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Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.
- Source :
-
Molecular and cellular probes [Mol Cell Probes] 2015 Dec; Vol. 29 (6), pp. 408-413. Date of Electronic Publication: 2015 Aug 31. - Publication Year :
- 2015
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Abstract
- Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be particularly useful to identify the main respiratory viruses directly from clinical samples, after nucleic acid extraction, and, also, to screen a large number of patients for epidemiological studies.<br /> (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Subjects :
- Adenoviridae classification
Coronavirus classification
Coronavirus isolation & purification
Enterovirus classification
Enterovirus isolation & purification
Human bocavirus classification
Humans
Influenza A virus classification
Influenza A virus isolation & purification
Influenza B virus classification
Influenza B virus isolation & purification
Metapneumovirus classification
Metapneumovirus isolation & purification
RNA Viruses classification
Respiratory Syncytial Viruses classification
Respiratory Syncytial Viruses isolation & purification
Respirovirus classification
Respirovirus isolation & purification
Rubulavirus classification
Rubulavirus isolation & purification
Adenoviridae isolation & purification
Human bocavirus isolation & purification
Multiplex Polymerase Chain Reaction methods
RNA Viruses isolation & purification
Respiratory Tract Infections virology
Subjects
Details
- Language :
- English
- ISSN :
- 1096-1194
- Volume :
- 29
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Molecular and cellular probes
- Publication Type :
- Academic Journal
- Accession number :
- 26334289
- Full Text :
- https://doi.org/10.1016/j.mcp.2015.08.006