Preparing the outbreak assistance laboratory network in the Netherlands for the detection of the influenza virus A(H1N1) variant

Protocols (N=14) used for influenza virus molecular detection in 11 outbreak assistance laboratories involved in preparing for surge diagnostic capacity for respiratory disease outbreaks. Flow diagram shows the variations in RNA extraction platforms, cDNA synthesis, PCR chemistry and amplification platforms, and amount of cDNA or RNA input into the PCR reaction. Weight of the lines reflects the number of laboratories using a particular step. Closed circles indicate steps that were shown to be critical, and dotted lines show steps for which no significant differences were observed in assay performance. Results of RT-PCR are expressed in the table for each of the specimens in the panel for the different targets used and split in one-step or two-step RT-PCR format, by showing the average Ct and standard deviation (SD) for the one-step M-gene based protocol using CDC primers and probe as the “gold standard”, and the delta average Ct and SD for all other targets by one- or two-step RT-PCR approach compared to the “gold standard”. Greyed cells indicate a deviation from the expected result.