tetano
Editor, Senior Moderator
J Virol Methods. 2012 Jul 11. [Epub ahead of print]
Optimization of one-step duplex real-time rt-pcr for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates.
de-Paris F, Beck C, Machado AB, Paiva RM, Menezes DD, Nunes LD, Kuchenbecker R, Barth AL.
Source
Hospital de Cl?nicas de Porto Alegre (HCPA), Servi?o de Patologia Cl?nica, Unidade de Pesquisa Biom?dica-Laborat?rio de Biologia Molecular. Rua Ramiro Barcelos 2350, Porto Alegre - RS, Brazil 90035-903; Universidade Federal do Rio Grande do Sul (UFRGS), Faculdade de Medicina, Programa de P?s-gradua??o em Ci?ncias M?dicas. Rua Ramiro Barcelos 2400, Porto Alegre - RS, Brazil 90035-903.
Abstract
Viruses are major contributors to acute respiratory infection-related morbidity and mortality worldwide. The influenza (IF) viruses and human respiratory syncytial virus (RSV) play a particularly important role in the aetiology of acute respiratory infections. This study sought to standardize a one-step duplex real-time RT-PCR technique to optimize diagnosis of IFA/IFB and RSVA/RSVB infection. Viral RNA was extracted with the commercially available QIAamp Mini Kit according to manufacturer instructions. RT-PCR was performed with primers to the matrix protein gene of IFA, the hemagglutinin gene of IFB and the N gene of RSVA and RSVB. The limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The specificity of RT-PCR was determined by comparison against a panel of several respiratory pathogens. RT-PCR and indirect immunofluorescence (IIF) were compared in a sample of 250 nasopharyngeal aspirates (NPAs) collected during the year 2010. RT-PCR was more sensitive than IIF and able to detect viral co-infections. In summary, RT-PCR optimized for IFA/IFB and RSVA/RSVB is sensitive and specific for these viral agents and is therefore useful for assessment of the aetiology of respiratory infections, whether for clinical or epidemiological purposes.
Copyright ? 2012. Published by Elsevier B.V.
PMID:
22796284
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22796284
Optimization of one-step duplex real-time rt-pcr for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates.
de-Paris F, Beck C, Machado AB, Paiva RM, Menezes DD, Nunes LD, Kuchenbecker R, Barth AL.
Source
Hospital de Cl?nicas de Porto Alegre (HCPA), Servi?o de Patologia Cl?nica, Unidade de Pesquisa Biom?dica-Laborat?rio de Biologia Molecular. Rua Ramiro Barcelos 2350, Porto Alegre - RS, Brazil 90035-903; Universidade Federal do Rio Grande do Sul (UFRGS), Faculdade de Medicina, Programa de P?s-gradua??o em Ci?ncias M?dicas. Rua Ramiro Barcelos 2400, Porto Alegre - RS, Brazil 90035-903.
Abstract
Viruses are major contributors to acute respiratory infection-related morbidity and mortality worldwide. The influenza (IF) viruses and human respiratory syncytial virus (RSV) play a particularly important role in the aetiology of acute respiratory infections. This study sought to standardize a one-step duplex real-time RT-PCR technique to optimize diagnosis of IFA/IFB and RSVA/RSVB infection. Viral RNA was extracted with the commercially available QIAamp Mini Kit according to manufacturer instructions. RT-PCR was performed with primers to the matrix protein gene of IFA, the hemagglutinin gene of IFB and the N gene of RSVA and RSVB. The limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The specificity of RT-PCR was determined by comparison against a panel of several respiratory pathogens. RT-PCR and indirect immunofluorescence (IIF) were compared in a sample of 250 nasopharyngeal aspirates (NPAs) collected during the year 2010. RT-PCR was more sensitive than IIF and able to detect viral co-infections. In summary, RT-PCR optimized for IFA/IFB and RSVA/RSVB is sensitive and specific for these viral agents and is therefore useful for assessment of the aetiology of respiratory infections, whether for clinical or epidemiological purposes.
Copyright ? 2012. Published by Elsevier B.V.
PMID:
22796284
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22796284