Development of Three Molecular Diagnostic Tools for the Identification of the False Codling Moth (Lepidoptera: Tortricidae)
| Autor: | Patrizia Sacchetti, Giovanni Cappellini, N Boersma, Domenico Rizzo, Fabrizio Pennacchio, Antonio Aronadio, Tommaso Bruscoli, D Del Nista, Elisabetta Rossi, Chiara Salemi, Linda Bartolini, Daniele Da Lio |
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| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Tortricidae qPCR SYBR Green Loop-mediated isothermal amplification quarantine pest molecular diagnostics qPCR TaqMan probe qPCR SYBR Green loop-mediated isothermal amplification qPCR TaqMan probe Moths 01 natural sciences Sensitivity and Specificity molecular diagnostics 03 medical and health sciences TaqMan False positive paradox Animals Pathology Molecular Detection limit Chromatography Ecology biology quarantine pest Reproducibility of Results General Medicine Repeatability Molecular diagnostics biology.organism_classification 010602 entomology 030104 developmental biology Molecular Diagnostic Techniques Insect Science False codling moth loop-mediated isothermal amplification Nucleic Acid Amplification Techniques |
| Zdroj: | Journal of economic entomology. 114(4) |
| ISSN: | 1938-291X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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