Department of Biology, Chiang Mai University, Chiang Mai 50200, Thailand.
Exp Parasitol. 2012 Oct;132(2):237-42. doi: 10.1016/j.exppara.2012.07.007. Epub 2012 Aug 1.
Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5'-GGCCAACGCAATCGTCATCC-3'and Hapt_R1 5'-CTCTCGACCTCCTCTAGAAT-3' which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5'-AATCGGGCTGCATATTGACCGAT-3' and OpV-1R: 5'-CGGTGTTGCTTATTTTGCAGACAA-3' which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68°C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl(2)). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand. The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs.
本研究利用 HAT-RAPD-PCR 方法，针对华支睾吸虫和肝片形吸虫设计了特异性引物。我们对 14 种随机引物（Operon Technologies）进行了筛选，以产生多态性 DNA 图谱。结果表明，OPA-04 引物产生的 319 bp 片段可能是华支睾吸虫的特异性片段，而 OPP-11 引物产生的 256 bp 片段可能是肝片形吸虫的特异性片段。基于每条序列数据，我们设计了两对特异性引物，其序列如下：肝片形吸虫，Hapt_F：5'-GGCCAACGCAATCGTCATCC-3'和 Hapt_R：5'-CTCTCGACCTCCTCTAGAAT-3'，扩增出 170 bp 的 PCR 产物。对华支睾吸虫，OpV-1F：5'-AATCGGGCTGCATATTGACCGAT-3'和 OpV-1R：5'-CGGTGTTGCTTATTTTGCAGACAA-3'，扩增出 319 bp 的 PCR 产物。这些特异性引物经过功效和特异性检测，可用于所有寄生虫 DNA 样本。结果表明，在肝片形吸虫和华支睾吸虫中，170 bp 和 319 bp 的特异性 PCR 产物可作为阳性结果，与所有检测的寄生虫均无交叉反应。PCR 条件推荐为 68°C 退火温度和 0.5 mM 氯化镁（MgCl(2)）。此外，本研究中开发的特异性引物可有效地检测鱼类和中间宿主蜗牛中两种寄生虫的存在，因为在泰国北部很少发现华支睾吸虫，因此在人工添加其 DNA 的情况下，也能检测到。本研究成功开发的肝片形吸虫和华支睾吸虫特异性引物可用于流行病学监测、预防管理和控制项目。
