Development of a droplet digital PCR for detection and quantitation of human parvovirus B19.

UNLABELLED: The objective of this study is to establish a droplet digital PCR (ddPCR) method for the detection of human parvovirus B19 (B19V) and provide accurate and reliable technical support for molecular biological diagnosis and epidemiological investigation of the virus. Specific primers and a TaqMan probe targeting the NS1 region of the B19V genome were designed, and a reaction system based on ddPCR was constructed and optimized. The methodology was validated through sensitivity, specificity, and repeatability tests. Subsequently, the method was applied to eight B19V-positive samples to evaluate its practical applicability. Methodological validation experiments demonstrated excellent sensitivity with a good linear relationship (R² = 0.9974) and a minimum detection limit of 1.013 × 10⁻¹ copies/μL. No cross-reactivity was observed with 11 respiratory pathogens, 5 pathogens presenting similar clinical manifestations, and 4 blood-borne pathogens. The inter- and intra-assay coefficients of variation for high-, medium-, and low-concentration samples were all ≤10%. Furthermore, this method was successfully applied to clinical samples, with stable detection of B19V in five high-concentration throat swab samples and three low-concentration blood samples. The ddPCR method established in this study exhibits high sensitivity, specificity, and repeatability, offering a robust tool for molecular diagnosis and epidemiological monitoring of B19V infection.

IMPORTANCE

Early human parvovirus B19 (B19V) infections were sporadic or occurred in small clusters, attracting little attention. Since late 2023, nine European Union/European Economic Area (EU/EEA) countries have reported a significant increase in B19 infections. Moreover, the virus has robust physicochemical tolerance and the potential to resist pathogen removal processes such as filtration, inactivation, and pasteurization, which have raised the close attention and vigilance of international organizations, governments, and the public. Despite existing qPCR and antigen/antibody tests, the growing number of infections in multiple countries highlights the need for a more accurate and efficient detection system. Based on this, our team carried out related research and built a system configuration based on the third-generation droplet digital polymerase chain reaction platform, with a view to updating the B19V detection method.