Abstract
Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.
Publication types
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Comparative Study
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Evaluation Study
MeSH terms
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Child
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Coronavirus 229E, Human / genetics
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Coronavirus 229E, Human / isolation & purification
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Coronavirus OC43, Human / genetics
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Coronavirus OC43, Human / isolation & purification
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Fluorescent Antibody Technique
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Gammainfluenzavirus / genetics
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Gammainfluenzavirus / isolation & purification
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Humans
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Influenza A virus / genetics
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Influenza A virus / isolation & purification
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Influenza B virus / genetics
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Influenza B virus / isolation & purification
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Metapneumovirus / genetics
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Metapneumovirus / isolation & purification
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Nasal Cavity / virology
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Parainfluenza Virus 1, Human / genetics
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Parainfluenza Virus 1, Human / isolation & purification
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Parainfluenza Virus 2, Human / genetics
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Parainfluenza Virus 2, Human / isolation & purification
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Parainfluenza Virus 3, Human / genetics
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Parainfluenza Virus 3, Human / isolation & purification
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Parainfluenza Virus 4, Human / genetics
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Parainfluenza Virus 4, Human / isolation & purification
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Quality Control
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RNA Viruses / genetics
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RNA Viruses / isolation & purification*
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RNA, Viral / analysis*
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RNA, Viral / genetics
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Respiratory Syncytial Viruses / genetics
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Respiratory Syncytial Viruses / isolation & purification
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Respiratory Tract Infections / diagnosis*
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Respiratory Tract Infections / virology*
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Reverse Transcriptase Polymerase Chain Reaction / methods*
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Reverse Transcriptase Polymerase Chain Reaction / standards
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Rhinovirus / genetics
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Rhinovirus / isolation & purification
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Sensitivity and Specificity
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Virus Cultivation