河南省三株人源隐孢子虫分离株鉴定

目的隐孢子虫病是一种人兽共患寄生虫病,在艾滋病病人和儿童中的感染率可分别高达48％和17．5％,可导致严重的水样腹泻甚至危及生命,免疫功能正常者也能感染出现急性自限性腹泻。本文通过不同软件对三株人源隐孢子虫分离株进行种类鉴定。方法参照Xiao L H 1999年发表的资料合成隐孢子虫18S rDNA特异性引物(上游引物:5’-AACCTGGTTGATCCTGCCAGTAGTC -3’,下游引物:5’-TGAGCCTTCTGCAGGTTCACCTACG-3’,由大连宝生物工程技术公司合成。),FCR 扩增,产物测序、序列分别用Paup4．0、rhylip3．6、DNAstar4．0等软件分析并与12株隐孢子虫相应序列进行同源性分析,绘制了系统发育进化树,分析鉴定。1、2号株采自非免疫抑制病人,3号株采自免疫抑制病人。结果①三株隐孢子虫均能扩增出1700bp左右的目的片段。②构建系统发生树;用DNAstar4．0软件进行分析:1与2号株的同源性为99．8％,1和2号株与 AF108864 C．parvum-cattle的同源性分别为99．5％和99．3％,与AF112569 C．hominis进化关系较远。3号株与AF112574 C． meleagridis(火鸡源 C．meleagridis)的同源性为99．8％,3号株与C．suis(AF108861)的同源性为98．9％,而与AF108864 C．parvum -cattle、AF112569 C．hominis、AF108865 C．parvum(Homo sapiens)、AY642591 C．muris、AB210854 C．canis、AF108862 C． felis等均不处于同一进化枝,亲缘关系较远。利用PAUP4．0、Phylip3.6的方法与DNAstar4．0 软件分析的结果基本一致。结论 1和2号分离株均为C．parvum牛基因型,3号分离株是C．meleagridis(火鸡隐孢子虫)。本文结果为深入研究人隐孢子虫病的分子流行病学打下基础。

Identification of molecule species of the three cryptosporidiosis in henan province

object:Cryptosporidiosis is paratise of the zoonoses. the rate of infection is respectively 48% and 17. 5% in AIDS and childrens, It can cause heavy watery diarrher and even threaten life . immunocompetent individuals can also infect and occur acute diarrhea . the topic is to identified the three species of cryptosporidium by using different software. . Methods:to synthesize 18S rDNA specific primer by referring to datum published in 1999 by xiao L H,(upstream primers:5'- AACCTGGTTGATGCTGCCAGTAGTC -3',downstream primers:5? TGAGCCTTCTGCAGGTTCACCTACG? 3', primer was synthesized by biology enginer technology company.) , PCR amplify、 production sequenced. The measured sequence was analysed by the software of Paup4.0、Phylip3.6、DNAstar4.0 and analysed homologily of 12 cryptosporidium corresponding sequence . and protract the cladogram of phylogenesis.results:①the three cryptosporidium can amplify aimed segment of about 1700bp.②construct the cladogram of phylogenesis was analysed by the software of DNAstar4.0:the homology of 1#2# is 99.8% , the homology of 1#2 # AF108864 C. parvum-cattle is respectively 99. 5% . 99. 3%,and relation of evolution of AF112569 C. hominis is very far. the homology of 3# and AF112574 C. meleagridis is 99. 8% , the homology of 3# C. suis is 98. 9%,and AF108864 C. parvum -cattle、AF112569 C. hominis、AF108865 C. parvum(Homo sapiens)、AY642591 C. muris、 AB210854 C. cam's、AF108862 C. felis is located differently tree of evolution, the relation is very far . the result analysed by using the method of PAUP4. 0、Phylip3. 6 and the software of DNAstar4. 0 are consistent . conclusion:1 # 2# are C. parvum, 3# is C. meleagridis. the result of the article lay good foundations for molecule epidemiological study of cryptosporidiosis.