【摘要】： 目的應用環(huán)境掃描電子顯微鏡(environmental scanning electronmicroscope，ESEM)技術觀察兩種人體蠕形螨的超微結構，同時與普通掃描電鏡掃描的山羊蠕形螨超微結構進行比較，做出形態(tài)學分類。用隨機擴增多態(tài)DNA(RAPD)技術確定三種蠕形螨的分類地位，，進行條帶分析并計算不同類蠕形螨的遺傳距離。判斷三者的親緣關系并與傳統(tǒng)的形態(tài)學分類做比較。 方法加壓刮取人額部皮脂，切開新鮮山羊皮下結節(jié)，將獲得物置于滴有296洗潔精溶液的載玻片上，在解剖鏡直視下，用自制挑螨針將毛囊蠕形螨、皮脂蠕形螨和山羊蠕形螨從皮脂中分離并清洗干凈。 將潔凈活螨按觀察部位的要求，在解剖鏡下擺上電鏡樣品托，立即掃描，拍攝照片。環(huán)境掃描電鏡工作參數(shù)：工作距離(WD)7.5-9.3mm、高電壓(HV)20.0KV、高真空探頭(Det)GSED、束斑(Spot)3.0、溫度5℃、壓力0.5 kpa、相對濕度60％。 將潔凈新鮮的三種蠕形螨分別在解剖鏡直視下，于自制微型研缽中研磨粉碎。用改進的小型昆蟲基因組提取方法，分別提取三種蠕形螨的基因組DNA，摸索聚合酶鏈反應(PCR)總體積、反應條件。選取12條不同的隨機排列堿基順序的多聚核苷酸單鏈為引物，進行RAPD-PCR擴增反應。將擴增產(chǎn)物通過瓊脂糖電泳，制備DNA多態(tài)性圖譜，比較三種蠕形螨基因組DNA多態(tài)性，分析三種蠕形螨兩兩間的遺傳距離，從DNA水平鑒定這三種蠕形螨。 結果經(jīng)過掃描電鏡觀察，發(fā)現(xiàn)三種蠕形螨在背基刺形狀及相對位置、口器形狀、須爪數(shù)目和排列等超微結構上，均有明顯的差異；足體上，三種蠕形螨的背部皮紋、背足體毛、足節(jié)形狀及爪、爪距、股距、雄性生殖孔及陰莖形狀、雌性陰門形狀都不相同；末體的形狀和紋形也不相同。 經(jīng)過RAPD電泳條帶分析，篩選出5個擴增產(chǎn)物較多、譜帶清晰、多態(tài)性較好的引物，用這5個引物共擴增出49條條帶，片段大小范圍為250—2500bp。其中山羊蠕形螨共有17個多態(tài)性片段，皮脂蠕形螨共有9個多態(tài)性片段，毛囊蠕形螨有23個多態(tài)性片段。山羊蠕形螨與毛囊蠕形螨有5條共享條帶。山羊蠕形螨與皮脂蠕形螨有4條共享條帶。毛囊蠕形螨與皮脂蠕形螨間僅有1條共享條帶。根據(jù)獲得的條帶進行遺傳距離分析，毛囊蠕形螨和皮脂蠕形螨兩個種間的平均遺傳距離(D1)為0.9375，毛囊蠕形螨和山羊蠕形螨之間的平均遺傳距離(D2)為0.8500，皮脂蠕形螨和山羊蠕形螨之間的平均遺傳距離(D3)0.6923。D1和D2均大于D3。 結論三種蠕形螨在超微結構上形態(tài)均有多處不同。三種蠕形螨RAPD-PCR電泳條帶表型有明顯不同，說明RAPD技術可在DNA水平上鑒別毛囊蠕形螨、皮脂蠕形螨、山羊蠕形螨這三種形態(tài)特征十分相似的種類。遺傳距離分析說明盡管毛囊蠕形螨和皮脂蠕形螨均寄生于人體，但它們之間的親緣關系卻較遠；皮脂蠕形螨反而與山羊蠕形螨的親緣關系反而較近，這可能跟它們的寄生部位及食性類似有關。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R382.5

【引證文獻】

相關期刊論文 前2條

1 趙亞娥;成慧;尋萌;吳李萍;;人體蠕形螨的DNA提取與隨機引物PCR檢測[J];昆蟲學報;2009年08期

2 崔立云;張霄霄;楊毅梅;;RAPD技術在寄生蟲分類和鑒定中的應用[J];中國人獸共患病學報;2012年04期

相關碩士學位論文 前1條

1 唐成;重慶地區(qū)牛附紅體病分子流行病學調(diào)查及黃牛附紅體RAPD-SCAR標記的建立[D];西南大學;2009年

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