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A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium).
Pest Manag Sci. 2021 Dec; 77(12):5509-5521.PM

Abstract

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 °C for Khapra LAMP test and 88.0 ± 1.0 °C for 18S. The new Khapra LAMP assay is sensitive to very low levels of DNA (1.02 × 10-6 ng μL-1). Additionally, we developed a gBlock double stranded DNA fragment for use as positive Khapra control with a different anneal derivative of 80 °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. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Links

Authors+Show Affiliations

Agriculture Victoria, AgriBio, Bundoora, VIC, Australia.

Agriculture Victoria, AgriBio, Bundoora, VIC, Australia.

Agriculture Victoria, AgriBio, Bundoora, VIC, Australia.

Department of Agriculture, Water and the Environment, Science and Surveillance Group, Canberra, ACT, Australia.

Agriculture Victoria, AgriBio, Bundoora, VIC, Australia.

Agriculture Victoria, AgriBio, Bundoora, VIC, Australia.

MeSH

AnimalsColeopteraInsect ControlMolecular Diagnostic TechniquesNucleic Acid Amplification Techniques

Pub Type(s)

Journal Article

Language

eng

PubMed ID

34363302

Citation

Rako, Lea, et al. "A LAMP (loop-mediated Isothermal Amplification) Test for Rapid Identification of Khapra Beetle (Trogoderma Granarium)." Pest Management Science, vol. 77, no. 12, 2021, pp. 5509-5521.

Rako L, Agarwal A, Semeraro L, et al. A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium). Pest Manag Sci. 2021;77(12):5509-5521.

Rako, L., Agarwal, A., Semeraro, L., Broadley, A., Rodoni, B. C., & Blacket, M. J. (2021). A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium). Pest Management Science, 77(12), 5509-5521. https://doi.org/10.1002/ps.6591

Rako L, et al. A LAMP (loop-mediated Isothermal Amplification) Test for Rapid Identification of Khapra Beetle (Trogoderma Granarium). Pest Manag Sci. 2021;77(12):5509-5521. PubMed PMID: 34363302.

* Article titles in AMA citation format should be in sentence-case

TY - JOUR T1 - A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium). AU - Rako,Lea, AU - Agarwal,Arati, AU - Semeraro,Linda, AU - Broadley,Adam, AU - Rodoni,Brendan C, AU - Blacket,Mark J, Y1 - 2021/08/18/ PY - 2021/07/20/received PY - 2021/08/06/accepted PY - 2021/8/8/pubmed PY - 2021/11/12/medline PY - 2021/8/7/entrez KW - 18S LAMP control assay KW - Dermestidae KW - Khapra beetle LAMP assay KW - Khapra beetle identification KW - Trogoderma granarium KW - field diagnostics KW - loop-mediated isothermal amplification (LAMP) SP - 5509 EP - 5521 JF - Pest management science JO - Pest Manag Sci VL - 77 IS - 12 N2 - 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 °C for Khapra LAMP test and 88.0 ± 1.0 °C for 18S. The new Khapra LAMP assay is sensitive to very low levels of DNA (1.02 × 10-6 ng μL-1). Additionally, we developed a gBlock double stranded DNA fragment for use as positive Khapra control with a different anneal derivative of 80 °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. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. SN - 1526-4998 UR - https://www.unboundmedicine.com/medline/citation/34363302/A_LAMP__loop_mediated_isothermal_amplification__test_for_rapid_identification_of_Khapra_beetle__Trogoderma_granarium__ DB - PRIME DP - Unbound Medicine ER -