作　　者： ; ; ; ; ; ;

机构地区： 中国农业大学动物医学院

出　　处： 《畜牧兽医学报》 2003年第5期509-514,共6页

摘　　要： 隐孢子虫病是一种重要的人畜共患原虫病。为了在临床样品中更准确、快速地检测隐孢子虫卵囊,从初步纯化的含有不同数量隐孢子虫卵囊的样品中和含有不同数量隐孢子虫卵囊的奶牛粪便中,直接提取DNA或用DNA纯化试剂盒对提取的奶牛粪便中卵囊DNA进行纯化之后用作起始PCR(Primary PCR)模板,以起始PCR的产物为模板进行巢式PCR(Nested PCR),用2对人工合成寡核苷酸分别作为两个PCR的引物,扩增片段大小分别为1325bp和820bp。优化了Mg2+浓度、引物浓度和dNTP浓度,并进行了特异性检验。建立的巢式PCR具有隐孢子虫属特异性,不仅扩增出新鲜样品DNA提取物中的目的片段,而且扩增出放置6年之久的DNA提取物中的目的片段。样品经过初步纯化之后,起始PCR和巢式PCR最低检测值卵囊分别为2 86×103个/ml和≤2 86个/ml;从含有隐孢子虫卵囊的奶牛粪便中提取DNA,尔后经过DNA纯化试剂盒纯化,其起始PCR和巢式PCR粪便中卵囊最低检测值分别为2 86×107个/g和≤2 86个/g。有望发展为试剂盒。 Cryptosporidiosis is a important protozonic zoonosis.DNA extracted directly from primarily purity oocysts samples or extracted from dairy fecal samples that was of differnet quantity oocyst,then was purified with DNA purity kit,that were used as the source of template for the primary PCR for detective cryptosporidia oocyst fast and accurately in clinical samples.The products of primary PCR was used as the source of template for the secondary PCR,two pair of oligonucleotide primers was synthesized and used to amplification of a 1325 bp and 820 bp.MgCl2 concentration and primer concentration and dNTP concentration were optimized,specificity of Cryptosporidium were tested.The nested PCR was of Cryptosporidium specificity.Not only target fragment of DNA extracted from fresh samples was amplified,but also target fragment of DNA extracted from samples that was preserved 6 years long was amplified.The threshold of detection in purified oocyst samples and livestock feces by the primary PCRbased test allowed the 286×103 oocysts/ml and 286×107 oocysts/g feces,by the secondary PCRbased test allowed the ≤286 oocyst/ml and ≤286 oocyst/g fecesIt will be developed to dignostic kit

关 键 词： 巢式 检测 隐孢子虫卵囊 人畜共患原虫病 临床样品 纯化 数量 奶牛粪便

领　　域： [农业科学] [农业科学] [农业科学]