Abstract
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.
Funder
Research of animal and plant quarantine
National Research Foundation of Korea