Design and optimisation of rapid real‐time PCR assays for the detection of key C ulicoides species
- 1SETU School of Science and Computing, South East Technological University, Cork Road, Waterford, Ireland;
- 2UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland;
- 3The Pirbright Institute, Ash Road, Woking, Surrey GU24 0NF, UK
Abstract
In extensive surveillance programmes of Culicoides biting midges (Diptera: Ceratopogonidae), morphological identification can be time-consuming and difficult, while DNA barcoding, although highly accurate, may not be cost-effective or suitable for rapid analysis, as it requires individual specimen processing. To address these limitations, we developed a rapid screening method using real-time PCR assays with either SYBR Green or hydrolysis probe-based detection chemistries. Species-specific primers and, where necessary, hydrolysis probes were designed based on the updated sequences of the ITS2 region of seven Culicoides species. The specificity and efficiency of these assays were validated both in silico and through real-time PCR testing on target and non-target Culicoides species, tested individually and as mixed-species samples. The new real-time PCR assays detect the presence of key species including C. obsoletus, C. scoticus, C. chiopterus, C. dewulfi, C. pulicaris, C. punctatus and C. impunctatus in pools of specimens. The molecular techniques developed in this study provide a valuable tool for accurate and high-throughput Culicoides surveillance, which can be used for year-round monitoring of adult midges in traps, larvae and in environmental samples, potentially revealing novel insights into the spatial and temporal turnover of Culicoides species. These methods can be applied to large-scale vector screening programmes involving pooled samples, addressing the limitations of previously described methods used in midge surveillance.
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BibTeX
@article{isiye2026design,
title = {{Design and optimisation of rapid real‐time PCR assays for the detection of key C ulicoides species}},
author = {Elsie Isiye and Angela Valcarcel Olmeda and Thomas G. Curran and David O’Neill and Theo D. De Waal and Gerald Barry and Aidan O’Hanlon and James O’Shaughnessy and Marion England and Annetta Zintl and Denise B. O’Meara},
journal = {Medical and Veterinary Entomology},
year = {2026},
month = jun,
doi = {10.1111/mve.70088},
url = {https://doi.org/10.1111/mve.70088},
note = {CC BY},
}
RIS
TY - JOUR
TI - Design and optimisation of rapid real‐time PCR assays for the detection of key C ulicoides species
AU - Elsie Isiye
AU - Angela Valcarcel Olmeda
AU - Thomas G. Curran
AU - David O’Neill
AU - Theo D. De Waal
AU - Gerald Barry
AU - Aidan O’Hanlon
AU - James O’Shaughnessy
AU - Marion England
AU - Annetta Zintl
AU - Denise B. O’Meara
PY - 2026
DA - 2026/06/19
JO - Medical and Veterinary Entomology
DO - 10.1111/mve.70088
UR - https://doi.org/10.1111/mve.70088
AB - In extensive surveillance programmes of Culicoides biting midges (Diptera: Ceratopogonidae), morphological identification can be time-consuming and difficult, while DNA barcoding, although highly accurate, may not be cost-effective or suitable for rapid analysis, as it requires individual specimen processing. To address these limitations, we developed a rapid screening method using real-time PCR assays with either SYBR Green or hydrolysis probe-based detection chemistries. Species-specific primers and, where necessary, hydrolysis probes were designed based on the updated sequences of the ITS2 region of seven Culicoides species. The specificity and efficiency of these assays were validated both in silico and through real-time PCR testing on target and non-target Culicoides species, tested individually and as mixed-species samples. The new real-time PCR assays detect the presence of key species including C. obsoletus, C. scoticus, C. chiopterus, C. dewulfi, C. pulicaris, C. punctatus and C. impunctatus in pools of specimens. The molecular techniques developed in this study provide a valuable tool for accurate and high-throughput Culicoides surveillance, which can be used for year-round monitoring of adult midges in traps, larvae and in environmental samples, potentially revealing novel insights into the spatial and temporal turnover of Culicoides species. These methods can be applied to large-scale vector screening programmes involving pooled samples, addressing the limitations of previously described methods used in midge surveillance.
SN - 0269-283X
SN - 1365-2915
ER -