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Updated: May 31, 2026
Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV
Published on: November 10, 2023
The laboratory reality of multiplex respiratory viral detection: findings from a comprehensive external quality
Rongxue Peng1, Xin Yang2, Huiying Lai3
1National Center for Clinical Laboratories, Beijing Hospital, National Center for Gerontology; National Clinical Research Center for Gerontology; The key Laboratory of Geriatrics of NHC; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, PR China; Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China.
Background:
Multiplex nucleic acid amplification tests (NAATs) are essential for managing respiratory viral infections. To robustly confirm the capability of laboratories for multiplex respiratory viral detection, a well-designed, large-scale external quality assessment study incorporating in-depth analysis was conducted.
Methods:
Laboratories performing multiplex viral NAATs in China were publicly recruited and provided with a reference panel to assess their testing performance. Multivariate analysis was performed on the collected results and detailed methodological data to identify potential sources of errors.
Results:
Among 914 participating laboratories, overall concordance was 81.6% (746/914). False-positives (FPs) were concentrated in 13 laboratories, suggesting the occurrence of laboratory contamination. False-negatives (FNs) were more prevalent, primarily occurring in samples with low viral loads. Multivariate analysis identified capillary electrophoresis-based multiplex PCR assay (aOR = 2.1, 95% CI: 1.1-3.8), certain commercial kit (Flu/RSV kit [Autobio], aOR = 102.2, 95% CI: 13.4-776.0), and laboratory-developed tests (aOR = 6.59, 95% CI: 2.2-19.8) as independent risk factors for FNs. Suboptimal laboratory performance also increased the odds of FNs.
Conclusions:
This study demonstrates the advances and limitations of multiplex viral NAATs: high adoption and acceptable concordance reflect the technology's maturity, yet the occurrence of FNs and FPs highlights a need for continued assay optimization, strengthened laboratory quality management, and increased clinician awareness of testing limitations.
