虫卵ITS-2序列分析鉴别华支睾吸虫和扇棘单睾吸虫的研究

目的建立以分子生物学技术为基础的华支睾吸虫与扇棘单睾吸虫的ITS-2序列分析鉴别方法。方法在华支睾吸虫流行区采集华支睾吸虫和扇棘单睾吸虫成虫,从成虫虫体分离并收集虫卵。以蛔虫、鞭虫、钩虫、蛲虫做对照虫种,按不同虫种和虫卵数分成5个实验组,各组分别提取DNA,并扩增ITS-2序列,对扩增产物进行测序并作同源性分析以确保为目标片段,根据PCR扩增产物电泳获得的条带判定虫种。结果以华支睾吸虫和扇棘单睾吸虫虫卵DNA为模板的PCR产物电泳图谱分别在400bp、500bp左右各显示一条明显条带;在以两种虫卵DNA混合液为模板的PCR产物中,电泳图谱在400bp左右和500bp左右各显示明显条带。将测序得到的两种核苷酸序列进行Blast比对后,显示与GenBank中相应的华支睾吸虫和扇棘单睾吸虫序列高度同源。混入四种肠道线虫虫卵DNA的PCR产物中,除目的条带外,无其他条带。结论该方法可以对华支睾吸虫及扇棘单睾吸虫虫卵进行鉴别,且结果不受常见四种肠道线虫虫卵干扰,敏感性和特异性较高,可作为两虫种的鉴别检测方法。

Study on identification of Clonorchis sinensis and Haplorchis taichui by analysis of Egg ITS-2 sequence

Objective To establish an identification method basis on molecular biology techniques for Clonorchis sinensis and Haplorchis taichui. Methods Adult flukes of C. sinensis and H. taichui were collected from Clonorchiasis endemic areas and the eggs were separated from the worms under stereomicroscope. Roundworm, Whipworm, Hookworm and Pinworm were served as control species, all kinds of eggs were divided into five experimental groups according to the species and the number of eggs. DNA from each group was extracted and amplified the sequence of ITS-2, and amplified products were sequenced and homology analyzed ensure that the products were the target fragments. The species of insect were determined according to the electrophoresis of PCR products. Results The electrophoresis of PCR products of C. sinensis and H. taichui display a distinct band at around 400bp, 500bp, respectively; electropherogram in PCR products of mixture DNA of two species eggs showed two obvious bands at around 400bp and 500bp. The two nucleotide sequencing which had sequenced were compared to Blast, show that both of the sequences are highly homologous with C. sinensis and H. taichi in GenBank , respectively. The PCR products with four kinds of Heligmosomoides polygyrus eggs have no other bands except the objective strips. Conclusion This method can be used to identify C. sinensis and H. taichui, and the results are not affected by four kinds of Heligmosomoides polygyrus eggs , is an identification method for Clonorchis sinensis and Haplorchis taichui with high sensitivity and specificity.