[Evaluation of reverse transcription loop-mediated isothermal amplification for detection of avian influenza A H5N1 virus].
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.
Li
Qi-Ming
QM
State Key Laboratory for Molecular Virology and Genetic Engineering, Beijing 100052, China.
Ma
Xue-Jun
XJ
Gao
Han-Chun
HC
Zhou
Rui
R
Kuang
Zhi-Zhou
ZZ
Hou
Yun-De
YD
D000818:Animals; D001717:Birds; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000302:isolation & purification; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity