Coinfection with human adenovirus (HAdV) and SARS-CoV-2 has been associated with acute hepatitis in children with unknown etiology. Similar cases have been reported in many countries, and HAdV 40 and HAdV 41 have been identified. The quantification method is established based on digital PCR (dPCR) for HAdV 40/41, which is more convenient for low-concentration virus detection. The limit of detections of HAdV 40/41 dPCR were 4 and 5 copies/μL. Pseudovirus reference material (RM) that contains the highly conserved gene was developed and quantified with the dPCR method. The assigned values with expanded uncertainty were (1.43 ± 0.35) × 10 copies/μL for HAdV 40 RM and (1.21 ± 0.28) × 10 copies/μL for HAdV 41 RM. The values could be reproduced on multiple platforms. The dPCR method and pseudovirus RMs contribute to the improved accuracy of HAdV 40/41 detection, which is crucial for clinical diagnosis.
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