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

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.