The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR.
J Clin Virol
; 48(2): 91-5, 2010 Jun.
Article
em En
| MEDLINE
| ID: mdl-20413345
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. STUDYDESIGN:
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.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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Viral
/
Sondas de Oligonucleotídeos
/
Primers do DNA
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Pareamento Incorreto de Bases
/
Influenza Humana
/
Vírus da Influenza A Subtipo H1N1
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
País/Região como assunto:
Asia
Idioma:
En
Revista:
J Clin Virol
Assunto da revista:
VIROLOGIA
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Tailândia