Lin R Q, Dong S J, Nie K, Wang C R, Song H Q, Li A X, Huang W Y, Zhu X Q
College of Veterinary Medicine, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou, Guangdong Province 510642, People's Republic of China.
Parasitol Res. 2007 Aug;101(3):813-7. doi: 10.1007/s00436-007-0512-0. Epub 2007 Mar 15.
In the present study, a polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) approach combined with DNA sequencing was used to characterise samples of Fasciola spp. from different host species and geographical locations in mainland China. The first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by PCR from individual Fasciola and analysed by SSCP. SSCP analyses displayed three different banding profiles that allowed the identification of all Fasciola samples examined into three groups: Fasciola hepatica, F. gigantica and the "intermediate" Fasciola. Then, the ITS-1 rDNA was sequenced from representative Fasciola samples, and analysis of the complete ITS-1 sequences supported the identification of all Fasciola samples by SSCP approach. The length of the ITS-1 sequences was 422 bp for all Fasciola samples sequenced. Although there was no variation in length or composition of the ITS-1 sequences among multiple specimens within each of the taxa, F. hepatica and F. gigantica differed by 1.2% in their ITS-1 sequences, whereas the "intermediate" Fasciola was unique, in which two different ITS-1 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is identical to that of F. gigantica. This study demonstrated that PCR-SSCP analysis of the ITS-1 rDNA followed by selective sequencing provides a reliable approach for the accurate identification of Fasciola spp., and also supports the existence of the "intermediate" Fasciola between F. hepatica and F. gigantica in mainland China.
在本研究中，采用聚合酶链反应-单链构象多态性（PCR-SSCP）方法并结合DNA测序，对来自中国大陆不同宿主物种和地理位置的片形吸虫样本进行特征分析。通过PCR从单个片形吸虫中扩增核糖体DNA（rDNA）的第一内部转录间隔区（ITS-1），并进行SSCP分析。SSCP分析显示出三种不同的条带图谱，可将所有检测的片形吸虫样本分为三组：肝片形吸虫、巨片形吸虫和“中间型”片形吸虫。然后，对代表性的片形吸虫样本进行ITS-1 rDNA测序，对完整ITS-1序列的分析支持通过SSCP方法对所有片形吸虫样本进行鉴定。所有测序的片形吸虫样本的ITS-1序列长度均为422 bp。虽然每个分类群内的多个样本之间ITS-1序列的长度或组成没有差异，但肝片形吸虫和巨片形吸虫的ITS-1序列差异为1.2%，而“中间型”片形吸虫则很独特，在单个片形吸虫虫体内的rDNA阵列中存在两种不同的ITS-1序列。其中一种序列与肝片形吸虫的相同，另一种与巨片形吸虫的相同。本研究表明，对ITS-1 rDNA进行PCR-SSCP分析并随后进行选择性测序，为准确鉴定片形吸虫提供了一种可靠的方法，也支持中国大陆存在介于肝片形吸虫和巨片形吸虫之间的“中间型”片形吸虫。
