tetano
Editor, Senior Moderator
Mol Cell Probes. 2015 Aug 31. pii: S0890-8508(15)30032-3. doi: 10.1016/j.mcp.2015.08.006. [Epub ahead of print]
[h=1]Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.[/h] Arvia R[SUP]1[/SUP], Corcioli F[SUP]2[/SUP], Ciccone N[SUP]3[/SUP], Della Malva N[SUP]4[/SUP], Azzi A[SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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[SUP]3[/SUP] copies/ml to 10[SUP]4[/SUP] copies/ml. For parainfluenza virus 2 only it was 10[SUP]5[/SUP] 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.
Copyright ? 2015 Elsevier Ltd. All rights reserved.
[h=4]KEYWORDS:[/h] Duplex real time PCR; Respiratory viruses detection
PMID: 26334289 [PubMed - as supplied by publisher]
[h=1]Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.[/h] Arvia R[SUP]1[/SUP], Corcioli F[SUP]2[/SUP], Ciccone N[SUP]3[/SUP], Della Malva N[SUP]4[/SUP], Azzi A[SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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[SUP]3[/SUP] copies/ml to 10[SUP]4[/SUP] copies/ml. For parainfluenza virus 2 only it was 10[SUP]5[/SUP] 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.
Copyright ? 2015 Elsevier Ltd. All rights reserved.
[h=4]KEYWORDS:[/h] Duplex real time PCR; Respiratory viruses detection
PMID: 26334289 [PubMed - as supplied by publisher]