牛源隐孢子虫上海分离株的巢式PCR鉴定

目的 用巢式PCR鉴定1株自然感染的上海牛源隐孢子虫。 方法 改良抗酸染色法检查含隐孢子虫卵囊的牛粪,油镜下观察卵囊形态,测量大小。以牛粪中的隐孢子虫卵囊DNA为模板,根据隐孢子虫18S rRNA序列设计2对引物,巢式PCR扩增目的片段,测序,同源性比对,并运用MEGA4.0软件构建系统发育树。 结果 上海牛源隐孢子虫分离株卵囊呈圆形或椭圆形,大小为（5.6±0.49）mm×（5.2±0.51）mm。扩增出的18S rRNA基因片段为812 bp。上海牛源隐孢子虫分离株的18S rRNA与巴西的牛隐孢子虫（Cryptospridium bovis,GenＢank登录号为151935628）序列一致性为100%,两者在种系发育树上为同一分支,亲缘关系最近;与中国青海、蒙古国、美国、突尼斯等地的牛隐孢子虫序列一致性均为99%。 结论 上海牛源隐孢子虫分离株为牛隐孢子虫（C. bovis）。

Identification of Calf-origin Cryptosporidium bovis Shanghai Isolate by Nested PCR

Objective To identify a strain of Cryptosporidium in the feces of naturally infected calf in Shanghai. Methods Stool sample was examined by modified acid-fast staining. The size and morphology of the oocysts were micros-copically determined. Genomic DNA was extracted from the oocysts isolated from feces of a naturally Cryptosporidium-infected calf. According to the sequence of Cryptosporidium 18S rRNA gene, two pairs of primers were designed and syn-thesized. The PCR products was amplified by nested PCR and sequenced in double directions. Homology searches were done over the Web using the program Blast. Phylogenetic tree was constructed with NJ method by MEGA4.0 software. Results Oocysts of the Shanghai isolate were round or elliptical with a size of （5.6±0.49） μm ×（5.2±0.51） μm. Nested PCR resulted in fragments of approximately 810 bp, and the 18S rRNA nucleotide sequence had 100% identity with C. bovis from Brazil （GenBank Accession No: 151935628）. This isolate was clustered in the same clade with C. bovis from Brazil. It showed an identity of 99% with the sequences of C. bovis from Qinghai Ｐrovince of China, Mongolia, USA, and Tunisia. Conclusion The calf-origin Cryptosporidium derived from Shanghai has been identified as C. bovis.