三种蠕形螨的形态学和RAPD研究
发布时间：2018-08-15 14:51
【摘要】： 目的应用环境扫描电子显微镜(environmental scanning electronmicroscope，ESEM)技术观察两种人体蠕形螨的超微结构，同时与普通扫描电镜扫描的山羊蠕形螨超微结构进行比较，做出形态学分类。用随机扩增多态DNA(RAPD)技术确定三种蠕形螨的分类地位，，进行条带分析并计算不同类蠕形螨的遗传距离。判断三者的亲缘关系并与传统的形态学分类做比较。 方法加压刮取人额部皮脂，切开新鲜山羊皮下结节，将获得物置于滴有296洗洁精溶液的载玻片上，在解剖镜直视下，用自制挑螨针将毛囊蠕形螨、皮脂蠕形螨和山羊蠕形螨从皮脂中分离并清洗干净。 将洁净活螨按观察部位的要求，在解剖镜下摆上电镜样品托，立即扫描，拍摄照片。环境扫描电镜工作参数：工作距离(WD)7.5-9.3mm、高电压(HV)20.0KV、高真空探头(Det)GSED、束斑(Spot)3.0、温度5℃、压力0.5 kpa、相对湿度60％。 将洁净新鲜的三种蠕形螨分别在解剖镜直视下，于自制微型研钵中研磨粉碎。用改进的小型昆虫基因组提取方法，分别提取三种蠕形螨的基因组DNA，摸索聚合酶链反应(PCR)总体积、反应条件。选取12条不同的随机排列碱基顺序的多聚核苷酸单链为引物，进行RAPD-PCR扩增反应。将扩增产物通过琼脂糖电泳，制备DNA多态性图谱，比较三种蠕形螨基因组DNA多态性，分析三种蠕形螨两两间的遗传距离，从DNA水平鉴定这三种蠕形螨。 结果经过扫描电镜观察，发现三种蠕形螨在背基刺形状及相对位置、口器形状、须爪数目和排列等超微结构上，均有明显的差异；足体上，三种蠕形螨的背部皮纹、背足体毛、足节形状及爪、爪距、股距、雄性生殖孔及阴茎形状、雌性阴门形状都不相同；末体的形状和纹形也不相同。 经过RAPD电泳条带分析，筛选出5个扩增产物较多、谱带清晰、多态性较好的引物，用这5个引物共扩增出49条条带，片段大小范围为250—2500bp。其中山羊蠕形螨共有17个多态性片段，皮脂蠕形螨共有9个多态性片段，毛囊蠕形螨有23个多态性片段。山羊蠕形螨与毛囊蠕形螨有5条共享条带。山羊蠕形螨与皮脂蠕形螨有4条共享条带。毛囊蠕形螨与皮脂蠕形螨间仅有1条共享条带。根据获得的条带进行遗传距离分析，毛囊蠕形螨和皮脂蠕形螨两个种间的平均遗传距离(D1)为0.9375，毛囊蠕形螨和山羊蠕形螨之间的平均遗传距离(D2)为0.8500，皮脂蠕形螨和山羊蠕形螨之间的平均遗传距离(D3)0.6923。D1和D2均大于D3。 结论三种蠕形螨在超微结构上形态均有多处不同。三种蠕形螨RAPD-PCR电泳条带表型有明显不同，说明RAPD技术可在DNA水平上鉴别毛囊蠕形螨、皮脂蠕形螨、山羊蠕形螨这三种形态特征十分相似的种类。遗传距离分析说明尽管毛囊蠕形螨和皮脂蠕形螨均寄生于人体，但它们之间的亲缘关系却较远；皮脂蠕形螨反而与山羊蠕形螨的亲缘关系反而较近，这可能跟它们的寄生部位及食性类似有关。
[Abstract]:Objective To observe the ultrastructure of two human Demodex species by environmental scanning electron microscopy (ESEM), and to classify them by comparing with the ultrastructure of goat Demodex scanned by ordinary scanning electron microscopy (SEM). The genetic distances of different Demodex species were calculated by band analysis, and their genetic relationships were judged and compared with the traditional morphological classification.
Methods Human forehead sebum was scraped under pressure and fresh goat subcutaneous nodules were incised. The obtained substances were placed on slide dripped with 296 detergent solution. Demodex folliculorum, Demodex sebum and Demodex goatum were separated and cleaned from sebum by self-made needle under microscope.
The working parameters of environmental scanning electron microscope are: working distance (WD) 7.5-9.3 mm, high voltage (HV) 20.0 KV, high vacuum probe (Det) GSED, Spot 3.0, temperature 5 C, pressure 0.5 kpa, relative humidity 60%.
The genomic DNA of three kinds of Demodex were extracted by improved small insect genome extraction method, and the total volume of polymerase chain reaction (PCR) and reaction conditions were explored. Twelve polynucleosides with different random base sequences were selected. DNA polymorphism maps were prepared by agarose electrophoresis. The genomic DNA polymorphisms of three Demodex species were compared. The genetic distances between the three species were analyzed and the three Demodex species were identified at DNA level.
