【作者】 严若峰；

【导师】 李培英； 周金林； 余为一；

【作者基本信息】 安徽农业大学 ， 预防兽医学， 2001， 硕士

【摘要】 从某奶牛场分离出隐孢子虫卵囊，用金胺酚-改良抗酸染色及饱和蔗糖漂浮后，通过显微镜检，根据其形态、结构、大小等特性鉴定为鼠隐孢子虫（Cryptosporidium muris）。 根据隐孢子虫18s rDNA序列，借助计算机分析，设计并合成出隐孢子虫属特异性和鼠隐孢子虫种特异性引物。在经优化的反应条件和扩增参数下进行聚合酶链式反应，扩增出540bp的片段。初始PCR产物进一步作NestedPCR，扩增出一条250bp的片段。结果表明，该片段是鼠隐孢子虫所特有，以艾美耳球虫、伊氏锥虫、贾第虫、大肠杆菌等病原的DNA为模板进行扩增反应，均无此片段。 Nested PCR具有高度敏感性，可以检测出1个纯化的隐孢子虫卵囊。当样品中卵囊密度高于10个/g（ml）时，该方法亦能很好的检测出。与常规PCR、饱和蔗糖溶液漂浮法及金胺酚改良抗酸染色法比较，Nested PCR的敏感性是常规PCR的10~3倍以上、是漂浮法和染色法的10~5倍以上。 本文还比较了4种卵囊DNA提取方法，结果表明2％二硫苏糖醇冻融法具有很好的敏感性，而且其操作简便，快速易行。 应用Nested PCR技术，对奶牛粪便、牛奶、污水等119个样品隐孢子虫感染及污染情况进行了检测，检出率分别为23.29％、11.11％和0.00％，均高于常规PCR、漂浮法和染色法的检测结果。 综上所述，新建立的Nested PCR技术是一种具有较高特异性和敏感性的微量隐孢子虫卵囊检测方法，具有较好的应用前景。更多还原

【Abstract】 Cryptosporidium oocysts were separated from the faeces in a cattle farm and identified as Cryptosporidium muris according to their morphology, structure arid size after a sheather’s floating and an auramine-phenol and acid-fast-staining (AP-AFS).Two sets of special primers to Cryptosporidium and Cryptosporidium muris were designed and synthesized respectively based on the sequence of 18s rDNA. A special fragment of 540bp was amplified after polymerase chain reaction (PCR) with a series of suitable reaction parameters and in an optimal concentration of Mg2+ and Taq polymerase. Then the products of primary PCR were used as templates, oligonucleotides special to Cryptosporidium muris as primers in the Nested PCR, a fragment of 250 bp was amplified, while no fragment was observed in the testing of other samples from disease pathogens such as Eimeria coccidian, Typansooma Evansi, giardias, E.col et al.A minimal number from 1 to 10 of oocysts could be detected by the Nested PCR. Compared with other methods, the Nested PCR might be provided with the highest sensitivity for the detection, whose sensitivity was 103 to 10s times higher than that of conventional PCR, sheather’s floating and AP-AFS.In order to enhance the practicability of the Nested PCR, four methods of extracting DNA from oocysts of Cryptosporidium muris were tested and evaluated. It was found that the following easier treatment of oocysts appeared higher sensitive: the oocysts were suspended in 2% DTT, fractured by 3 cycles of freezing in liquid nitrogen and thawing in room temperature or 8 cycles freezing at -20癈 and thawing at room temperature, then centrifuged at 13 OOOg.In addition, the Nested PCR was used in examining 119 of clinic samples, a positive rate of 23.29%, 11.11% and 0.00% for Cryptosporidium spp. respectively in faeces, milk and polluted water was gained, that showed its sensitivity was higher than that of conventional PCR, sheather’s floating and AP-AFS.Briefly, the new Nested PCR has a bright prospect of application in the microdetection of Cryptosporidium oocysts because of its high specificity and sensitivity.更多还原