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.
Article
in Chinese
| 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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Swine
/
Swine Diseases
/
Temperature
/
Virology
/
Chemistry
/
Sensitivity and Specificity
/
DNA Primers
/
Colorimetry
/
Orthomyxoviridae Infections
/
Hemagglutinin Glycoproteins, Influenza Virus
Type of study:
Diagnostic study
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Chinese Journal of Virology
Year:
2010
Type:
Article
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