Diagnostic Testing for Pandemic Influenza in Singapore

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Diagnostic Testing for Pandemic Influenza in Singapore Hong Kai Lee, Chun Kiat Lee, Tze Ping Loh, Julian Wei-Tze Tang, Lily Chiu, Paul A. Tambyah, Sunil K. Sethi, Evelyn Siew-Chuan Koay The Journal of Molecular Diagnostics Volume 12, Issue 5, Pages 636-643 (September 2010) DOI: 10.2353/jmoldx.2010.100010 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 A and C show the locations of the hemagglutinin (HA) and nucleoprotein (NP) primer and probe sequences for the dual-gene duplex pandemic influenza A/H1N1/2009-specific assay. B and D show the locations of the corresponding HA and NP primer and probe sequences for the CDC/World Health Organization website method (http://www.who.int/csr/resources/publications/swineflu/CDCrealtimeRTPCRprotocol_20090428.pdf, last accessed April 29, 2009). The Journal of Molecular Diagnostics 2010 12, 636-643DOI: (10.2353/jmoldx.2010.100010) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Amplification curves of duplicates of serially diluted RNA transcripts (in RNase-free water). The gene copy number for each of the six dilutions is indicated. Standard curves were plotted with threshold cycle (Ct) values against log10 concentrations of RNA transcripts. A: Representative amplification profile of the 10-fold serially diluted NP RNA transcript. The PCR efficiency of 1.818 was calculated by the LC v2.0 software. B: Corresponding amplification profile of the 10-fold serially diluted HA RNA transcript, with calculated PCR efficiency of 1.772. The Journal of Molecular Diagnostics 2010 12, 636-643DOI: (10.2353/jmoldx.2010.100010) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 A: Boxplot for Ct values of 15 consecutive runs for the six-point standard curve of the 10-fold serially diluted NP RNA transcript (5 × 102–5 × 107 copies per PCR). Percentage coefficients of variation (%CV) for all six standards were less than 3.5%, and SD, less than 1.5. B: Boxplot for Ct values of 15 consecutive runs for the corresponding six-point standard curve of the 10-fold serially diluted HA RNA transcript (5 × 102–5 × 107 copies per PCR), with %CV for all six standards of less than 3%, and SD, less than 0.8. Boxes in both charts contain the middle 50% of the Ct values, with the upper hinge of the box indicating the 75th percentile of the Ct values, the lower hinge indicating the 25th percentile, and the line in the box denoting the median value of the data. The ‘whiskers’ indicate the minimum and maximum Ct values for each set of 15 replicates. X-Y (scatter) plots of Ct values in 15 consecutive runs against the log10 viral gene copies/PCR were drawn. The best fit lines for the amplification of NP (C) and HA (D) transcripts gave R2 = 0.988 for both. The Journal of Molecular Diagnostics 2010 12, 636-643DOI: (10.2353/jmoldx.2010.100010) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 X-Y (scatter) plots of the Ct values of the initial diagnostic testing against the Ct values of the repeat testing of the same 286 archived patient specimens. The two runs were performed on different days, by different operators, using different reaction mixes. The best fit lines for NP (A) and HA (B) gene target scatter plots were derived, with slopes/gradients of 0.9806 and 0.9341, respectively. The Journal of Molecular Diagnostics 2010 12, 636-643DOI: (10.2353/jmoldx.2010.100010) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions