Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification.
- Author:
Kai NIE
1
;
Da-Yan WANG
;
Meng QIN
;
Rong-Bao GAO
;
Miao WANG
;
Shu-Mei ZOU
;
Feng HAN
;
Xiang ZHAO
;
Xi-Yan LI
;
Yue-Long SHU
;
Xue-Jun MA
Author Information
1. State Key Laboratory for molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Colorimetry;
methods;
DNA Primers;
genetics;
Electrophoresis, Agar Gel;
Hemagglutinin Glycoproteins, Influenza Virus;
genetics;
Humans;
Influenza A Virus, H1N1 Subtype;
genetics;
Influenza A Virus, H3N2 Subtype;
genetics;
Influenza, Human;
diagnosis;
virology;
Naphthalenesulfonates;
chemistry;
Nucleic Acid Amplification Techniques;
methods;
Orthomyxoviridae Infections;
diagnosis;
veterinary;
virology;
Reverse Transcriptase Polymerase Chain Reaction;
methods;
Sensitivity and Specificity;
Swine;
Swine Diseases;
diagnosis;
virology;
Temperature
- From:
Chinese Journal of Virology
2010;26(2):81-87
- CountryChina
- Language:Chinese
-
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.
