Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification / 病毒学报
Chinese Journal of Virology
;
(6): 81-87, 2010.
Artigo
em Chinês
| WPRIM
| ID: wpr-297902
ABSTRACT
A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Suínos
/
Doenças dos Suínos
/
Temperatura
/
Virologia
/
Química
/
Sensibilidade e Especificidade
/
Primers do DNA
/
Colorimetria
/
Infecções por Orthomyxoviridae
/
Glicoproteínas de Hemaglutininação de Vírus da Influenza
Tipo de estudo:
Estudo diagnóstico
Limite:
Animais
/
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Virology
Ano de publicação:
2010
Tipo de documento:
Artigo
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