A single-tube multiplex qPCR assay for identification of Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species Mediterranean and Asia II-1

The Bemisia tabaci (Gennadius) species complex comprises numerous cryptic species that differ significantly in invasiveness, host range, virus transmission, and insecticide resistance. Accurate and rapid identification of high-risk cryptic species is therefore essential for biosecurity surveillance and diagnostic responses. In Australia, the Mediterranean (MED) and Asia II-1 cryptic species are not present but pose significant incursion risks due to their global spread and biological characteristics. In this study, a novel qPCR assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) region was developed for specific detection of cryptic species Asia II-1 and was multiplexed with an existing MED-specific qPCR assay into a single-tube reaction. The multiplex assay was optimized and evaluated using synthetic gBlock DNA and extensively validated against a broad panel of whitefly specimens, including 13 B. tabaci cryptic species and 10 nontarget whitefly species present in Australia. Both assays demonstrated high sensitivity, efficiency, and specificity in singleplex and multiplex formats, with no cross-reactivity detected in nontarget species. Assay robustness was further confirmed through an interlaboratory ring test conducted across two independent diagnostic laboratories, which achieved complete concordance in species identification and minimal variation in Ct values. This multiplex qPCR assay provides a rapid, reliable, and transferable diagnostic tool for simultaneous detection of two high-risk exotic B. tabaci cryptic species in Australia. Its strong specificity, reproducibility, and compatibility with portable qPCR platforms make it well suited for routine diagnostics and biosecurity surveillance, enabling faster decision-making in response to potential incursions.