CAJ | 학술논문

目的对来源于哈尔滨某奶牛场一头3月龄奶牛的一个隐孢子虫分离株进行了分子鉴定.选择隐孢子虫基因分型常用的18SrRNA和actin基因位点,设计引物,用巢氏PCR方法分别扩增出约840 bp和1 066bp目的片段.PCR产物经双向测序后用ClustlX、DNAstar和PHLIP软件进行序列分析.结果表明在18SrRNA基因位点上,此分离株与报道的牛源C.Ryanae分离株(DQ871345、AY587166、EU410344)同源性最高,达到100%.在actin位点,此分离株和美国牛源C.Ryanae分离株(EU410345)和隐孢子虫鹿样基因型(AY741309)同源性也为100%.种系发育分析显示所获得的分离株在两个基因位点上均和C.Ryanae处于同一进化分支.这些分析表明此次分离获得的隐孢子虫分离株为C.Ryanae.鉴定结果为研究我国牛源隐孢子虫种类分布以及评价其公共卫生意义提供了重要参考.
목적 대래원우합이빈모내우장일두3월령내우적일개은포자충분리주진행료분자감정.선택은포자충기인분형상용적18SrRNA화actin기인위점,설계인물,용소씨PCR방법분별확증출약840 bp화1 066bp목적편단.PCR산물경쌍향측서후용ClustlX、DNAstar화PHLIP연건진행서렬분석.결과 표명재18SrRNA기인위점상,차분리주여보도적우원C.Ryanae분리주(DQ871345、AY587166、EU410344)동원성최고,체도100%.재actin위점,차분리주화미국우원C.Ryanae분리주(EU410345)화은포자충록양기인형(AY741309)동원성야위100%.충계발육분석현시소획득적분리주재량개기인위점상균화C.Ryanae처우동일진화분지.저사분석표명차차분리획득적은포자충분리주위C.Ryanae.감정결과위연구아국우원은포자충충류분포이급평개기공공위생의의제공료중요삼고.
One Cryptopsoridium isolate from a 3 month-old calf in a dairy cow farm of Harbin was identified by molecular method, in which the common genes of 18S rRNA and actin used in Cryptosporidium genotyping were selected, the primers were designed, and the approximate 840 bp and 1066 bp fragments of these two genes were amplified respectively by nested PCR. The PCR products were sequenced by two directions, and then the sequences were analyzed by using the soft wares of ClustlX, DNAstar, and PHLIP. The experimental result showed that in the 18SrRNA gene, there was the highest identities compared to other reported isolates of cow-derived C. ryanae (DQ871345, AY587166, and EU410344), with 100% of similarity. Likewise, at actin locus, the present isolate also had 100% similarity with an American cow-derived C. ryanae isolate (EU410345) and a deer-like genotype isolate (AY741309). Phylogenetic analysis indicated that the present isolate shared the same evolution branch in the two gene loci. These analyses demonstrated that the present isolate belonged to C. ryanae. These results provide a significant reference for the study on the species distribution and assessment in the public health significance of Cryptosporidium in cow milk in China.