Probe-based quantitative PCR and RPA-Cas12a molecular diagnostics for detection of the tomato pest Phthorimaea absoluta (Lepidoptera: Gelechiidae)

Kyle M Lewald; Wenqi Song; Daniel Eweis-LaBolle; Cindy Truong; Kristine E Godfrey; Joanna C Chiu

doi:10.1093/jee/toad079

Probe-based quantitative PCR and RPA-Cas12a molecular diagnostics for detection of the tomato pest Phthorimaea absoluta (Lepidoptera: Gelechiidae)

J Econ Entomol. 2023 Jun 13;116(3):993-1001. doi: 10.1093/jee/toad079.

Authors

Kyle M Lewald¹, Wenqi Song¹, Daniel Eweis-LaBolle¹, Cindy Truong¹, Kristine E Godfrey², Joanna C Chiu¹

Affiliations

¹ Department of Entomology and Nematology, College of Agricultural and Environmental Sciences, University of California Davis, One Shields Ave, Davis, CA 95616, USA.
² Contained Research Facility, University of California, Davis, 555 Hopkins Rd, Davis, CA 95616, USA.

PMID: 37177893
DOI: 10.1093/jee/toad079

Abstract

The tomato pest Phthorimaea absoluta Meyrick is highly invasive but has not yet invaded North America. However, several morphologically similar species are already present, making detection of P. absoluta presence and invasion challenging. We designed a quantitative PCR molecular diagnostic to differentiate P. absoluta, P. operculella (Zeller), or Keiferia lycopersicella (Walsingham) (Lepidoptera: Gelechiidae) DNA. Additionally, we developed an RPA-Cas12a molecular diagnostic that allows for the isothermal detection of P. absoluta DNA, eliminating the need for a thermocycler. The results of the RPA-Cas12a diagnostic can be visualized simply using a UV light source and cell phone camera. We expect these diagnostics to improve quarantine and prevention measures against this serious agricultural threat.

Keywords: Tuta absoluta; CRISPR; invasion biology; quarantine; tomato leafminer.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
CRISPR-Cas Systems
Lepidoptera*
Moths* / genetics
Pathology, Molecular
Polymerase Chain Reaction
Solanum lycopersicum* / genetics