Development of Three Molecular Diagnostic Tools for the Identification of the False Codling Moth (Lepidoptera: Tortricidae)

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.

Quantitative Real-Time PCR Based on SYBR Green Technology for the Identification of Philaenus italosignus Drosopoulos & Remane (Hemiptera Aphrophoridae)

“…Our qPCR assay was demonstrated to be specific for the target, allowing its unambiguous identification from non-target species, either not taxonomically related to P. italosignus or very close to it, like P. spumarius and N. campestris . This is in agreement with other studies where specific SYBR Green qPCR assays were developed for the identification of insect pests, also considering congeneric non-target species [ 15 , 16 ]. High specificity granted by this technology may be useful when dealing with lesser-studied insects in all fields of application, as in the case of non-pest species (e.g., Philaenus spp.…”

Diagnostic Tool for the Identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) Using Real-Time PCR

“…The results, both in silico (for over 200 Bactrocera spp.) and in vivo, displayed favorable results when compared to similar frugivorous pests and demonstrated an analytical sensitivity (LoD) of 0.128 pg/µL, denoting a limit of detection in line with similar work [49].…”

“…The latter, however, offers the benefit of a simpler design and execution, along with cost-effectiveness. It should be noted that these advantages sometimes result in reduced specificity within qPCR assays employing SYBR Green, as documented in prior studies [48,49]. Test reliability is right now of the utmost importance because, despite the implementation of various measures and eradication attempts in the infested areas (such as the Campania Region in Italy), the population currently present in Italy certainly represents the greatest potential threat to the entire European territory [26,28].…”

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolařik) in Infected Walnut

“…Our molecular toolkit can be exploited both in routine screening (vLAMP, rtLAMP) and in diagnostic confirmation efforts (qPCR Sybr Green, rtLAMP). As previously observed, technical and operational parameters can also be assessed from a qualitative point of view to compare the efficacy of each developed method [31]. vLAMP is the fastest and cheapest method, the easiest to perform, and the most user-friendly; however, it suffers from potentially unclear positive identifications and a greater risk of contamination (false positives).…”