A multiplex RT-PCR for the detection of parainfluenza viruses 1-3 in clinical samples.
Parainfluenza viruses (PIV) are an important cause of respiratory morbidity. Conventional diagnostic methods for detection of PIV are time consuming or lack sensitivity. A multiplex PCR that detects PIV 1-3 was developed using novel primers for PIV viruses 1 and 2 and primers for PIV 3 described previously. Following RNA extraction a single multiplex reverse transcription was undertaken using antisense primers specific for each virus type. This was followed by a 40-cycle multiplex PCR using primers directed towards the haemagglutinin-neuraminidase coding region of each virus type. Products were probed with type-specific fluorescein labelled internal probes and detected by chemiluminescence. Cultured PIV viruses were detectable to a sensitivity of 1 TCID50. The technique was applied to 57 nasal aspirates taken from children presenting with various acute respiratory conditions and analysed previously by culture, immunofluorescence and/or serology. It was possible to detect PIV 1, 2 or 3 in 13/13 samples found previously positive for PIV by tissue culture, 13/15 found previously positive by immunofluorescence and 6/10 that coincided with positive serology. None of the samples found previously positive for other viruses (26) or negative to virus detection (6) were found positive by RT-PCR. It is concluded that this method is as sensitive as combined immunofluorescence and tissue culture for the detection of the PIV viruses 1-3 and should be useful for rapid diagnosis of PIV 1-3 infections.
Corne
J M
JM
University Medicine, Southampton General Hospital, UK.
Green
S
S
Sanderson
G
G
Caul
E O
EO
Johnston
S L
SL
D000818:Animals; D002460:Cell Line; D002648:Child; D003440:Croup / Q000473:pathology / Q000821:virology*; D046508:Culture Techniques / Q000379:methods; D005455:Fluorescent Antibody Technique; D006801:Humans; D008253:Macaca mulatta; 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*; D012367:RNA, Viral / Q000032:analysis; D010253:Respirovirus Infections / Q000473:pathology / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral