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. 2012 Aug;54(4):371-5.
doi: 10.1016/j.jcv.2012.05.005. Epub 2012 Jun 6.

Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus

Jennifer Klemenc  1 , S Asad Ali, Monika Johnson, Sharon J Tollefson, H Keipp Talbot, Tina V Hartert, Kathryn M Edwards, John V Williams
Affiliations

Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus

Jennifer Klemenc et al. J Clin Virol. 2012 Aug.

Abstract

Background: Human metapneumovirus (HMPV) is a paramyxovirus with multiple genetic lineages that is a leading cause of acute respiratory disease. Several RT-PCR assays have been described based on limited available sequence data.

Objectives: To develop a broadly reactive real-time RT-PCR assay for HMPV that allows for a rapid, sensitive, and specific detection in a clinical or research setting.

Study design: Three published assays for HMPV were modified based on analysis of multiple HMPV sequences obtained from GenBank. Original and modified assays were tested against prototype HMPV strains from each genetic sublineage, multiple isolates of HMPV from different years, a collection of clinical specimens, and commercial validation panels.

Results: A number of potential sequence mismatches with diverse HMPV strains were identified. Modifications were made to oligonucleotides to improve annealing efficiency. Primers and probes based on newer sequence data offered enhanced detection of all subgroups, especially for low titer specimens. The new primers and probe detected multiple clinical isolates of HMPV collected over a twenty-year period. The modified assay improved detection of HMPV in a panel of clinical specimens, and correctly identified HMPV samples in two commercial validation sets.

Conclusions: We report a modified real-time RT-PCR assay for HMPV that detects all genetic lineages with high sensitivity.

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Figures

Figure 1
Figure 1. Performance of assays tested against prototype HMPV strains
Plots of cycle threshold (CT) versus RNA serial 10-fold dilutions. A) TN96-12 A1. B) TN94-49 A2. C) TN98-242 B1. D) TN99-419 B2. Y axis = CT, x-axis = dilution.
See this image and copyright information in PMC

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