Barcoding Quantitative PCR Assay to Distinguish Between and .

The accurate identification of mosquito species is critical for effective mosquito surveillance and control, especially when presented with morphologically similar species like and . Damaged specimens and morphologically similar life stages such as eggs and larvae make it difficult to distinguish from using microscopy and taxonomic keys. To address this, the ID-qPCR assay, a multiplex quantitative PCR assay that utilizes single-nucleotide polymorphisms within the gene, was developed to distinguish between these two species. The assay was tested on DNA extracted from the eggs, larvae, and adults of both species, as well as from environmental DNA (eDNA) collected from natural mosquito reproduction sites. It demonstrated a high diagnostic accuracy across multiple life stages, with a sensitivity exceeding 95% for most groups and specificity exceeding 90%, except for field-collected adult (75%). For eDNA samples, the assay achieved 100% sensitivity and 94% specificity for samples classified as and 91% sensitivity and 86% specificity for . A two-graph receiver operating characteristic analysis was also used as an alternate method with which to establish Ct thresholds for interpreting results from unknown samples. The ID-qPCR assay enables the rapid and sensitive identification of and from specimens and eDNA, and may be of use in mosquito surveillance programs.