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Updated: Aug 17, 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
Two RT-PCR based assays to detect human metapneumovirus in nasopharyngeal aspirates
María Rosa López-Huertas1, Inmaculada Casas, Belsy Acosta-Herrera
1Laboratorio de Gripe y Virus Respiratorios, Servicio de Virología, Centro National de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, km 2, 28220 Majadahonda, Madrid, Spain.
Abstract:
Two sensitive and specific RT-PCR assays were standardised for testing the presence of human metapneumovirus. A total of 300 nasopharyngeal aspirates collected from infants suffering from bronchiolitis since October 2000 to June 2003 and shown previously as negative to common respiratory viruses were examined. Matrix and polymerase viral genes, which show a low rate of variation, were chosen to design amplification assays to ensure that any genotype of the human metapneumovirus could be detected. A RT-PCR followed by a reverse line blotting hybridisation was developed for viral polymerase gene. For the matrix gene, after the RT-PCR assay, a subsequent nested PCR was carried out. Both assays had similar sensitivity, equivalent to 0.1 TCID50 of human metapneumovirus strain NL/1/99 which was used as the positive control. The human metapneumovirus was present in 16.6% of the specimens studied. The approaches described below are not only a robust method for rapid diagnosis of the human metapneumovirus, but also to establish an etiological surveillance tool for epidemiological studies. Based on the results obtained, human metapneumovirus infections in Madrid followed a seasonal pattern, with most of the infections occurring between February and April.
Insights
Two new RT-PCR assays accurately detect human metapneumovirus in infants with bronchiolitis. This study identified human metapneumovirus in 16.6% of cases, revealing a seasonal pattern in Madrid.
Area of Science:
- Virology
- Molecular Biology
- Pediatrics
Background:
- Human metapneumovirus (hMPV) is a significant respiratory pathogen in infants.
- Bronchiolitis is a common respiratory illness in infants, often caused by viruses.
- Accurate diagnostic tools are crucial for understanding hMPV epidemiology.
Purpose of the Study:
- To develop and standardize sensitive and specific RT-PCR assays for hMPV detection.
- To investigate the prevalence of hMPV in infants with bronchiolitis.
- To establish a tool for etiological surveillance and epidemiological studies of hMPV.
Main Methods:
- Standardization of two RT-PCR assays targeting conserved matrix and polymerase genes of hMPV.
- Testing of 300 nasopharyngeal aspirates from infants with bronchiolitis.
- Assay validation using hMPV strain NL/1/99, achieving sensitivity equivalent to 0.1 TCID50.
Main Results:
- Both RT-PCR assays demonstrated high sensitivity and specificity for hMPV detection.
- Human metapneumovirus was detected in 16.6% of the examined infant specimens.
- Infections showed a seasonal pattern in Madrid, peaking between February and April.
Conclusions:
- The developed RT-PCR assays provide a robust method for rapid hMPV diagnosis.
- These assays serve as valuable tools for epidemiological surveillance of hMPV.
- hMPV is a notable cause of bronchiolitis in infants, with distinct seasonal occurrence.
