Loop-mediated isothermal amplification (LAMP) assays for detection of the New Guinea fruit fly Bactroceratrivialis (Drew) (Diptera: Tephritidae)

The cue-lure-responding New Guinea fruit fly, Bactroceratrivialis, 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., Bactrocerabreviaculeus 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 degrees C and 83.3 +/- 1.3 degrees C, respectively, detecting down to 1 x 10(1) copies/mu L and 1 x 10(3) copies/mu L, 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 degrees C and 85.8 +/- 0.2 degrees C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped B.trivialis, with BtrivEIF3L used for secondary confirmation when required.

Automated summary

The New Guinea fruit fly, Bactroceratrivialis, poses a biosecurity risk to neighboring countries, and a quicker molecular diagnostic assay was developed to detect this species within 25 minutes.