A multiplex PCR assay for the identification of fruit flies (Diptera: Tephritidae) of economic importance in South Africa

Andrews, Kelsey J; Bester, Rachelle; Manrakhan, Aruna; Maree, Hans J.

doi:10.1038/s41598-022-17382-x

Cited by 5 publications

(6 citation statements)

References 44 publications

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“…Additionally, the experimental designs of LAMP assays may, at times, lead to assays with lower sensitivity or specificity compared to a qPCR assay. Moreover, a multiplex PCR assay [52] and a single-gene TaqMan real-time PCR [53] were developed and assessed for specific species or, in the case of [54], exclusively for B. zonata.…”

Section: Discussionmentioning

confidence: 99%

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

Rizzo,

Zubieta,

Sacchetti

et al. 2024

Insects

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Accurate identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), commonly known as the Oriental fruit fly, is a significant challenge due to the morphological convergence and taxonomic uncertainties of species belonging to the same genus. This highly polyphagous species poses a significant threat to fruit crops. With its potential establishment in Europe becoming a growing concern, there is an urgent need for rapid and efficient diagnostic methods. The study presented here introduces a diagnostic protocol based on real-time PCR using a TaqMan probe for the early and reproducible identification of B. dorsalis. Specimens representing the genetic diversity of the Italian population were collected and analyzed. Specific primers and probe were designed based on the conserved regions and an in silico analysis confirmed their specificity. The assay conditions were optimized, and analytical sensitivity, specificity, repeatability, and reproducibility were evaluated. The protocol showed high sensitivity and specificity, accurately detecting low DNA concentrations of B. dorsalis. This standardized method provides a reliable tool for routine diagnostics, enhancing the accuracy and efficiency of identifying the Oriental fruit fly at all stages of its development, thereby facilitating effective pest management measures. The development of this diagnostic protocol is crucial for monitoring and supporting efforts to prevent the passive spread of B. dorsalis in Europe, particularly in light of the recent active infestations detected in Italy.

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Section: Discussionmentioning

confidence: 99%

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

Rizzo,

Zubieta,

Sacchetti

et al. 2024

Insects

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“…Colony adult males and wild trap-collected specimens underwent morphological identification and identity confirmation using BLASTn analysis querying the COI gene, which was amplified using the primer pair CI-J2183 and TL2-N3014 [34]. All adult specimens used in this study were identified to species level through morphological identification using published keys [17], as well as molecular identification using a multiplex PCR previously developed for use at Ports of Entry [27]. COI could differentiate C. capitata, C. cosyra and B. dorsalis to species level.…”

Section: Species Identity Confirmationmentioning

confidence: 99%

“…All specimens in this study, colony larvae, colony adults, and wild adults underwent identification using a multiplex PCR assay developed for the identification of these five fruit fly species of economic importance to South Africa following the protocol outlined in the study [27].…”

Section: Dna Extraction and Species Identificationmentioning

confidence: 99%

“…A molecular assay that can differentiate between all five species simultaneously would be valuable for routine monitoring and pest surveillance. A multiplex assay has recently been developed for use at PoE in cases of larval interception [27]. While this assay is useful for time-sensitive identification matters, routine pest monitoring and surveillance may benefit from a sequencing-based assay as the resources are readily available at these facilities, and diversity seen within or between species can be studied further.…”

Section: Introductionmentioning

confidence: 99%

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Utilisation of a mitochondrial intergenic region for species differentiation of fruit flies (Diptera: Tephritidae) in South Africa

