Novel multiplex Magpix assay platforms for simultaneous detection of different serotypes of BTV circulating in India

Bluetongue (BT), a significant economic disease affecting domestic and wild ruminants, requires rapid and precise diagnostic methods. The diversity of BTV serotypes, coupled with their high genetic and antigenic variability, poses substantial challenges for disease control and prevention. To address this, multiplex Magpix assays were developed for the simultaneous and accurate detection of BTV serotypes and topotypes. Primers and probes were designed to target segment 2 (Seg-2) of the BTV genome, a highly variable region that enables serotype-specific identification using probes conjugated to magnetic beads.The developed Magpix assays facilitate the identification of multiple BTV serotypes from a single sample. Five multiplex Magpix assays were created to detect eastern and western strains of 12 distinct currently circulating BTV serotypes (1, 2, 3, 4, 5, 9, 10, 12, 16, 21, 23, and 24) in India to align with the current epidemiological landscape. These assays were categorized as follows: eastern assays-E1 (1e, 2e, 4e), E2 (3e, 9e), and E3 (16e, 21e, 23e); and western assays-W1 (1w, 10w, 12w) and W2 (2w, 5w, and 24w). The detection limits varied across assays, with E1 showing a higher detection limit (500 pg) compared to E2 (50 pg), E3 (50 pg), W1 (5 pg), and W2 (5 pg). Importantly, the assays exhibited no cross-reactivity with other related viruses.These five multiplex Magpix assays provide an effective diagnostic tool for identifying circulating BTV strains in India. Additionally, the system offers flexibility for expansion to include more serotypes as needed, enhancing its utility for BT surveillance and control. In summary, the introduction of these advanced diagnostic methods presents a significant opportunity for more strategic and effective management of BTV, thereby ensuring better protection for both livestock and the livelihoods dependent on them.