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Updated: Jul 19, 2025
Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification
Published on: October 11, 2024
Establishment of Digital PCR Method and Reference Material for Adenoviruses 40 and 41
Guifang Chen1, Huijie Li2, Yunhua Gao1
1Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.
Insights
Digital PCR (dPCR) offers a sensitive method for detecting human adenovirus (HAdV) 40/41, crucial for diagnosing hepatitis in children. Developed pseudovirus reference materials ensure accurate quantification for improved clinical diagnosis.
Area of Science:
- Virology
- Molecular Diagnostics
- Pediatric Infectious Diseases
Background:
- Coinfection with human adenovirus (HAdV) and SARS-CoV-2 is linked to unexplained acute hepatitis in children.
- HAdV 40 and HAdV 41 have been identified as common culprits in these cases.
- Accurate detection of HAdV is critical for diagnosis and management.
Purpose of the Study:
- To establish a digital PCR (dPCR) quantification method for HAdV 40/41.
- To develop and quantify pseudovirus reference materials (RMs) for HAdV 40/41.
- To improve the accuracy of HAdV 40/41 detection in clinical settings.
Main Methods:
- Digital PCR (dPCR) was employed for sensitive detection of HAdV 40/41.
- Limit of detection for HAdV 40/41 dPCR was determined to be 4 and 5 copies/μL, respectively.
- Pseudovirus RMs containing the conserved HEXON gene were created and quantified using dPCR.
Main Results:
- The dPCR method demonstrated high sensitivity for low-concentration virus detection.
- Quantified values for HAdV 40 RM were (1.43 ± 0.35) × 103 copies/μL and for HAdV 41 RM were (1.21 ± 0.28) × 103 copies/μL.
- The developed RMs showed reproducibility across multiple platforms.
Conclusions:
- The established dPCR method provides a convenient and accurate approach for HAdV 40/41 detection.
- The pseudovirus RMs enhance the reliability and accuracy of HAdV quantification.
- These advancements are vital for precise clinical diagnosis of HAdV-associated hepatitis in children.
Abstract:
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 HEXON gene was developed and quantified with the dPCR method. The assigned values with expanded uncertainty were (1.43 ± 0.35) × 103 copies/μL for HAdV 40 RM and (1.21 ± 0.28) × 103 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.
