Optimization of one-step duplex real-time RT-PCR for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates

• Published in: Journal of virological methods Vol. 186; no. 1-2; pp. 189 - 192
• Main Authors: de-Paris, Fernanda, Beck, Caroline, Machado, Alice Beatriz Mombach Pinheiro, Paiva, Rodrigo Minuto, da Silva Menezes, Denise, de Souza Nunes, Luciana, Kuchenbecker, Ricardo, Barth, Afonso Luís
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.12.2012; Elsevier
• Subjects: Biological and medical sciences; DNA Primers - genetics; Etiology; Fundamental and applied biological sciences. Psychology; Hemagglutinins; Human respiratory syncytial virus; Humans; Immunofluorescence; Infection; Influenza; Influenza virus; Influenza, Human - diagnosis; Influenza, Human - virology; matrix protein; Microbiology; Molecular diagnostics; Molecular epidemiology; Morbidity; Mortality; Multiplex Polymerase Chain Reaction - methods; Multiplex Polymerase Chain Reaction - standards; N gene; Nasopharynx - virology; One-step real-time PCR; Orthomyxoviridae - isolation & purification; Pathogens; Polymerase chain reaction; Primers; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Respiratory syncytial virus; Respiratory Syncytial Virus Infections - diagnosis; Respiratory Syncytial Virus Infections - virology; Respiratory Syncytial Virus, Human - isolation & purification; Respiratory Tract Infections - virology; RNA; RNA, Viral - genetics; RNA, Viral - isolation & purification; Sensitivity and Specificity; Techniques used in virology; Virology; Molecular epidemiology; Molecular diagnostics; Respiratory syncytial virus; Influenza virus; One-step real-time PCR; Orthomyxoviridae; Genotype; Method; Real time; Influenzavirus; Optimization; Virus; Polymerase chain reaction; Detection; Reverse transcription polymerase chain reaction
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.07.008

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 etiology 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 1copy/μL for IFA, 10copies/μL for IFB, 5copies/μL for RSVA, and 250copies/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 etiology of respiratory infections, whether for clinical or epidemiological purposes.