人体蠕形螨的DNA提取与随机引物PCR检测  

Extraction and random primer PCR detection of genomic DNA of parasitic mites Demodex folliculorum and Demodex brevis(Acari:Demodicidae)

作　　者：赵亚娥[1] 成慧[1] 寻萌[1] 吴李萍[1] 

机构地区：[1]西安交通大学医学院免疫学与病原生物学系,西安710061

出　　处：《昆虫学报》2009年第8期929-933,共5页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(30872199);陕西省自然科学基金资助项目(2006C247)

摘　　要：【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞,选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法,分别提取冻存时间在5个月内和8～10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA,并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示,试剂盒法提取的DNA纯度较高、量较多,明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱,两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量,但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似,试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰,碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的,蠕形螨冻存时间不宜超过6个月,试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。[ Objective ] To explore the extraction methods of genomic DNA of parasitic mites Demodex folliculorum （D.f.） and Demodex brevis （D. b. ）. [ Methods ] Mites were disrupted by repeated freezing and thawing in liquid nitrogen. Then, the improved DNA extraction method of mini-insects, alkaline lysis and the commercial extraction kit were used respectively to extract genomic DNA of D.f. and D. b., which had been kept freezing for 〈 5 or 8 - 10 months. [ Results ] The results of protein and nucleic acid radiometer showed the purity and quantity of genomic DNA extracted by the commercial kit were obviously better than those by other methods of mini-insects and alkaline lysis. Clear DNA fingerprints were found in the amplification results of random amplified polymorphic DNA （RAPD）. There were obviously different banding patterns between D.f. and D. b. The freezing time affected the quantity of DNA extraction, but had only small effect on the purity and RAPD fingerprints of genomic DNA. The same species of mites had similar banding pattern even using different extraction methods. More and clear bands were detected by the commercial extraction kits and improved DNA extraction method of mini-insects. The bands detected by alkaline lysis method were less and obscure. [ Conclusions ] The application of repeated freezing-thawing in liquid nitrogen acts effectively to disrupt mites. The mites had better not be kept frozen more than 6 months. The commercial extraction kit is an effective approach to extract genomic DNA of mites. RAPD technique provides a simple method for detecting and classifying D.f. and D. b.

关 键 词：毛囊蠕形螨 皮脂蠕形螨 DNA提取 随机引物PCR检测 

分 类 号：Q966[生物学—昆虫学]