title: Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7.
abstract: A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7.
authors.affiliation: Animal Influenza Laboratory of the Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Bao
authors.forename: Xiurong
authors.initials: X
authors.lastname: Wang
authors.forename: Yuhui
authors.initials: Y
authors.lastname: Zhao
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Sun
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Li
authors.forename: Yongzhong
authors.initials: Y
authors.lastname: Xiong
authors.forename: Hualan
authors.initials: H
authors.lastname: Chen
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002645:Chickens; D002988:Cloaca / Q000821:virology; D009980:Influenza A virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010614:Pharynx / Q000821:virology; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D017201:Virus Shedding