Improved detection of human influenza A and B viruses in respiratory tract specimens by hemi-nested PCR

RT-PCR is the most sensitive assay for diagnosis of influenza, due to enhanced rapidity and sensitivity as compared to classical methods. Hemi-nested RT-PCR was developed, targeting NP gene for influenza A and NS gene for influenza B, based on a previous single round RT-PCR method. The new method wa...

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Bibliographic Details
Published in:Journal of virological methods 2007-05, Vol.141 (2), p.225-228
Main Authors: Minosse, Claudia, Selleri, Marina, Zaniratti, Maria S., Lauria, Francesco N., Puro, Vincenzo, Carletti, Fabrizio, Cappiello, Giuseppina, Gualano, Gina, Bevilacqua, Nazario, Capobianchi, Maria R.
Format: Article
Language:English
Subjects:
Biological and medical sciences
Disease Outbreaks
DNA Primers
Fundamental and applied biological sciences. Psychology
Genes, Viral
Humans
Influenza
Influenza A virus - genetics
Influenza A virus - isolation & purification
Influenza A Virus, H1N1 Subtype - genetics
Influenza A Virus, H1N1 Subtype - isolation & purification
Influenza A Virus, H3N2 Subtype - genetics
Influenza A Virus, H3N2 Subtype - isolation & purification
Influenza B virus - genetics
Influenza B virus - isolation & purification
Influenza, Human - diagnosis
Influenza, Human - epidemiology
Italy - epidemiology
Microbiology
Molecular diagnosis
Nucleoproteins - genetics
Respiratory infections
Respiratory System - virology
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA-Binding Proteins - genetics
RT-PCR
Seasons
Sensitivity and Specificity
Techniques used in virology
Viral Core Proteins - genetics
Viral Nonstructural Proteins - genetics
Virology
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Summary:RT-PCR is the most sensitive assay for diagnosis of influenza, due to enhanced rapidity and sensitivity as compared to classical methods. Hemi-nested RT-PCR was developed, targeting NP gene for influenza A and NS gene for influenza B, based on a previous single round RT-PCR method. The new method was compared with the previous technique for analytical sensitivity and specificity, and was applied to clinical samples from the lower and upper respiratory tract. The analytical sensitivity of hemi-nested RT-PCR was 10 (influenza A) and 4 times (influenza B) higher than the previous method. A high specificity of the new hemi-nested RT-PCR assay was observed by using whole respiratory viruses. When applied to lower respiratory tract specimens, the new method showed an increased rate of positivity as compared to the previous technique (9.3% versus 0.7% for influenza A, and 0.9% versus 0.2% for influenza B). Screening of upper respiratory tract samples collected during the seasonal 2005–2006 outbreak indicated 26.4% and 5.8% positivity for influenza A and B, respectively. The results were confirmed by sequence analysis: apart from influenza B, both influenza A subtypes H3N2 and H1N1, associated with the seasonal outbreak, were detected.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2006.12.010