title: Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay.
abstract: BACKGROUND
  Recent epidemiological investigation of different HA subtypes of avian influenza viruses (AIVs) shows that the H3 subtype is the most predominant among low pathogenic AIVs (LPAIVs), and the seasonal variations in isolation of H3 subtype AIVs are consistent with that of human H3 subtype influenza viruses. Consequently, the development of a rapid, simple, sensitive detection method for H3 subtype AIVs is required. The loop-mediated isothermal amplification (LAMP) assay is a simple, rapid, sensitive and cost-effective nucleic acid amplification method that does not require any specialized equipment.
  
  
  RESULTS
  A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect the H3 subtype AIVs visually. Specific primer sets target the sequences of the hemagglutinin (HA) gene of H3 subtype AIVs were designed, and assay reaction conditions were optimized. The established assay was performed in a water bath for 50 minutes, and the amplification result was visualized directly as well as under ultraviolet (UV) light reflections. The detection limit of the RT-LAMP assay was 0.1pg total RNA of virus, which was one hundred-fold higher than that of RT-PCR. The results on specificity indicated that the assay had no cross-reactions with other subtype AIVs or avian respiratory pathogens. Furthermore, a total of 176 clinical samples collected from birds at the various live-bird markets (LBMs) were subjected to the H3-subtype-specific RT-LAMP (H3-RT-LAMP). Thirty-eight H3 subtype AIVs were identified from the 176 clinical samples that were consistent with that of virus isolation.
  
  
  CONCLUSIONS
  The newly developed H3-RT-LAMP assay is simple, sensitive, rapid and can identify H3 subtype AIVs visually. Consequently, it will be a very useful screening assay for the surveillance of H3 subtype AIVs in underequipped laboratories as well as in field conditions.
authors: Department of Biotechnology, Guangxi Veterinary Research Institute, 51 You Ai Road, Nanning, Guangxi 530001, China.
authors: Yi
authors: Y
authors: Peng
authors: Zhixun
authors: Z
authors: Xie
authors: Jiabo
authors: J
authors: Liu
authors: Yaoshan
authors: Y
authors: Pang
authors: Xianwen
authors: X
authors: Deng
authors: Zhiqin
authors: Z
authors: Xie
authors: Liji
authors: L
authors: Xie
authors: Qing
authors: Q
authors: Fan
authors: Jiaxun
authors: J
authors: Feng
authors: Mazhar I
authors: MI
authors: Khan
mesh_terms: D000818:Animals; D001717:Birds; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus