Diagnostic testing for pandemic influenza in Singapore: a novel dual-gene quantitative real-time RT-PCR for the detection of influenza A/H1N1/2009

Abstract

With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant. Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009. The assay design includes a primer-probe set specific to only the hemagglutinin (HA) gene of this novel influenza A variant and a second set capable of detecting the nucleoprotein (NP) gene of all swine-origin influenza A virus. In silico analysis of the specific HA oligonucleotide sequence used in the assay showed that it targeted only the swine-origin pandemic strain; there was also no cross-reactivity against a wide spectrum of noninfluenza respiratory viruses. The assay has a diagnostic sensitivity and specificity of 97.7% and 100%, respectively, a lower detection limit of 50 viral gene copies/PCR, and can be adapted to either a qualitative or quantitative mode. It was first applied to 3512 patients with influenza-like illnesses at a tertiary hospital in Singapore, during the containment phase of the pandemic (May to July 2009).

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).

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.

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.

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.