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


OBJECTIVE
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.


METHODS
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.
Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, United States.
Jennifer
J
Klemenc
S
S
Asad Ali
Monika
M
Johnson
Sharon J
SJ
Tollefson
H Keipp
HK
Talbot
Tina V
TV
Hartert
Kathryn M
KM
Edwards
John V
JV
Williams
D017931:DNA Primers; D006801:Humans; D029121:Metapneumovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D018184:Paramyxoviridae Infections / Q000175:diagnosis* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012141:Respiratory Tract Infections / Q000175:diagnosis / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral