Development and clinical validation of a PCR assay for human parvovirus B19 detection with applications in challenging diagnostic scenarios.

Publication date: Jul 03, 2026

Human parvovirus B19 (HB19V) infection poses significant clinical challenges in resource-limited settings, particularly regarding blood safety surveillance and diagnosis of associated syndromes. Limited access to validated diagnostic assays restricts clinical management and transfusion safety protocols. The objective is to develop and validate an in-house duplex real-time PCR assay for HB19V detection and evaluate its clinical utility in a resource-limited setting. A duplex real-time PCR assay targeting the conserved NS1 gene of HB19V with RNase P as endogenous control was developed. Analytical validation assessed amplification efficiency, limit of detection (LOD), precision (intra- and inter-assay coefficients of variation), and cross-reactivity. Clinical validation compared qualitative agreement with a CE-certified commercial kit using Cohen’s kappa statistic. Positive samples underwent Sanger sequencing for genotype characterization. Clinical specimens from suspected HB19V infections were tested and results correlated with patient presentations. The assay demonstrated 98. 82% amplification efficiency with LOD of 6 copies/uL. Precision metrics showed excellent reproducibility (CoV 

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