Anses, Honey bee Pathology Unit, European Reference Laboratory for Bee Health, route des Chappes, CS, Sophia Antipolis, France.
J Econ Entomol. 2021 Apr 13;114(2):520-529. doi: 10.1093/jee/toaa330.
Tropilaelaps mite (Mesostigmata: Laelapidae) is an ectoparasite of bees present, to date, only on the Asian continent. In the context of the threat of Tropilaelaps's introduction into new regions, accurate, rapid, and sensitive detection of the Tropilaelaps spp. is essential. In the present study, we developed a novel molecular method for bee mite's identification, which consists of a new real-time PCR method. A high-resolution melting analysis (HRM) was then performed on the amplified products to differentiate the species. PCR amplification was applied on the cytochrome c oxidase subunit I gene (580 bp). Short fragments from the most variable regions of this gene were identified in silico to amplify and discriminate among the four Tropilaelaps species. Four reference plasmids were synthesized to characterize species by well-distinguished melting curves. The method was then validated in terms of its specificity and sensitivity using a panel of 12 specimens. The results showed that an HRM method can be applied for the intended objective: for rapid and simultaneous identification of Tropilaelaps species. To our knowledge, this study reports the first direct HRM assay developed for the genome of a bee mite, specific for Tropilaelaps species. This COI barcode-HRM technique could be a promising tool for mite species identification.
嗜革螨（真螨目：革螨科）是一种仅存在于亚洲大陆的蜜蜂外寄生虫。在 Tropilaelaps 被引入新地区的威胁背景下，准确、快速和敏感地检测 Tropilaelaps spp. 至关重要。在本研究中，我们开发了一种新的蜜蜂螨鉴定的分子方法，该方法包括一种新的实时 PCR 方法。然后对扩增产物进行高分辨率熔解分析（HRM），以区分物种。PCR 扩增应用于细胞色素 c 氧化酶亚基 I 基因（580 bp）。从该基因最具变异性的区域中鉴定出短片段，以在计算机上进行扩增并区分四种 Tropilaelaps 物种。合成了四个参考质粒，以通过区分明显的熔解曲线来表征物种。然后使用 12 个标本的小组对该方法的特异性和敏感性进行了验证。结果表明，HRM 方法可用于快速且同时鉴定 Tropilaelaps 物种。据我们所知，本研究首次报道了针对蜜蜂螨基因组开发的直接 HRM 检测方法，专门用于鉴定 Tropilaelaps 物种。这种 COI 条码-HRM 技术可能是一种有前途的螨种鉴定工具。
