前报道的标准序列进行相似性比较。[结果]1.除C号虫体未见明显的生殖孔以外,其他三条片形吸虫都具有片形吸虫的典型结构:口吸盘、腹吸盘、生殖孔以及遍布虫体的体棘。口吸盘直径在310μm-540μm之间,腹吸盘直径在450μm-600μm之间。对这些虫体的口、腹吸盘放大后显示具有两种形式的乳突:圆锥形乳突和纤毛样乳突。将虫体部分体棘高倍放大后发现其呈现横向生长于虫体表面的具有基底较宽,端部较窄的圆弧状结构,端部生长有锯齿样结构。2.8条片形吸虫进行分子生物学鉴定后显示:其中2条经PCR扩增及琼脂糖凝胶电泳后显示大片形吸虫的目的条带,因此这两条鉴定为大片形吸虫;另外6条经PCR扩增及琼脂糖凝胶电泳后显示肝片形吸虫的目的条带,这6条鉴定为肝片形吸虫。3.PCR扩增云南大理片形吸虫的线粒体cox1基因部分序列测序显示其种内分歧度为0.9%-2.3%,种间分歧度3.0%-10.0%;线粒体nad5基因部分序列其种内分歧度为0.9%-1.2%,种间分歧度为 5.0%-13.0%。[结论]1.云南大理片形吸虫部分结构的扫描电镜结果显示四条片形吸虫有三条是肝片形吸虫,一条是大片形吸虫。除一条片形吸虫未观察到明显的生殖孔以外,其余三条片形吸虫都具有口腹吸盘、生殖孔,且虫体表面均生长一些致密的体棘,而且口吸盘处体棘比腹吸盘处致密。2.在以核糖ITS-2基因为分类遗传标记的研究基础之上,云南大理存在两种片形吸虫:肝片形吸虫与大片形吸虫。3.通过对这8条片形吸虫样品的核糖体ITS-2基因和线粒体cox1、nad5基因部分序列进行测序显示该地区片形吸虫之间呈现种间差异大于种内差异的特点。
[Abstract]:[Objective] To observe the ultrastructure of the cone part (mouth suction cup, abdomen suction cup, hole and body spine) of Fasciola paragonimus in Yunnan Province, and to identify the insect species of the Fasciola paragonimus by molecular biology method. To provide basic information for the biological characteristics and genetic variation of paragonimus yunnanensis.[Method] 1. Scanning electron microscope (SEM) of the partial structure of paragonimiasis in Yunnan Province: 10 pieces of Fasciola paragonimus were obtained from 4 Yellow Cattle Hepatobiliary Tubes of the Natural Infection Tablets of Taixing Agricultural Trade Market. After washing the blood with physiological saline, they were separately packed into glass bottles filled with physiological saline. Mark 1, 2, 3, 4 in sequence. From bottle No. 4, select two more complete bottles numbered A and B, and select two more complete numbers from No. 3 bottle to be C, D. The specimens were collected, fixed, dehydrated, embedded, sectioned, stained and observed under an electron microscope after cold storage overnight. The genomic DNA of adult adult worm was extracted: 1 bottle, 6 with no number 1-6, 2 in bottle 2, 7-8 No. 7-8, washed with physiological saline, flushed with ultrapure water for 4 to 5 times, and contained in sterilized 1. 5ml centrifuge tube. The DNA of the paragonimus paragonimus is extracted by a hand-held grinder, and the DNA concentration of 8 samples is detected by a nucleic acid quantitative instrument by referring to the DNA of the TaKaRa MiniBEST Universal Genomeic DNA Extraction KitVer. 5. 0 kit specification. The PCR amplification and sequencing are carried out to amplify the ribosomal ITS-2 gene fragment of the slice-shaped paragonimus, mitochondrial cox1 partial gene fragment and mitochondrial nad5 partial gene fragment, and the obtained amplified product is sent to Dalian Bao biological company for sequencing. Sequence analysis: The obtained sequence was compared with the previously reported standard sequence using DNAStar7.0 software.[Results] 1. The other three tablets have the typical structure of the paragonimus except that the C No. 48 is not seen in the obvious maxillary foramen: the mouth sucker, the belly sucker, the foramen magnum, and the body spine spread all over the body. The diameter of the mouth sucker is between 310. m u.m and 540. m u.m, and the diameter of the abdominal suction disc is between 450. m u.m and 600. m Two forms of mastoid, conical mastoid and ciliated mastoid, were displayed after enlargement of the abdominal suction cups. it is found that it has a wider base and a narrower end part, and the end part grows with sawtooth-like structure. Two of them were amplified by PCR and agarose gel electrophoresis to show the target bands of large-scale paragonimus, so the two were identified as large-scale paragonimus, and 6 were amplified by PCR and agarose gel electrophoresis to show the objective bands of the Fasciola paragonimus. The mitochondrial cox1 gene partial sequence of paragonimus paragonimus was amplified by PCR, and the difference between species was 0. 9% -2. 3%, the difference between species was 3.0%-10.0%, and the divergence of mitochondrial nad5 gene partial sequence was 0. 9%-1. 2%, and the difference between species was 5.0%-13.0%.[Conclusion] 1. The scanning electron microscope (SEM) of the partial structure of paragonimiasis in Yunnan Province showed that there were three pieces of paragonimiasis, one of which was a large piece of paragonimus. except that one piece of paragonimus was not observed, the other three pieces of paragonimus had an abdominal suction cup and a buccal cavity, and a compact body was grown on the surface of the mouth, and the body spine at the mouth sucker was denser than that at the abdominal suction cup. On the basis of the research on the classification of genetic markers with ribose ITS-2 gene, there are two kinds of paragonimus in Yunnan Province: Fasciola paragonimus and large-scale paragonimus. By sequencing the ribosomal ITS-2 gene and mitochondrial cox1, nad5 gene partial sequences of the 8 pieces of paragonimiasis, the differences between species and species in the region were found to be more than that of intraspecific differences.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R383.2

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