【摘要】 为了检测样品中的微量隐孢子虫 ,从含有不同数量隐孢子虫卵囊的奶牛粪便中 ,直接提取DNA用作初始PCR(Initial PCR)模板 ,以稀释的初始PCR的产物为模板进行套式PCR(Nested PCR) ,用两对人工合成寡核苷酸分别作为两PCR的引物 ,扩增大小分别为 540pb、2 58pb的特异片段。PCR产物经电泳鉴定 ,可从阳性粪便标本DNA抽提物中扩增出目的片段 ,而阴性对照不能扩增目的片段 ;初始PCR、套式PCR的敏感小生最低可分别检测到含卵囊1 0 0、5个 /g粪便。初步应用结果表明某奶牛场的奶牛自然感染率为 1 6 4%。试验表明套式PCR的敏感性比普通PCR约高 1 0 0倍 ,能用于奶牛隐孢子虫感染情况的调查更多还原

【Abstract】 To develop a technique for the detection of few number of Cryptosporidium muris in sample.DNA extracted directly from fecal sample with different number of C.muris oocytis in dary cattle was used as the source of template for the Initial PCR,the dilute production of Initial PCR was used as the source of template for the Nested PCR,two pair of oligonucleotide primers was synthesized and used to prime the amplification of a 540pb?258pb target fragment specific for C.muris respectively.The results showed that the target fragment was present in DNA extracts of fecal sample with C.muris,but not in DNA from native sample,the level of detection by initial PCR was 100 oocysts per gram of feces,that by nested PCR was 5 oocysts per gram of feces,The preliminary application indicated that there is 16 4% of cattle infected by.C.muris.This study showed that Nested PCR was about 100 times more sensitive than ordinary PCR for detection of C.muris and could serve as a valuable tool for epidemiological survey.更多还原