Portable Molecular Diagnostics for Cetacean Morbillivirus: Development of a Reverse Transcription Insulated...

Cetacean morbillivirus (CeMV) drives recurrent unusual-mortality events, yet surveillance is uneven where laboratory capacity is limited. We developed a portable reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting a conserved phosphoprotein (P)-gene segment and evaluated analytical performance, tissue-level clinical sensitivity, and concordance with reverse transcription-quantitative PCR (RT-qPCR) under low-copy conditions that resemble challenging strandings. Using synthetic RNA, RT-iiPCR achieved 100% detection from 62,560 to 513 copies µL^-1, and probit analysis estimated a 95% limit of detection (LOD₉₅) of 139 copies µL^-1. Clinical sensitivity was assessed with two spiking regimes (RNA added after or before extraction) in cerebrum and lung. Singleplex RT-iiPCR maintained 100% positivity to approximately cycle threshold (Ct) 33 irrespective of spiking order, indicating that near-limit failures are governed by template scarcity rather than extraction loss. Duplex RT-iiPCR co-amplifying β2-microglobulin (B2M) provided process control with a slight sensitivity cost, sustaining perfect detection to ~Ct 30-31. Across low-copy panels, agreement with RT-qPCR was substantial (overall κ = 0.68-0.76) and very good in cerebrum (singleplex κ = 0.85; duplex κ = 0.87), while duplex lung showed lower concordance (κ = 0.55) driven solely by Ct >33 false-negative calls, with no false positives. The assay detected five lineages (dolphin morbillivirus [DMV], pilot whale morbillivirus [PWMV], beaked whale morbillivirus [BWMV], Guiana DMV [GDMV], and Fraser's DMV [FDMV]) in formalin-fixed, paraffin-embedded tissues archived up to 28 years, and sequence alignments indicate expected coverage of additional lineages. Lyophilized reagents, compact hardware, and a quick, simple workflow support deployment in hot, humid, resource-limited settings. A strain-agnostic, field-ready RT-iiPCR can underpin transboundary CeMV surveillance, enable rapid carcass triage and sequencing, and provide early warning where diagnostic gaps currently exist.