Development of a plasmid reference material to improve the accuracy of quantitative detection of Bovine parvovirus†

Abstract

Reference materials (RMs) serve as essential standards for the quantitative analysis of nucleic acid molecules using quantitative real-time PCR (qPCR), a widely employed method for pathogen detection. However, the lack of universal reference materials traceable to the International System of Units (SI) compromises the accuracy and comparability of quantitative results. To address this gap, we developed a plasmid RM for the quantitative detection of the Bovine Parvovirus (BPV) VP1 gene. To determine the reference value of the plasmid RM, we established a highly accurate and specific digital PCR (dPCR) method. This dPCR assay demonstrated excellent linearity across five orders of magnitude (10⁰–10⁵ copies/reaction, R² > 0.999). The limits of detection (LOD) and quantification (LOQ) were determined to be 9 copies/reaction and 30 copies/reaction, respectively. Using the validated dPCR method, the reference value of the BPV plasmid RM was quantified as (2.21 ± 0.25) × 10⁶ copies per μL, with an expanded uncertainty (coverage factor k = 2). The developed plasmid RM provides a reliable standard for the quantitative detection of BPV using qPCR, offering improved accuracy and traceability in diagnostic applications.