一种从蠕虫组织切片中提取DNA作PCR鉴定虫种的简便方法  

A Simple Method of PCR Identifying the Species of Helminthes by Using DNA Extracted from Paraffin-embedded Tissue Sections

作　　者：胡勉娟[1] 曾庆仁[1] 粟占三[2] 兰智华[2] 贺美[1] 余权[1] 张祖萍[1] 蔡力汀[1] 

机构地区：[1]中南大学基础医学细胞与分子生物学实验中心,湖南长沙410013 [2]中南大学湘雅三医院病理科

出　　处：《实用预防医学》2013年第3期350-353,共4页Practical Preventive Medicine

基　　金：中央高校基金前沿研究计划重点项目资助(2010QZZD027)

摘　　要：目的建立一种实用于临床病理鉴别诊断组织切片内蠕虫虫种的分子病理学方法。方法制作已知的和从临床收集的多种蠕虫和宿主组织病理切片标本,用改良蛋白K消化法对各种蠕虫切片的白片、HE染色片和免疫组化片组织提取DNA;设计合成检测并殖吸虫、曼氏裂头蚴和猪囊虫各自特异性基因片段的引物,分别对各DNA提取物作PCR扩增,经凝胶电泳观察目的基因片段;比较分析三种蠕虫各自最小组织量检测的敏感性和特异性。结果对三种蠕虫所选用的引物均可扩增出清晰的特异性目的条带。用改良蛋白K消化法提取蠕虫DNA可PCR扩增出目的片段的敏感性均优于商品化试剂盒提取法,可提出DNA的最小组织面及厚度为:肺吸虫的为1.50 mm2,10μm;曼氏裂头蚴的为4.87 mm2,5μm;猪囊虫的为5.80 mm2,5μm。经10个临床标本检测均获得理想结果。结论本研究成功地探索出一种对病理切片中微量组织提取DNA作PCR鉴定虫种的简便方法,为病理诊断提供了进一步鉴别此3种蠕虫的分子病理学手段。Objective To establish a molecular pathology method which can be used for clinical pathological diagnosis on the species identification of helminthes in paraffin-embedded tissue sections.Methods The specimens known and from the clinical variety of worms and host tissues were collected and made.The DNA was extracted from the white thin slices,HE stained slices and immunohistochemical staining slices of the specimens with modified proteinase K digestion,respectively.The primers were designed and synthesized for detecting the specific gene fragments of Paragonimus,Sparganum mansoni and cysticercus cellulosae of Taenia solium and conducting PCR amplification.PCR products were analyzed by gel electrophoresis for observing the target gene strip.The sensitivity and specificity of detecting and identifying minimum tissue amount of the three species of worms were analyzed.Results The specific target gene fragments were amplified from the primers of the genes of three species of worms detected.The results of PCR amplification of the target gene fragments showed that the sensitivity of extracting the worm DNA with modified proteinase K digestion was significantly better than that of commercial kits extraction method.A smallest area and thickness of the worm organizations which could submit DNA with modified proteinase K digestion in Paragonimus,Sparganum mansoni,and cysticercus cellulosae were 1.50 mm2 and 10 μm,4.87 mm and 5 μm,5.80 mm and 5 μm,respectively.The results of 10 clinical specimens detected with the above methods were all very ideal.Conclusions This study successfully obtains a simple method of PCR identifying the species of helminthes by using DNA extracted from paraffin-embedded tissue sections,and it provides a means of molecular pathology for clinical pathological differential diagnosis of these three kinds of worms.

关 键 词：蠕虫 石蜡切片 蛋白酶K消化法 DNA提取 聚合酶链反应 

分 类 号：R383.2[医药卫生—医学寄生虫学]