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 su btyping of influenza virus genomes in clinical specimens. The TaqMan-PCR em ploys a probe technology that exploits the endogenous 5'-3' nuclease activi ty of the Tag DNA polymerase to allow direct detection of the amplicon by r elease of a fluorescent reporter during the PCR. Therefore, post-PCR analys is is avoided since hybridization with the fluorogenic probe and quantifica tion of the amplified product is performed simultaneously during PCR cyclin g. The specificity of the method was evaluated on 86 influenza A (25 H1N1 a nd 61 H3N2) and 19 influenza B virus reference strains and isolates. The se nsitivity of the technique was found to be at the level of 0.1 50% tissue c ulture infective dose. This TaqMan-PCR was applied prospectively to surveil lance work by community-based sampling in Germany during the last two influ enza seasons. Seven hundred five throat swabs were analyzed during the wint er 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 19 98-1999 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 differentia tion of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997-1998 and 1998-1999. The TaqMa n-PCR was much more sensitive than culture and revealed an excellent correl ation for typing and subtyping of influenza viruses when samples were posit ive by both methods.