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 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.
Minosse
Claudia
C
Laboratory of Virology, National Institute of Infectious Diseases L. Spallanzani, Rome, Italy.
Selleri
Marina
M
Zaniratti
Maria S
MS
Lauria
Francesco N
FN
Puro
Vincenzo
V
Carletti
Fabrizio
F
Cappiello
Giuseppina
G
Gualano
Gina
G
Bevilacqua
Nazario
N
Capobianchi
Maria R
MR
D017931:DNA Primers; D004196:Disease Outbreaks; D005814:Genes, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000302:isolation & purification; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000453:epidemiology; D007558:Italy / Q000453:epidemiology; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D012137:Respiratory System / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012621:Seasons; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics
D017931:DNA Primers; C048842:NP protein, Influenza A virus; C108309:NS protein, influenza virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins