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Starkie, Melissa L.; Fowler, Elizabeth V.; Zhu, Xiaocheng; Agarwal, Arati; Rako, Lea; Schneider, Isarena C.; Schutze, Mark K.; Royer, Jane E.; Gopurenko, David; Gillespie, Peter; Blacket, Mark J.
Scientific reports, 07/2022, Letnik: 12, Številka: 1Journal Article
Abstract 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 1 copies/µL and 1 × 10 3 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.
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