et al. 2022

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Background Fruit flies (Diptera: Tephritidae) comprise species of agricultural and economic importance. Five such fruit fly species are known to affect commercial fruit production and export in South Africa: Ceratitis capitata, Ceratitis cosyra, Ceratitis rosa, Ceratitis quilicii, and Bactrocera dorsalis. Management practices for these pests include monitoring, application of pest control products, post-harvest disinfestation measures and inspection of consignments both prior to shipment and at ports of entry. In activities relating to monitoring and inspection, accurate identification of these pests to species level is required. While morphological keys for adult stages of these fruit fly species have been well developed, morphological keys for earlier life stages remain problematic. In instances where closely related species cannot be reliably distinguished morphologically, there is a need for molecular tools to assist in identifying these five fruit fly species during surveillance practices, where sequencing-based approaches would be beneficial. Results Two complete mitochondrial genomes were assembled for each fruit fly species investigated using high throughput sequencing data generated in this study. A single primer set was designed to amplify a region between tRNAile and tRNAmet. The amplicon consists of a partial segment of tRNAile, intergenic region I (tRNAile - tRNAgln), the complete sequence of tRNAgln, intergenic region II (tRNAgln - tRNAmet), and a partial segment of tRNAmet. PCR amplicons were generated for 20 specimens of each species, five of which were colony adult males, five colony larvae, and 10 wild, trap-collected specimens. Upon analysis of the amplicon, intergenic region I was identified as the most informative region, allowing for unambiguous identification of the five fruit fly species. The similarity in intergenic region II was too high between C. rosa and C. quilicii for accurate differentiation of these species. Conclusion The identity of all five fruit flies investigated in this study can be determined through sequence analysis of the mitochondrial intergenic regions. Within the target amplicon, intergenic region I (tRNAile - tRNAgln) shows interspecific variation sufficient for species differentiation based on multiple sequence alignment. The variation in the length of intergenic region I is proposed as a potential tool for accurately identifying these five fruit flies in South Africa.

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“…), molecular methods are especially useful and valuable [9]. When dealing with the need for a precise, specific, and reliable determination and identification of agricultural pests, beneficial organisms, or endangered species, molecular methods have frequently proven to be both reliable and the most cost-effective option (e.g., [10][11][12][13][14]). In addition, the overall limited availability of traditional taxonomists' expertise on the identification of specific groups of organisms or species has been cited repeatedly as one of the primary reasons for the use of molecular identification methods, DNA barcoding, associations, are evidenced as vectors of 'Ca.…”

Section: Introductionmentioning

confidence: 99%

Multiplex PCR for Discriminating Host Plant Associations of Hyalesthes obsoletus (Hemiptera: Cixiidae), a Key Vector and Driver of ‘Ca. Phytoplasma solani’ Epidemiology

Jović

Toševski

2023

Agronomy

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Given the ecological and epidemiological specialization of Hyalesthes obsoletus, the principle vector of ‘Candidatus Phytoplasma solani’, the primary objective of this study was to develop molecular tools for discriminating three host plant associations of the vector populations: (i) Convolvulus arvensis–Urtica dioica (Ca–Ud), (ii) Vitex agnus-castus (Vac), and (iii) Crepis foetida (Cf). The genetic diversity of the nearly full-length mitochondrial COI gene (1467 bp) was analyzed and compared among previously reported and newly collected individuals of the three host plant associations on a wide geographic range. Multiplex PCR was designed and evaluated for discriminating H. obsoletus host plant associations based on the size of amplified fragments: 1084 bp for the Cf association, 645 bp for the Ca–Ud association, and 355 bp for the Vac association. Examples of the epidemiological value of combining data on the genetic characteristics of the vector and the pathogen are provided. The method is intended to facilitate an accurate identification of the vector’s phylogenetic lineage, natural host plant preference, and epidemiological transmission routes of ‘Ca. P. solani’. When applied to H. obsoletus specimens collected from cultivated plants within an agroecosystem and combined with ‘Ca. P. solani’ genotyping, the method should provide valuable information on disease epidemiology, source(s) of emergence, and transmission routes.

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A multiplex PCR assay for the identification of fruit flies (Diptera: Tephritidae) of economic importance in South Africa

Cited by 5 publications

References 44 publications

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

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

Utilisation of a mitochondrial intergenic region for species differentiation of fruit flies (Diptera: Tephritidae) in South Africa

Multiplex PCR for Discriminating Host Plant Associations of Hyalesthes obsoletus (Hemiptera: Cixiidae), a Key Vector and Driver of ‘Ca. Phytoplasma solani’ Epidemiology

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