Results Scanning electron microscopy showed that there were obvious differences among the three Demodex in the shape and relative position of the dorsal basal spine, the shape of the mouth organ, the number and arrangement of the claws. The shape and shape of the last body are different.
By RAPD electrophoretic band analysis, five primers with more amplified products, clear bands and better polymorphism were screened out. Forty-nine bands were amplified with these primers. The fragments ranged from 250 BP to 2500 bp. Among them, 17 polymorphic fragments were found in Demodex goatum, 9 polymorphic fragments were found in Demodex sebaceous mite and 23 polymorphic fragments in Demodex folliculorum. There were 5 shared bands between Demodex folliculorum and Demodex folliculorum. There were 4 shared bands between Demodex folliculorum and Demodex sebaceous mite. There was only one shared band between Demodex folliculorum and Demodex sebaceous mite. The average genetic distance (D2) between Demodex cysticercus and Demodex goat was 0.8500. The average genetic distance (D3) between Demodex sebaceous and Demodex goat was 0.6923.D1 and D2 were greater than D3.
Conclusion There are many differences in the ultrastructure of the three demodex. The RAPD-PCR electrophoretic band phenotypes of the three Demodex are obviously different, indicating that the RAPD technique can be used to identify Demodex folliculorum, Demodex sebaceous mite and Demodex goat mite at the DNA level. Genetic distance analysis shows that Demodex folliculorum and Demodex folliculorum mite have similar morphological characteristics. Demodex sebaceous mites are parasitic on human beings, but their genetic relationship is far from each other. Demodex sebaceous mites are closely related to Demodex goats, which may be related to their parasitic location and feeding habits.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R382.5
本文编号：2184541
[Abstract]:Objective To observe the ultrastructure of two human Demodex species by environmental scanning electron microscopy (ESEM), and to classify them by comparing with the ultrastructure of goat Demodex scanned by ordinary scanning electron microscopy (SEM). The genetic distances of different Demodex species were calculated by band analysis, and their genetic relationships were judged and compared with the traditional morphological classification.
Methods Human forehead sebum was scraped under pressure and fresh goat subcutaneous nodules were incised. The obtained substances were placed on slide dripped with 296 detergent solution. Demodex folliculorum, Demodex sebum and Demodex goatum were separated and cleaned from sebum by self-made needle under microscope.
The working parameters of environmental scanning electron microscope are: working distance (WD) 7.5-9.3 mm, high voltage (HV) 20.0 KV, high vacuum probe (Det) GSED, Spot 3.0, temperature 5 C, pressure 0.5 kpa, relative humidity 60%.
The genomic DNA of three kinds of Demodex were extracted by improved small insect genome extraction method, and the total volume of polymerase chain reaction (PCR) and reaction conditions were explored. Twelve polynucleosides with different random base sequences were selected. DNA polymorphism maps were prepared by agarose electrophoresis. The genomic DNA polymorphisms of three Demodex species were compared. The genetic distances between the three species were analyzed and the three Demodex species were identified at DNA level.
Results Scanning electron microscopy showed that there were obvious differences among the three Demodex in the shape and relative position of the dorsal basal spine, the shape of the mouth organ, the number and arrangement of the claws. The shape and shape of the last body are different.
By RAPD electrophoretic band analysis, five primers with more amplified products, clear bands and better polymorphism were screened out. Forty-nine bands were amplified with these primers. The fragments ranged from 250 BP to 2500 bp. Among them, 17 polymorphic fragments were found in Demodex goatum, 9 polymorphic fragments were found in Demodex sebaceous mite and 23 polymorphic fragments in Demodex folliculorum. There were 5 shared bands between Demodex folliculorum and Demodex folliculorum. There were 4 shared bands between Demodex folliculorum and Demodex sebaceous mite. There was only one shared band between Demodex folliculorum and Demodex sebaceous mite. The average genetic distance (D2) between Demodex cysticercus and Demodex goat was 0.8500. The average genetic distance (D3) between Demodex sebaceous and Demodex goat was 0.6923.D1 and D2 were greater than D3.
Conclusion There are many differences in the ultrastructure of the three demodex. The RAPD-PCR electrophoretic band phenotypes of the three Demodex are obviously different, indicating that the RAPD technique can be used to identify Demodex folliculorum, Demodex sebaceous mite and Demodex goat mite at the DNA level. Genetic distance analysis shows that Demodex folliculorum and Demodex folliculorum mite have similar morphological characteristics. Demodex sebaceous mites are parasitic on human beings, but their genetic relationship is far from each other. Demodex sebaceous mites are closely related to Demodex goats, which may be related to their parasitic location and feeding habits.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R382.5
【引证文献】
相关期刊论文 前2条
1 赵亚娥;成慧;寻萌;吴李萍;;人体蠕形螨的DNA提取与随机引物PCR检测[J];昆虫学报;2009年08期
2 崔立云;张霄霄;杨毅梅;;RAPD技术在寄生虫分类和鉴定中的应用[J];中国人兽共患病学报;2012年04期
相关硕士学位论文 前1条
1 唐成;重庆地区牛附红体病分子流行病学调查及黄牛附红体RAPD-SCAR标记的建立[D];西南大学;2009年
本文编号：2184541
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