Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses.
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.
Bellau-Pujol
S
S
Laboratory of Human and Molecular Virology, University Hospital, av. Georges Clemenceau, 14033 Caen, France.
Vabret
A
A
Legrand
L
L
Dina
J
J
Gouarin
S
S
Petitjean-Lecherbonnier
J
J
Pozzetto
B
B
Ginevra
C
C
Freymuth
F
F
D002648:Child; D028941:Coronavirus 229E, Human / Q000235:genetics / Q000302:isolation & purification; D028962:Coronavirus OC43, Human / Q000235:genetics / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification; D009982:Gammainfluenzavirus / Q000235:genetics / Q000302:isolation & purification; D029121:Metapneumovirus / Q000235:genetics / Q000302:isolation & purification; D009296:Nasal Cavity / Q000821:virology; D010222:Parainfluenza Virus 1, Human / Q000235:genetics / Q000302:isolation & purification; D010223:Parainfluenza Virus 2, Human / Q000235:genetics / Q000302:isolation & purification; D010224:Parainfluenza Virus 3, Human / Q000235:genetics / Q000302:isolation & purification; D029482:Parainfluenza Virus 4, Human / Q000235:genetics / Q000302:isolation & purification; D011786:Quality Control; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000302:isolation & purification; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012229:Rhinovirus / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014776:Virus Cultivation
D012367:RNA, Viral