Development and validation of a national reference material system for quality control of chikungunya virus nucleic...

The absence of a World Health Organization (WHO) international standard for Chikungunya virus (CHIKV) nucleic acid testing has long impeded the comparability of diagnostic results across different assays and platforms. To address this critical gap in quality assurance, we developed and rigorously characterized a national reference material system consisting of a quantified standard and a validation panel for CHIKV molecular detection. These materials were developed using intact, inactivated CHIKV particles to closely mimic clinical extraction and amplification. The assigned value of the standard was determined through a robust multi-laboratory study using eight digital polymerase chain reaction (dPCR) platforms, yielding a traceable concentration of 7.60 ± 0.14 log₁₀ copies/mL (k = 2). Extensive evaluation using nine kits demonstrated 100% concordance for both positive and negative samples. The materials exhibited excellent repeatability (coefficient of variation, CV <5%) and consistent detectability (≥95% positivity rate) at the claimed limit of detection across all kits. In summary, this study developed a well-characterized reference material system to support the standardization, development, and quality control of CHIKV nucleic acid assays. The availability of this reference material system addresses a longstanding limitation of results across different platforms and geographical regions. Their implementation is expected to facilitate assay optimization and improve the consistency of surveillance data, thereby strengthening preparedness and the accurate identification of cases, especially in non-endemic areas at risk of importation.IMPORTANCEThe absence of a World Health Organization (WHO) international standard for Chikungunya virus (CHIKV) nucleic acid testing has long compromised result comparability and global outbreak response. Here, we established a national reference material system derived from intact, inactivated CHIKV particles-authentically mirroring the entire clinical workflow. Covering both Asian and East/Central/South African (ECSA) genotypes, this system mitigates false-negative risks caused by viral genetic diversity. Assigned via multi-laboratory dPCR and validated across nine commercial kits, it demonstrated 100% concordance, ≥95% detectability at LoD, and coefficient of variation (CV) <5%. This traceable, genotype-inclusive, workflow-authentic benchmark enables, for the first time, unified performance alignment for regulatory review, manufacturer quality control, and inter-laboratory comparison. It fills a critical gap in domestic quality assurance while providing a technical template for future WHO standardization efforts. Widespread adoption will accelerate reliable diagnostics, strengthen surveillance comparability, and support timely clinical decisions in both endemic and importation-prone settings.