Related Experiment Videos
A multiplex RT-PCR for the detection of parainfluenza viruses 1-3 in clinical samples
J M Corne1, S Green, G Sanderson
1University Medicine, Southampton General Hospital, UK.
Abstract:
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.
Insights
A new multiplex PCR method accurately detects parainfluenza viruses (PIV) 1-3 in respiratory samples. This rapid diagnostic tool offers sensitivity comparable to existing methods for PIV detection.
Area of Science:
- Virology
- Molecular Diagnostics
- Respiratory Infections
Background:
- Parainfluenza viruses (PIV) are significant contributors to respiratory morbidity.
- Current diagnostic methods for PIV detection are often slow or lack sufficient sensitivity.
- There is a need for rapid and sensitive diagnostic tools for PIV infections.
Purpose of the Study:
- To develop and evaluate a novel multiplex PCR assay for the simultaneous detection of Parainfluenza virus types 1, 2, and 3 (PIV 1-3).
- To assess the sensitivity and clinical utility of the developed multiplex PCR assay compared to conventional diagnostic techniques.
Main Methods:
- A multiplex PCR assay was designed using novel primers for PIV 1 and 2, and previously described primers for PIV 3.
- The assay involved RNA extraction, multiplex reverse transcription, and a 40-cycle multiplex PCR targeting the haemagglutinin-neuraminidase gene.
- Detection utilized type-specific fluorescein-labeled probes and chemiluminescence, with sensitivity assessed using cultured PIV viruses (1 TCID50).
Main Results:
- The multiplex RT-PCR assay demonstrated high sensitivity, detecting PIV 1-3 in clinical samples previously positive by tissue culture (13/13), immunofluorescence (13/15), and serology (6/10).
- The assay correctly identified no PIV in samples positive for other viruses (26) or negative samples (6).
- The limit of detection for cultured PIV viruses was as low as 1 TCID50.
Conclusions:
- The developed multiplex PCR method provides sensitive and specific detection of PIV 1-3.
- This assay is comparable in sensitivity to combined immunofluorescence and tissue culture methods.
- The multiplex RT-PCR assay is a valuable tool for the rapid diagnosis of PIV 1-3 infections in clinical settings.