title: Reverse-transcription, loop-mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype avian influenza viruses.
abstract: BACKGROUND
  The H10 subtype avian influenza viruses (H10N4, H10N5 and H10N7) have been reported to cause disease in mammals, and the first human case of H10N8 subtype avian influenza virus was reported in 2013. Recently, H10 subtype avian influenza viruses (AIVs) have been followed more closely, but routine diagnostic tests are tedious, less sensitive and time consuming, rapid molecular detection assays for H10 AIVs are not available.
  
  
  METHODS
  Based on conserved sequences within the HA gene of the H10 subtype AIVs, specific primer sets of H10 subtype of AIVs were designed and assay reaction conditions were optimized. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the rapid detection of H10 subtype AIVs. The specificity was validated using multiple subtypes of AIVs and other avian respiratory pathogens, and the limit of detection (LOD) was tested using concentration gradient of in vitro-transcribed RNA.
  
  
  RESULTS
  The established assay was performed in a water bath at 63 °C for 40 min, and the amplification result was visualized directly as well as under daylight reflections. The H10-RT-LAMP assay can specifically amplify H10 subtype AIVs and has no cross-reactivity with other subtypes AIVs or avian pathogens. The LOD of the H10-RT-LAMP assay was 10 copies per μL of in vitro-transcribed RNA.
  
  
  CONCLUSIONS
  The RT-LAMP method reported here is demonstrated to be a potentially valuable means for the detection of H10 subtype AIV and rapid clinical diagnosis, being fast, simple, and low in cost. Consequently, it will be a very useful screening assay for the surveillance of H10 subtype AIVs in underequipped laboratories as well as in field conditions.
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. luosisi2011@126.com.
authors.forename: Sisi
authors.initials: S
authors.lastname: Luo
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. xiezhixun@126.com.
authors.forename: Zhixun
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. xie3120371@163.com.
authors.forename: Liji
authors.initials: L
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. jiaboliu@163.com.
authors.forename: Jiabo
authors.initials: J
authors.lastname: Liu
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. xzqman2002@sina.com.
authors.forename: Zhiqin
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. dengxianwen@126.com.
authors.forename: Xianwen
authors.initials: X
authors.lastname: Deng
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. lhuang405@126.com.
authors.forename: Li
authors.initials: L
authors.lastname: Huang
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. huangjiaoling728@126.com.
authors.forename: Jiaoling
authors.initials: J
authors.lastname: Huang
authors.affiliation: Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, 530001, P.R. China. tingtingzeng1986@163.com.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zeng
authors.affiliation: Department of Pathobiology and Veterinary Science, University of Connecticut, 61 North Eagleville Road Storrs, Connecticut, 06269-3089, USA. mazhar.khan@uconn.edu.
authors.forename: Mazhar I
authors.initials: MI
authors.lastname: Khan
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D015047:Zoonoses / Q000175:diagnosis* / Q000821:virology
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus