Research Platform of Agricultural Biotechnology, S. Seifullin Kazakh Agrotechnical University, Nur-Sultan, Kazakhstan.
2Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Georgian Med News. 2021 Jan(310):176-182.
Opisthorchis felineus and Metorchis bilis are two common small worms that parasitize in the gallbladder and bile ducts of the liver of humans and carnivores. These parasites have a severe impact on health and are considered pathogens of serious diseases worldwide, such as cholangiocarcinoma. However, there are still no commercially available molecular diagnostic kits capable of simultaneously detecting these parasites in humans. Therefore, the study aimed to develop a multiplex PCR analysis that will differentially determine these two opisthorchiasis infections in one reaction. Two specific primer pairs for a multiplex polymerase chain reaction (PCR) were designed based on corresponding mitochondrial genome sequences. The multiplex assay detection limit was assessed by serial dilutions of the genomic DNAs of trematode worms examined. Naturally, infected samples of human bile and feces were tested using the developed assay. A multiplex PCR assay was developed based on mitochondrial DNA that accurately and simultaneously identifies two trematode species in one reaction using specific fragment sizes of 307 and 252 bp for O. felineus and M. bilis, respectively. The optimal reaction conditions, specificity, and sensitivity of the multiplex PCR assay were investigated. The lowest DNA concentration detected was 100 pg for M. bilis and O. felineus in a 25μl reaction system. This study provides an efficient tool for the simultaneous detection of O. felineus and M. bilis. The proposed multiplex PCR assay will be potentially useful in epidemiological studies, diagnosis, and treatment of this mixed opisthorchiasis infection.
肝片形吸虫和双腔吸虫是两种常见的寄生在人类和肉食动物的胆囊和胆管中的小型蠕虫。这些寄生虫对健康有严重影响，被认为是全世界严重疾病的病原体，如胆管癌。然而，目前仍然没有商业上可用的分子诊断试剂盒能够同时检测人类体内的这些寄生虫。因此，本研究旨在开发一种多重 PCR 分析方法，能够在一个反应中区分这两种肝片形吸虫感染。根据相应的线粒体基因组序列，设计了两对用于多重聚合酶链反应（PCR）的特异性引物。通过对检查的吸虫基因组 DNA 的连续稀释来评估多重检测的检测限。使用开发的检测方法对自然感染的人胆汁和粪便样本进行了测试。基于线粒体 DNA 开发了一种多重 PCR 检测方法，该方法使用分别为 307 和 252 bp 的 O. felineus 和 M. bilis 的特异性片段大小，在一个反应中准确且同时鉴定两种吸虫。研究了多重 PCR 检测方法的最佳反应条件、特异性和敏感性。在 25μl 反应体系中，检测到的最低 DNA 浓度为 100 pg。本研究为同时检测 O. felineus 和 M. bilis 提供了一种有效的工具。该多重 PCR 检测方法有望在这种混合性肝片形吸虫感染的流行病学研究、诊断和治疗中得到应用。
