A rapid, accurate, and on-site molecular diagnostic protocol for red imported fire ants (Solenopsis invicta, Si) was developed using loop-mediated isothermal amplification (LAMP) assays. Si11977 (GenBank accession no. MK986826) was confirmed to be a Si-specific gene. Four-primer Si11977-LAMP (4pSi-LAMP) and six-primer Si11977-LAMP (6pSi-LAMP) assays specifically detected Si. The reaction time of 6pSi-LAMP assays was reduced by 5 min compared with 4pSi-LAMP assays. The optimal amount of polymerase and the detection limit for the 6pSi-LAMP assays were 0.1 unit/μL and 5 fg/μL, respectively. In addition, a method for extracting genomic DNA from ant tissues within 2 to 3 min and a protocol for performing on-site LAMP assays using a car heating mug and a LAMP observation box were described. The on-site Si detection protocol used in this study was possible within 30 min from DNA extraction to species identification.
Keywords: loop-mediated isothermal amplification; molecular diagnostics; on-site; species identification.