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目的对采自云南、贵州两省的38条人体带绦虫进行分子鉴别,了解当地猪带绦虫、牛带绦虫和亚洲带绦虫的分布状况。方法对云南大理和贵州都匀及从江地区有排节片史的病人以槟榔-南瓜子法驱虫,虫体经形态学初步鉴别后,以组织DNA提取试剂盒提取虫体基因组DNA,分别采用亚洲带绦虫、牛带绦虫和猪带绦虫线粒体细胞色素C氧化酶亚单位1基因(cox1)片段的特异性引物对各DNA样品进行常规PCR扩增和琼脂糖凝胶电泳检测;PCR阴性样品采用带绦虫cox1片段PCR通用引物进行扩增,并选择1份亚洲带绦虫特异性PCR阳性的样品作为对照。从3种带绦虫PCR阳性的扩增产物中各选择1份进行核酸序列测定,并用NCBIBlast将各核酸序列与GenBank数据库中带绦虫的cox1进行比对分析。结果采自大理的4条带绦虫(DL1～4)的猪带绦虫cox1片段PCR为阳性,扩增产物约980bp;其余25条大理带绦虫(DL5～29)和5条都匀带绦虫(DY1～5)的亚洲带绦虫cox1片段PCR为阳性,扩增产物约260bp;各样品的牛带绦虫cox1片段PCR均为阴性。DL2PCR产物的核酸序列与GenBank中猪带绦虫cox1的同源性为99%～100%,DY1PCR产物的核酸序列与亚洲带绦虫cox1的同源性为100%。特异性PCR均阴性的4条从江带绦虫(CJ1～4)和亚洲带绦虫特异性PCR阳性的DL7以带绦虫cox1片段通用引物的PCR扩增出约1kb的产物。测序表明,CJ1片段的核酸序列与牛带绦虫cox1的同源性为99%,DL7与亚洲带绦虫cox1的同源性为99%～100%。结论cox1片段PCR扩增和核酸测序分析表明,采集的云南大理人体带绦虫为猪带绦虫和亚洲带绦虫,贵州都匀和从江人体带绦虫分别为亚洲带绦虫和牛带绦虫。
Objective To identify 38 Taenia solium strains collected from Yunnan and Guizhou provinces to understand the distribution of Taenia solium, Taenia saginata and Taenia saginata. Methods The genital DNA of parasites was extracted from the patients with the history of ganglios in Dali, Yunnan Province and Congjiang district of Guizhou Province by the method of betel nut - The DNA samples were subjected to conventional PCR and agarose gel electrophoresis using specific primers for the Taenia saginata, Taenia saginata and Taenia capsici mitochondrial cytochrome C oxidase subunit 1 genes (cox1) fragments. PCR-negative samples A TaqMan-based cox1 fragment PCR universal primer was used for amplification, and a sample of Asian Taenia saginata specific PCR was selected as a control. One of the three TaqMan-PCR-positive amplification products was selected for nucleic acid sequence analysis, and each nucleic acid sequence was compared with the cox1 of the tapeworms in the GenBank database by NCBI Res. Results PCR amplification of cox1 of 4 Taenia solium (DL1 ~ 4) from Dali of Dali was positive and the amplified product was about 980bp. The remaining 25 Taenia saginata (DL5 ~ 29) and 5 Taenia solium ~ 5) of the tapeworm Asia cox1 PCR fragment was positive, the amplification product of about 260bp; each sample Taenia saginata cox1 fragment PCR were negative. The nucleotide sequence of DL2PCR gene was 99% -100% homologous to Taenia solium cox1 in GenBank, and the homology of the DY1 PCR product with that of Taenia saginata cox1 was 100%. Specific PCR products were amplified by PCR from four Taenia solium (CJ1-4) and Taenia saginata specific PCR-positive DL7 Taenia solium cox1 fragment universal primers. Sequencing showed that the nucleotide sequence of CJ1 fragment was 99% homologous to Taenia saginata cox1, and the homology between DL7 and Taenia saginata cox1 was 99% -100%. Conclusion The cox1 fragment was amplified by PCR and sequenced. The results showed that Taenia solium and Taenia saginata collected from Dali, Yunnan Province, Taenia saginata and Taenia saginata from Dujiang, Guizhou were Taenia asiatis and Taenia saginata, respectively.