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25 June 2021 Development of Three Molecular Diagnostic Tools for the Identification of the False Codling Moth (Lepidoptera: Tortricidae)
D. Rizzo, D. Da Lio, L. Bartolini, G. Cappellini, T. Bruscoli, C. Salemi, A. Aronadio, D. Del Nista, F. Pennacchio, N. Boersma, E. Rossi, P. Sacchetti
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Abstract

Three molecular protocols using qPCR TaqMan probe, SYBR Green, and loop-mediated isothermal amplification (LAMP) methods were set up for the identification of larvae and adults of an African invasive moth, Thaumatotibia leucotreta (Meyrick, 1913) (Lepidoptera: Tortricidae). The DNA extracts from larval and adult samples of T. leucotreta were perfectly amplified with an average Ct value of 19.47 ± 2.63. All assays were demonstrated to be inclusive for T. leucotreta and exclusive for the nontarget species tested; the absence of false positives for nontarget species showed a 100% of diagnostic specificity and diagnostic sensitivity for all assays. With the SYBR Green protocol, the Cq values were only considered for values less than 22 (cutoff value) to prevent false-positive results caused by the late amplification of nonspecific amplicons.The limit of detection (LoD) for the qPCR probe protocol was equal to 0.02 pg/µl while a value equal to 0.128 pg/µl for the qPCR SYBR Green assay and LAMP method were established, respectively.The intrarun variabilities of reproducibility and repeatability in all the assays evaluated as CV%, ranged between 0.21 and 6.14, and between 0.33 and 9.52, respectively; the LAMP values were slightly higher than other assays, indicating a very low interrun variability. In order for an operator to choose the most desirable method, several parameters were considered and discussed. For future development of these assays, it is possible to hypothesize the setup of a diagnostic kit including all the three methods combined, to empower the test reliability and robustness.

© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
D. Rizzo, D. Da Lio, L. Bartolini, G. Cappellini, T. Bruscoli, C. Salemi, A. Aronadio, D. Del Nista, F. Pennacchio, N. Boersma, E. Rossi, and P. Sacchetti "Development of Three Molecular Diagnostic Tools for the Identification of the False Codling Moth (Lepidoptera: Tortricidae)," Journal of Economic Entomology 114(4), 1796-1807, (25 June 2021). https://doi.org/10.1093/jee/toab103
Received: 27 February 2021; Accepted: 29 April 2021; Published: 25 June 2021
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KEYWORDS
loop-mediated is othermal amplification
molecular diagnostics
qPCR SYBR Green
qPCRTaqMan probe
quarantine Pest
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