Development and validation of a TaqMan SNP genotyping assay for the molecular identification of the malaria mosquitoes Anopheles messeae and Anopheles daciae

The Anopheles maculipennis complex comprises closely related mosquito species of epidemiological importance across the Palearctic region. Anopheles messeae and Anopheles daciae are among its most widely distributed members but are morphologically indistinguishable, hindering species-specific vector surveillance and ecological and epidemiological studies. This limitation highlights the need for rapid, sensitive, specific, and high-throughput molecular methods for their identification. We report the development and validation of a TaqMan SNP genotyping assay targeting the Internal Transcribed Spacer 2 (ITS2) locus to discriminate between two closely related members of the Anopheles maculipennis complex, An. messeae and An. daciae . The assay targets a diagnostic single-nucleotide polymorphism (SNP) within the ITS2 region of ribosomal DNA. The optimized TaqMan SNP genotyping assay was applied to 293 field-collected mosquitoes. The assay showed high analytical sensitivity, with a detection limit of 0.1 ng of genomic DNA, and high specificity, with no false-positive results among the non-target species tested. The TaqMan assay showed 98% overall agreement with PCR–RFLP (Cohen’s κ = 0.96) and reliably identified An. messeae , An. daciae , and interspecific heterozygotes. Compatible with rapid DNA extraction protocols, the assay provides a rapid and high-throughput approach for species identification in evolutionary and epidemiological studies. The TaqMan SNP genotyping assay was developed and validated to successfully differentiate An. messeae and An. daciae . The assay provides a rapid and scalable approach for species identification and may facilitate large-scale surveillance of An. messeae and An. daciae populations.