Development of reverse transcription loop-mediated isothermal amplification for rapid detection of H9 avian influenza virus.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 45 min at 63 degrees C. In this study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H9 subtype of avian influenza virus (AIV). The experiment results from the reference strains demonstrated that the established RT-LAMP sensitivity was 10-fold higher than that of RT-PCR, with the detection limit of 10 copies per reaction, and no cross-reactivity was observed from the samples of other related viruses including H5N1, H3N2 subtype of AIV and Newcastle disease virus. Furthermore, a total of 112 clinical samples were tested by RT-LAMP, RT-PCR, and virus isolation, respectively. All of the 85 positive specimens identified by virus isolation were also positive by RT-LAMP, while 7 of these samples were missed by RT-PCR. These results suggest that the present RT-LAMP system may provide a new avenue for the recognition of H9 subtype virus, and may be employed to screen for potential carriers in wild and domestic birds.
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Hao-Tai
HT
Chen
Jie
J
Zhang
De-Hui
DH
Sun
Li-Na
LN
Ma
Xiang-Tao
XT
Liu
Xue-Peng
XP
Cai
Yong-Sheng
YS
Liu
D000818:Animals; D001483:Base Sequence; D001717:Birds; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics* / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral