title: The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR.
abstract: BACKGROUND
  In response to the 2009 H1N1 pandemic the US CDC and WHO rapidly developed and distributed 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.
  
  
  METHODS
  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 4.66Ct P<0.0001). Phylogenetic tree revealed that the viruses in this study clustered into two groups, coinciding with RT-PCR sensitivity.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand.
authors.forename: Chonticha
authors.initials: C
authors.lastname: Klungthong
authors.forename: Piyawan
authors.initials: P
authors.lastname: Chinnawirotpisan
authors.forename: Kittinun
authors.initials: K
authors.lastname: Hussem
authors.forename: Thipwipha
authors.initials: T
authors.lastname: Phonpakobsin
authors.forename: Wudtichai
authors.initials: W
authors.lastname: Manasatienkij
authors.forename: Chuanpis
authors.initials: C
authors.lastname: Ajariyakhajorn
authors.forename: Kamonthip
authors.initials: K
authors.lastname: Rungrojcharoenkit
authors.forename: Robert V
authors.initials: RV
authors.lastname: Gibbons
authors.forename: Richard G
authors.initials: RG
authors.lastname: Jarman
mesh_terms: D001210:Asia, Southeastern; D020137:Base Pair Mismatch; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral