Department of Agriculture and Fisheries, Biosecurity Queensland, Brisbane, QLD, Australia.
New South Wales Department of Primary Industries, Orange, NSW, Australia.
Sci Rep. 2022 Jul 23;12(1):12602. doi: 10.1038/s41598-022-16901-0.
The cue-lure-responding New Guinea fruit fly, Bactrocera trivialis, poses a biosecurity risk to neighbouring countries, e.g., Australia. In trapping programs, lure caught flies are usually morphologically discriminated from non-target species; however, DNA barcoding can be used to confirm similar species where morphology is inconclusive, e.g., Bactrocera breviaculeus and B. rufofuscula. This can take days-and a laboratory-to resolve. A quicker, simpler, molecular diagnostic assay would facilitate a more rapid detection and potential incursion response. We developed LAMP assays targeting cytochrome c oxidase subunit I (COI) and Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L); both assays detected B. trivialis within 25 min. The BtrivCOI and BtrivEIF3L assay anneal derivatives were 82.7 ± 0.8 °C and 83.3 ± 1.3 °C, respectively, detecting down to 1 × 10 copies/µL and 1 × 10 copies/µL, respectively. Each assay amplified some non-targets from our test panel; however notably, BtrivCOI eliminated all morphologically similar non-targets, and combined, the assays eliminated all non-targets. Double-stranded DNA gBlocks were developed as positive controls; anneal derivatives for the COI and EIF3L gBlocks were 84.1 ± 0.7 °C and 85.8 ± 0.2 °C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped B. trivialis, with BtrivEIF3L used for secondary confirmation when required.
新几内亚实蝇，Bactrocera trivialis，是一种对邻国（例如澳大利亚）具有生物安全风险的 cue-lure-responding 水果蝇。在诱捕计划中，诱饵捕获的苍蝇通常通过形态学区分非目标物种；然而，DNA 条形码可用于确认形态学不确定的相似物种，例如 Bactrocera breviaculeus 和 B. rufofuscula。这可能需要几天时间和一个实验室来解决。更快、更简单的分子诊断检测方法将有助于更快速地检测和潜在的入侵响应。我们开发了针对细胞色素 c 氧化酶亚基 I（COI）和真核翻译起始因子 3 亚基 L（EIF3L）的 LAMP 检测；这两种检测方法都在 25 分钟内检测到了 B. trivialis。BtrivCOI 和 BtrivEIF3L 检测的退火衍生物分别为 82.7±0.8°C 和 83.3±1.3°C，分别检测到低至 1×10 拷贝/µL 和 1×10 拷贝/µL。每个检测方法都从我们的测试面板中扩增了一些非目标物；然而，值得注意的是，BtrivCOI 消除了所有形态相似的非目标物，并且联合使用时，这两种检测方法消除了所有非目标物。双链 DNA gBlocks 被开发为阳性对照；COI 和 EIF3L gBlocks 的退火衍生物分别为 84.1±0.7°C 和 85.8±0.2°C。我们建议使用 BtrivCOI 检测来确认可疑的 cue-lure 诱捕到的 B. trivialis，当需要时，使用 BtrivEIF3L 进行二次确认。
