Development and Validation of a Multiplex Molecular Diagnostic Platform for Simultaneous Detection of Seven Priority Viral Pathogens in Nigeria

Nigeria's viral disease surveillance is constrained by single-pathogen diagnostic approaches that are inadequate for managing co-infections and atypical presentations of priority viral diseases in resource-limited settings. This study develops and validates a novel multiplex RT-PCR panel for the simultaneous detection of seven priority viral pathogens: Lassa mammarenavirus, Ebola virus, SARS-CoV-2, Dengue virus (all serotypes), Yellow fever virus, Monkeypox virus, and Hepatitis E virus. Panel design will use bioinformatics-guided primer and probe design (Primer3, Beacon Designer) targeting conserved genomic regions for each pathogen, with in silico specificity testing against the RefSeq viral database. Analytical validation will assess sensitivity, specificity, limit of detection, and cross-reactivity on a blinded panel of 300 archived clinical samples from NCDC reference laboratories in Lagos and Abuja. Clinical validation will enrol 500 febrile patients presenting with undifferentiated febrile illness at three tertiary hospitals across South West (Lagos), South East (Enugu), and North Central (FCT) zones, comparing multiplex panel performance against singleplex reference assays. Implementation feasibility in resource-limited settings will be assessed on a portable qPCR platform (Bio-Rad CFX Opus). This doctoral study makes an original contribution to diagnostic virology through the development of an indigenous, contextually optimised diagnostic tool, reducing diagnostic turnaround time and expanding the differential diagnosis capacity of Nigerian reference laboratories. Keywords: multiplex RT-PCR, diagnostic platform, priority pathogens, Nigeria, validation