Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand.
Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand; Center of Excellence in Animal, Plant and Parasitic Biotechnology, Srinakharinwirot University, Bangkok 10110, Thailand.
Acta Trop. 2021 Feb;214:105795. doi: 10.1016/j.actatropica.2020.105795. Epub 2020 Dec 10.
This study aimed to investigate the prevalence of cercarial infections in freshwater snails collected from the Chai Nat province of Central Thailand. Moreover, we aimed to develop a duplex PCR technique, using the ITS2 and candidate MT-ND1 gene, to determine the dissemination of H. taichui and H. pumilio, respectively. Six types of cercariae were discovered with an overall prevalence of 4.71% (59/1,252). The parapleurolophocercous cercariae were demonstrated to be the dominant type, infecting only Melanoides tuberculata snails. The duplex PCR was optimized for specific amplification of ITS2 for H. taichui (115 bp) and MT-ND1 for H. pumilio (335 bp). Both specific primers confirmed the specificity of the duplex PCR reaction, with no cross-reactivity with other heterophyids or related species. In addition, this duplex PCR could be used to detect co-infection at a concentration of 3.0 ng/µL. For the molecular identification, 9 of 22 parapleurolophocercous cercaria specimens in Chai Nat province generated the specific DNA fragment of H. pumilio. These results proved that the MT-ND1 gene is a species-specific method for heterophyid detection and provides a rapid method for identification based on larval and adult stages of H. taichui and H. pumilio in their intermediate and/or definitive hosts in the infected area.
本研究旨在调查泰国中北部猜纳府采集的淡水螺中尾蚴感染的流行情况。此外，我们旨在开发一种双重 PCR 技术，使用 ITS2 和候选 MT-ND1 基因，分别确定中华肝吸虫和后睾吸虫的传播。发现了 6 种尾蚴，总感染率为 4.71%（59/1,252）。证明具pleurolophocercous 的尾蚴是优势类型，仅感染 Melanoides tuberculata 螺。双重 PCR 针对 ITS2 特异性扩增进行了优化，用于检测中华肝吸虫（115 bp），针对 MT-ND1 特异性扩增用于检测后睾吸虫（335 bp）。两种特异性引物均证实了双重 PCR 反应的特异性，与其他异形吸虫或相关物种无交叉反应。此外，该双重 PCR 可用于检测 3.0ng/µL 浓度下的混合感染。对于分子鉴定，在猜纳府的 22 个具 pleurolophocercous 的尾蚴标本中，有 9 个产生了后睾吸虫的特异性 DNA 片段。这些结果证明 MT-ND1 基因是一种用于异形吸虫检测的种特异性方法，并为在感染地区的中间宿主和/或终末宿主中鉴定中华肝吸虫和后睾吸虫的幼虫和成虫阶段提供了一种快速方法。
