Pilot Evaluation of RT-PCR/Electrospray Ionization Mass Spectrometry (PLEX-ID/Flu assay) on Influenza-Positive Specimens

Cordey, Samuel; Thomas, Yves; Suter, Patricia; Kaiser, Laurent

The typing performance of the PLEX-ID/Flu assay versus the Sanger-based sequencing method was then compared for all influenza specimens with low viral loads (C T values 30; 19 influenza A and 18 influenza B-positive NPS; Table 2 ). Briefly, for the Sanger-based sequencing method, reverse transcription was performed in the presence of Uni12w or BUni11w primers updated from [20] and [21] , respectively, provided by Prof. Rod Daniels (MRC, London). Part of the influenza HA-1 gene was then amplified as follows: for influenza A viruses, a first PCR reaction was performed with cswHAF1 and cswHAR1264 primers, followed by two nested PCR reactions with cswHAF31/cswHAR873 and cswHAF451/cswHAR1264 primer pairs (supplementary Table 1 ). For influenza B viruses, BHA1F1 and BHA1R1 primers were used for the first PCR reaction, followed by a nested PCR using the BHA25/BHAF primer pair. Sequencing was performed with ABI Prism 3130XL DNA Sequencer (Applied Biosystems, Rotkreuz, Switzerland) and analyzed with the Geneious program (Biomatters, Auckland, New Zealand). The same 29 negative controls are used in both comparisons (marked *). ND: not done.