Optimization of one-step duplex real-time RT-PCR for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates.
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 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 etiology of respiratory infections, whether for clinical or epidemiological purposes.
Hospital de Clínicas de Porto Alegre (HCPA), Serviço de Patologia Clínica, Unidade de Pesquisa Biomédica - Laboratório de Biologia Molecular, Rua Ramiro Barcelos 2350, Porto Alegre, RS 90035-903, Brazil. fparis@hcpa.ufrgs.br
Fernanda
F
de-Paris
Caroline
C
Beck
Alice Beatriz Mombach Pinheiro
AB
Machado
Rodrigo Minuto
RM
Paiva
Denise
D
da Silva Menezes
Luciana
L
de Souza Nunes
Ricardo
R
Kuchenbecker
Afonso Luís
AL
Barth
D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009305:Nasopharynx / Q000821:virology; D009975:Orthomyxoviridae / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis* / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000302:isolation & purification*; D012141:Respiratory Tract Infections / Q000821:virology*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral