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印囊,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PcR模板,用1对人工合成寡核苷酸作为PCR引物,扩增片段大小为452bp。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,最低检测值100个卵囊/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,PCR最低检测值为105个卵囊/g粪便。
Cryptosporidiosis is a important protozonic zoonosis. DNA extracted directly from primarily purity oocysts samples or extracted from dairy fecal samples that was of different quantity oocyst , then was purified with DNA purity kit ,that were used as the source of template for the PCR for detective cryp-tosporidia oocyst fast and accurately in clinical samples. A pair of oligonucleotide primers was synthesized and used to amplification of a 452bp. MgCl2 concentration and primer concentrition and dNTP concentration were optimized , specificity of Cryptosporidium were tested . The 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 PCR-based test allowed the 100 oocysts/ml and 10 X 10s oocysts/g feces respectively.
出处
《中国兽医杂志》
CAS
北大核心
2003年第7期3-7,共5页
Chinese Journal of Veterinary Medicine
基金
河南省杰出青年科学基金资助课题(0112000600)
