摘要
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 molecularly differentiate 38 human taeniid cestodes and to ascertain the local prevalence of Taenia solium,Taenia asiatica,and Taenia saginata.Methods Taenia tapeworms were collected from patients given areca nuts and pumpkin seeds in Dali,Yunnan and Duyun,Guizhou.After preliminary morphological identification,DNA of the 38 isolates was extracted with a tissue DNA extraction kit,and mitochondrial cytochrome C oxidase subunit 1 gene（cox1）fragments of all DNA samples were amplified by PCR with primers specific for T.solium,T.asiatica,and T.saginata,respectively.Isolates which were negative in PCR with all specific primers were then amplified by general primers for the cox1 gene of Taenia spp and a sample which was positive for T.asiatica was used as a control.In all positive PCR products,a sample of each Taenia spp was then sequenced and the sequences were analyzed using BLAST on the NCBI website.Results PCR specific for T.solium was positive for 4 isolates（DL1-4）collected from Dali,and the products were about 980 bp.The other 25 isolates from Dali（DL5-29）and 5 isolates from Duyun（DY1-5）were positive in PCR specific for T.asiatica,and the products were about 260 bp.All samples were negative in PCR specific for T.saginata.The sequences of DL2 had 99%100% homology with those of T.solium cox1 in GenBank,and the sequences of DY1 had 100% homology with those of T.asiatica.Fragments of roughly 1 kb were amplified from the 4 Congjiang isolates,which were negative in specific PCR amplifications,and DL7 which was positive in PCR specific for T.asiatica.The sequences of CJ1 cox1 fragments had 99% homology with T.saginata and the sequences of DL7 had 99%-100% homology with T.asiatica.Conclusion PCR and sequencing of cox1 fragments indicated that taeniid cestodes from Dali,Yunnan were T.solium and T.asiatica,while those from Duyun and Congjiang,Guizhou were T.asiatica and T.saginata,respectively.
出处
《中国病原生物学杂志》
CSCD
2010年第4期263-265,共3页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.30660168)
关键词
亚洲带绦虫
猪带绦虫
牛带绦虫
分子鉴别
Taenia asiatica
Taenia solium
Taenia saginata
molecular differentiation
