Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples.
A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5'-3' nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997-1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998-1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997-1998 and 1998-1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.
Schweiger
B
B
Robert Koch-Institut, Berlin, Germany. Schweiger@rki.de
Zadow
I
I
Heckler
R
R
Timm
H
H
Pauli
G
G
D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D005453:Fluorescence; D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000302:isolation & purification; D009981:Influenza B virus / Q000145:classification* / Q000302:isolation & purification; D007251:Influenza, Human / Q000453:epidemiology / Q000821:virology*; D008969:Molecular Sequence Data; D010614:Pharynx / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012367:RNA, Viral / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity; D019914:Taq Polymerase
D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D012367:RNA, Viral; D019914:Taq Polymerase