Agriculture Victoria Research, AgriBio - Centre for AgriBioscience, 5 Ring Road, Bundoora, VIC, 3083, Australia.
Commonwealth Scientific and Industrial Research Organisation, Canberra, ACT, 2601, Australia.
Sci Rep. 2023 Jul 24;13(1):11931. doi: 10.1038/s41598-023-38860-w.
Varroa mites are serious pests of European honeybees (Apis mellifera). For detection of Varroa mite, a new molecular LAMP-based assay has been developed, which retains the body of the mite intact for morphological identification. Six novel Varroa LAMP primers were designed from existing DNA sequences of the COI locus to target V. destructor and V. jacobsoni, providing the ability to tell them apart from other non-target beehive associated mite and insect species. This LAMP assay is specific in detecting these Varroa species and has been tested on specimens originating from multiple countries. It produces amplification of V. destructor and V. jacobsoni in 16 ± 3.4 min with an anneal derivative of 78 ± 0.5 °C whilst another Varroa species,V. underwoodi, showed late amplification. A gBlock gene fragment, used here as a positive control has a different anneal derivative of 80 °C. Three non-destructive DNA extraction methods (HotShot, QuickExtract and Xtract) were tested and found to be suitable for use in the field. The LAMP assay was sensitive to very low levels of Varroa DNA, down to 0.24 picogram (~ 1 × 10 copies/µL of Varroa gBlock). This is a new molecular tool for rapid and accurate detection and identification of Varroa mites for pest management, in areas where these mites do not occur.
瓦螨是欧洲蜜蜂（Apis mellifera）的严重害虫。为了检测瓦螨，开发了一种新的基于分子 LAMP 的检测方法，该方法保留了螨体的完整性，以便进行形态学鉴定。从 COI 基因座的现有 DNA 序列设计了 6 个新型瓦螨 LAMP 引物，以靶向 V. destructor 和 V. jacobsoni，能够将它们与其他非目标蜂巢相关螨和昆虫物种区分开来。该 LAMP 检测方法特异性检测这些瓦螨物种，并已在多个国家的标本上进行了测试。它在 16±3.4 分钟内产生 V. destructor 和 V. jacobsoni 的扩增，退火衍生物为 78±0.5°C，而另一种瓦螨 V. underwoodi 则显示晚期扩增。在这里用作阳性对照的 gBlock 基因片段具有不同的退火衍生物 80°C。测试了三种非破坏性 DNA 提取方法（HotShot、QuickExtract 和 Xtract），发现它们适用于野外使用。LAMP 检测方法对非常低水平的瓦螨 DNA 敏感，低至 0.24 皮克（~1×10 个瓦螨 gBlock 拷贝/µL）。这是一种新的分子工具，用于在这些螨类不发生的地区进行快速准确的检测和鉴定，以进行害虫管理。
