人体混合感染巨片吸虫和巨片吸虫与肝片吸虫的中间型

Faschiola gigantica and Intermediate Form between Faschiola gigantica and Faschola hepatica from a Mixed Infected Body

目的通过线粒体nad1和细胞核ITS1序列,对从云南省玉溪地区一女性片形吸虫病患者体内取出的2条片形吸虫成虫进行虫种鉴定,获知寄生于人体的片形吸虫种类.方法片形吸虫标本保存于75%乙醇溶液,提取全基因组DNA,PCR扩增线粒体基因NADH脱氢酶亚基1(nad1)和细胞核r DNA的第一内转录间隔区(ITS1)序列,正反引物双向测序.所测序列与Gen Bank中Fasciola属相关同源序列组成DNA数据集,进行ITS1单倍型分析和构建nad1的NJ系统发育树.结果测得片形吸虫标本的ITS1和nad1序列长度分别是422 bp和542bp.2条片形吸虫标本的nad1序列完全一致,属于F.gigantica(Fg型).但是ITS1单倍型不一致,标本YXGD01在该基因中属于F.gigantica(Fg型),标本YXGD02属于F.hepatica(Fh型).基于nad1 DNA数据集构建的NJ系统发育树,明显分为F.gigantica和F.hepatica 2个类群.结论基于线粒体nad1和细胞核ITS1序列,从云南省玉溪地区一片形吸虫病患者体内取出的2条片形吸虫属于不同类型,标本YXGD01鉴定为F.gigantica,标本YXGD02可能是F.gigantica与F.hepatica杂交生成的中间型个体,但需要更多的实验证据.

Objective To identify two Fasciola spp., Sample YXGD01 and YXGD02, collected from one female fasciolosis patient living in Yuxin, Yunnan Province in China using DNA marker nad1 and ITS1, and to investigate causative agents of Zoonoses fasciolosis in humans. Me thods Two Fasciola fluckes collected from one female patient were preserved in 75% ethyl alcohol. The mitochondrial NADH dehydrogenase subunit 1（nad1）gene and first internal transcribed spacer（ITS1） of nuclear ribosomal DNA（r DNA） of individual trematodes were amplified by polymerase chain reaction（PCR）. Then the PCR product were sequenced in forward and reverse direction. The sequences analyzed by comparing with related sequences of Fasciola available in Genbank database.Using the DNA database, to identify Fasciola spp. by haplotype analysis for ITS1 and construct phylogenetic tree by NJ method for nad1. Re s ults 422 bp ITS1 sequences and 542 bp nad1 sequences were obtained respectively for thetwo Fasciola fluckes sequenced in our study. Using haplotype analysis method, both Fasciola fluckes with the identical nad1 haplotype similar toF. gigiantica（Fg） haplogoup, showed Fg type to sample YXGD01 and Fh type to sample YXGD02 in ITS1 respectively. The NJ phylogenetic tree inferred from nad1 displays distinct F. gigantica goup and F. hepatica goup. Conclusion This study identified the two Fasciola spp. from one fasciolosis patient living in Yuxi, Yunnan Province usingmitochondria nad1 and nuclear ITS1. The sample YXGD01 should belong toF.gigantica in molecular characteristics. The confusion result between ITS1 and nad1 haplotype of sample YXGD02 infers that it may belong to one special type of intermediate form Fasciola between F. hepatica and F. gigantica, but more experimental proof is needed.