Journal of Clinical Virology 48 (2010) 91–95 Contents lists available at ScienceDirect Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR Chonticha Klungthong, Piyawan Chinnawirotpisan, Kittinun Hussem, Thipwipha Phonpakobsin, Wudtichai Manasatienkij, Chuanpis Ajariyakhajorn, Kamonthip Rungrojcharoenkit, Robert V. Gibbons, Richard G. Jarman ∗ Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand article info Article history: Received 11 March 2010 Accepted 12 March 2010 Keywords: Novel influenza A (H1N1) 2009 Real-time RT-PCR Primer Probe Mismatches abstract Background: In response to the 2009 H1N1 pandemic the US CDC and WHO rapidly developed and dis- tributed a real-time RT-PCR kit to detect this strain in clinical samples. The results from the WHO swH1 primer and probe set exhibited diverse sensitivities for the 2009 influenza A/H1N1 strains in Southeast Asia (SEA). Objective: Investigate the primer and probe-template mismatches among the 2009 influenza A/H1N1 strains in SEA that reduced the real-time RT-PCR sensitivity. Study design: Thirty-seven swH1 positive samples categorized into sensitive and insensitive groups based on real-time RT-PCR results were selected for hemagglutinin (HA) gene sequencing. The sequence in swH1 primer and probe binding regions of the viruses was examined for mismatches. Phylogenetic analysis was performed to investigate the diversity among these viruses. Primers and probe were redesigned to match each of our sequences and tested to determine the impact on sensitivity. Results: HA sequencing of the viruses isolated from patients with high and low sensitivities revealed that a single mismatch at the 3rd base of the probe reduced sensitivity in 23/37 viruses. Homologous primers and probes increased the sensitivity (mean difference 4.66 Ct P < 0.0001). Phylogenetic tree revealed that the viruses in this study clustered into two groups, coinciding with RT-PCR sensitivity. Conclusion: Results obtained indicate that at least two variants of the novel H1N1 transmitting in SEA and the mutations in HA gene have a direct effect on the detection by using WHO swH1 primer and probe set. Published by Elsevier B.V. 1. Background Since the emergence of a novel influenza A/H1N1 in April 2009 in Mexico and the United States, sustained transmission worldwide occurred rapidly. The World Health Organization (WHO) declared pandemic alert stage 6 on 11 June 2009. 1 The urgent production of the 2009 H1N1 vaccines remains inadequate. 2,3 Early detection of the novel influenza A/H1N1/2009 virus is crucial for control and prevention of the pandemic. To support laboratories world- wide, the WHO provides a real-time RT-PCR (rRT-PCR) protocol for the detection of A/H1N1/2009. The kit contains primers and probe sets for detection of influenza A viruses (InfA), swine influenza A (swInfA), swine H1 (swH1), and a RNaseP control. 4 The Armed Forces Research Institute of Medical Sciences conducts influenza surveillance in Southeast Asia (SEA) using this WHO recommended ∗ Corresponding author. Tel.: +66 2 696 1756; fax: +66 2 644 4760. E-mail address: richard.jarman@afrims.org (R.G. Jarman). assay for detection of novel influenza A/H1N1/2009 virus. We observed that the swH1 primer and probe exhibited diverse sen- sitivities for SEA virus strains. We hypothesized that mutation in hemagglutinin (HA) gene contained primer and/or probe-template mismatches reducing rRT-PCR sensitivity. 2. Objective Objective of the study was to investigate the swH1 primers and probe binding region in HA gene sequences of the 2009 influenza A/H1N1 strains in SEA to determine if primer and probe-template mismatches reduced rRT-PCR sensitivity 3. Study design 3.1. Clinical specimens Nasal and/or throat swabs collected from Thailand, Bhutan, and the Philippines patients with influenza like illness (ILI) were placed 1386-6532/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jcv.2010.03.012