The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR.
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.
Klungthong
Chonticha
C
Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand.
Chinnawirotpisan
Piyawan
P
Hussem
Kittinun
K
Phonpakobsin
Thipwipha
T
Manasatienkij
Wudtichai
W
Ajariyakhajorn
Chuanpis
C
Rungrojcharoenkit
Kamonthip
K
Gibbons
Robert V
RV
Jarman
Richard G
RG
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
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral