Matsuda Tomoko, Sakamoto Hironori, Kayukawa Takumi, Kitashima Yasuki, Kozaki Toshinori, Gotoh Tetsuo
Research and Development Department, Nihon BioData Corporation, Kawasaki, Kanagawa, Japan.
Biodiversity Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan.
PLoS One. 2025 Jun 9;20(6):e0321199. doi: 10.1371/journal.pone.0321199. eCollection 2025.
Using PCR to distinguish closely related species can be difficult because they may have very similar genomes. Advances in bioinformatics make it possible to design PCR primers that are species-specific. In this study, we developed a bioinformatics method for extracting species-specific primer candidate sequences (i.e., unpaired primers that were specific to a single species) from RNA-Seq data sets of 19 species of spider mites (Acari, Tetranychidae). Using k-mer counting, we obtained between 257 and 48,621 species-specific unpaired primer candidates for the 19 species. We then manually obtained a second primer that was also species-specific. The primer pairs were then confirmed to work in the target species and not to work in the non-target species. Finally, species-specific primer pairs were obtained for 17 of the 19 species tested. Such species-specific primers may be used for practical species discrimination by optimizing multiplex PCR. Our primer design method is expected to be applicable to other taxa.
使用PCR区分亲缘关系密切的物种可能会很困难，因为它们的基因组可能非常相似。生物信息学的进展使得设计物种特异性的PCR引物成为可能。在本研究中，我们开发了一种生物信息学方法，用于从19种植食性螨类（蜱螨亚纲，叶螨科）的RNA测序数据集中提取物种特异性引物候选序列（即对单个物种具有特异性的不成对引物）。通过k-mer计数，我们为这19个物种获得了257至48,621个物种特异性不成对引物候选序列。然后我们手动获得了另一个同样具有物种特异性的引物。随后证实这些引物对在目标物种中有效，而在非目标物种中无效。最后，在所测试的19个物种中，为其中17个物种获得了物种特异性引物对。通过优化多重PCR，此类物种特异性引物可用于实际的物种鉴别。我们的引物设计方法有望适用于其他分类群。
