|RASZICK, TYLER - Texas A&M University
|GODOY, ALEJANDRA - Animal And Plant Health Inspection Service (APHIS)
|XANTHE, SHIRLEY - Animal And Plant Health Inspection Service (APHIS)
|WRIGHT, KAREN - Texas A&M University
|MARTIN, PAXTON - Texas A&M University
|RUIZ-ARCE, RAUL - Animal And Plant Health Inspection Service (APHIS)
|SWORD, GREGORY - Texas A&M University
Submitted to: Insects
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2023
Publication Date: 10/31/2023
Citation: Raszick, T.J., Perkin, L.C., Godoy, A., Shirley, X.A., Wright, K., Martin, P.T., Suh, C.P.-C., Ruiz-Arce, R., Sword, G.A. 2023. A new qPCR assay for the rapid diagnosis of Anthonomus grandis subspecies. Insects. 14(11):845. https://doi.org/10.3390/insects14110845.
Interpretive Summary: Although the boll weevil has been eradicated from all cotton production areas in the U.S. except from the southernmost production area of Texas, eradication programs continue to operate pheromone traps in eradicated areas to monitor for boll weevil re-infestations. These traps frequently capture other weevil species such as the thurberia weevil, which is difficult to differentiate from the boll weevil. Because misidentification of weevil species could result in unnecessary mitigation actions and costs or, conversely, preclude or delay necessary remedial actions, rapid and accurate identification of captured weevils is critically important to eradication programs. This work developed a laboratory diagnostic assay that takes less than one day to complete and is 98% accurate in distinguishing the boll weevil and thurberia weevil. This tool has recently been adopted by USDA-APHIS and is currently being used to identify suspect boll weevils captured in traps as well as those intercepted at ports of entry.
Technical Abstract: Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication programs. Current diagnostic methods have limitations in terms of ambiguity and time to diagnosis (up to 7 days). Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate diagnosis of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay could be expected to amplify; (2) a non-target specificity test to ensure no amplification in non-target weevils; and (3) an accuracy test. These experiments were carried out in parallel at three different facilities to confirm the robustness of the assay. We used boll weevil DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity, specificity, and accuracy in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday. The deployment of this assay as a diagnostic tool will greatly benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for diagnosing unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts.