A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium)

BACKGROUND Khapra beetle (Trogoderma granarium Everts) is a significant pest of food products around the world, causing great losses of stored grain and produce, with export restrictions imposed on countries with established beetle populations. Khapra beetle is a high-priority exotic invertebrate pest in many countries requiring a rapid quarantine/biosecurity response when incursions occur. To address this, we developed a novel Khapra LAMP (loop-mediated isothermal amplification) assay using a portable real-time fluorometer and an additional 18S ribosomal DNA (18S) insect control LAMP assay for confirmation of the presence of insect DNA. Both LAMP tests can be performed either in a portable real-time fluorometer or using simple, visual colorimetric technique. RESULTS Both the Khapra and 18S LAMP tests amplify positive samples within <= 25 min, with an anneal derivative temperature of 77.7 +/- 0.7 degrees C for Khapra LAMP test and 88.0 +/- 1.0 degrees C for 18S. The new Khapra LAMP assay is sensitive to very low levels of DNA (1.02 x 10(-6) ng mu L-1). Additionally, we developed a gBlock double stranded DNA fragment for use as positive Khapra control with a different anneal derivative of 80 degrees C. Both assays are simple to use in the field and are capable of amplifying DNA from target beetles, even when samples are partially degraded which is typically found during surveillance activities. By screening a broad panel of Dermestidae species we demonstrate that our new assay is species-specific, with no detections of false positives. Also, we evaluated multiple DNA extraction methods, with both QuickExtract and HotSHOT extraction methods proving suitable for in-field use. CONCLUSION The novel Khapra and 18S LAMP assays should improve speed, accuracy and confidence of detection of Khapra beetle at incursion points and aid rapid biosecurity responses in any country affected, especially as the assays described here are portable and easy to implement in the field conditions where resources are limited.

Automated summary

The novel Khapra and 18S LAMP assays are able to amplify target beetle DNA quickly and sensitively, even from partially degraded samples, improving speed, accuracy, and confidence of detection at incursion points. The assays are portable and easy to implement in field conditions with limited resources, aiding rapid biosecurity responses in countries affected by Khapra beetle infestations.