Evaluation of reverse transcription loop-mediated isothermal amplification for detection of avian influenza A H5N1 virus.
- Author:
Qi-Ming LI
1
;
Xue-Jun MA
;
Han-Chun GAO
;
Rui ZHOU
;
Zhi-Zhou KUANG
;
Yun-De HOU
Author Information
1. State Key Laboratory for Molecular Virology and Genetic Engineering, Beijing 100052, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Birds;
Influenza A Virus, H5N1 Subtype;
genetics;
isolation & purification;
Influenza in Birds;
diagnosis;
virology;
Nucleic Acid Amplification Techniques;
methods;
Reproducibility of Results;
Reverse Transcriptase Polymerase Chain Reaction;
methods;
Sensitivity and Specificity
- From:
Chinese Journal of Virology
2008;24(3):178-184
- CountryChina
- Language:Chinese
-
Abstract:
A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was established to provide a new alternative for clinical diagnosis of Avian influenza A H5N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the target for amplification of nucleic acid under isothermal conditions. In current study, fifty-one experimentally infected animal specimens and viral cultures that had been tested were analyzed by RT-LAMP for NA gene and HA gene, respectively. The amplification process of LAMP was monitored in real-time by the addition of SYBR Green dye. Meanwhile, the result showed high correlation between nested PCR and RT-LAMP. The specificity of the RT-LAMP assay was confirmed by restriction enzyme digestion analysis and sequencing of the amplified product. When the sensitivity of this assay was tested by serial 10-fold dilutions of RNA molecules from specimens, it was found that the RT-LAMP method achieved theoretically a sensitivity of 10 RNA molecules. Thus, we concluded that the RT-LAMP assay has potential usefulness for rapid detection of the Avian influenza A H5N1 virus.
