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Updated: Jun 26, 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
Comparison of automated microarray detection with real-time PCR assays for detection of respiratory viruses in
Frédéric Raymond1, Julie Carbonneau, Nancy Boucher
1Infectious Disease Research Center of the CHUQ-CHUL and Laval University, Sainte-Foy, Quebec, Canada.
Insights
Two new assays accurately detect 23 common respiratory viruses in children, aiding diagnosis and public health. These molecular diagnostic methods improve etiological assessment for hospitalized children with respiratory infections.
Area of Science:
- Medical diagnostics
- Virology
- Molecular biology
Background:
- Respiratory virus infections are a significant cause of illness, hospitalization, and testing in young children.
- Accurate and rapid identification of causative agents is crucial for effective patient management and public health strategies.
Purpose of the Study:
- To develop and compare two distinct molecular assays for the simultaneous detection of up to 23 common respiratory viruses in pediatric patients.
- To evaluate the performance of these assays in identifying viral pathogens from nasopharyngeal aspirates of hospitalized children.
Main Methods:
- Development of single TaqMan quantitative real-time PCR assays in a 96-well-plate format.
- Development of a multiplex PCR assay coupled with primer extension and microarray hybridization on the Infiniti analyzer.
- Application of both assays to 221 nasopharyngeal aspirates from children with respiratory tract infections.
Main Results:
- Respiratory viruses were detected in 81.4% of the specimens using at least one of the methods.
- Human respiratory syncytial virus (HRSV) B (38.0%) and A (10.0%), and influenzavirus A (13.1%) were among the most prevalent viruses identified.
- Multiple viral infections were present in 13.1% of cases, with 94.1% concordance between the two developed assays.
Conclusions:
- The developed molecular assays provide a comprehensive etiological assessment of respiratory viruses in hospitalized children.
- These diagnostic tools can significantly aid in guiding public health interventions and clinical management of pediatric respiratory infections.
Abstract:
Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions.
