Two different kinds of red blood cells were used since the actual H3N2 influenza strains did not react with chicken red blood check details cells. Material from the highest log10 inoculum dilution, which showed a clearly positive HA reaction after the previous passage, was used for the following passage. Extraction of viral DNA or RNA from clinical specimens and culture supernatants was performed with the Nucleic Acid Isolation Kit I in the MagNA Pure compact extraction system (Roche) or with the QIAsymphony® Virus/Bacteria Midi Kit (Qiagen) in the QIAsymphony robotic system. The ResPlex II

v2.0 multiplex PCR panel (Qiagen) was used according to the manufacturer’s instructions. The test applies a RT-PCR (reverse transcription and PCR reaction) by the OneStep RT PCR Kit (Qiagen) in combination with two pairs of specific primers for each target. The enzyme mix contains the Omniscript™ and Sensiscript™ reverse transcriptase and the HotStarTaq™ DNA polymerase. The dNTP mix contained 10 mM of each dNTP. The primer mix consisted of a mixture of individual primers for each viral target, carrying a tail with the target sequence for the superprimers, and the forward and backwards superprimers. Results of the multiplex PCRs were read with the LiquiChip detection system, which consists of microspheres coated with target-specific hybridization molecules and a steptavidin–biotin JQ1 based fluorescence

detection reaction giving an individual fluorescence color pattern for each viral target. Result readings were evaluated with the QIAplex MDD-RVO Beta software. According to the manufacturer’s instructions signals above values of 150 are positive, values below 100 are negative and values between 100 and 150 are considered as questionable results. The method’s results are given as counts (median fluorescence intensity, MFI) but the method is not intended

or designed to be used quantitatively. The ResPlex II v2.0 method is designed to detect 18 different virus species or virus subgroups simultaneously. These pathogens and the target genes used are summarized in Table 1. Independent, conventional in-house qRT-PCRs or commercially available PCR methods were used to confirm ResPlex results with clinical Sclareol specimens. These methods and according references are summarized in Table 5. The total number of samples investigated was 468. Positive results with the ResPlex II v2.0 PCR were obtained with 370 (79%) samples. Due to 21 double and one triple infection in the same sample the total number of virus-positive results was 393 in the 370 samples. Of the positive results 317 (85.7%) were positive for influenza virus with an almost equal distribution between A and B subtypes. 76 positive results with 66 samples indicated the presence of other respiratory viruses. The proportion of the different viruses found by the multiplex PCR is shown in Table 2.