Portable Molecular Diagnostics for Cetacean Morbillivirus: Development of a Reverse Transcription Insulated Isothermal PCR (RT‐iiPCR) for Global Surveillance

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 (LOD 95 ) 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.