Authors : You-Ling Wang, Jin-Xing Yang, Xiao -Yuan Yuan, Wen-Bao Qi, Ming Liao and Wu Ai
Abstract: Avian Influenza Viruses (AIV) have been isolated from a wide range of avian species through out the world. And the H4 subtype AIV is one of the most predominant subtypes among Low Pathogenic AIVs (LPAIVs). Recently some H4 subtype AIVs can be found and isolated in South China from chicken or duck and have become the main popular LPAIVs subtype. Consequently, the development of a rapid, simple, Sensitive Detection Method for H4 subtype AIVs is required for us. A Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) assay was developed to detect the H4 subtype AIVs visually. Specific primer sets targeting the sequences of the Hemagglutinin (HA) gene of H4 subtype AIVs were designedand RT-LAMP assay reaction conditions were optimized, ordinary RT-PCR and virus isolation assays were compared. The established LAMP assay was performed for 50 min in a water bathand the amplifications were visualized directly Under ultraviolet (UV) light. The detection limit of the RT-LAMP assay was 10 EID 50 mL-1 RNA of virus which was more sensitive than that of ordinary one-step RT-PCR (100 EID50 mL-1 RNA of virus). The specificity results indicated that the assay had no cross-reactions with other subtype AIVs. Furthermore, the result of clinical samples by the H4-subtype-specific RT-LAMP were consistent with that of virus isolation which was superior to ordinary RT-PCR. In this study, the established RT-LAMP assay with high sensitivity was performed completely within only 50 minand the amplification results were visualized by adding fluorescence reagent. The newly developed simple assay can be used as one important method for detecting H4 subtype AIVs in clinical conditions with no need for specialized equipment and high techinique.
You-Ling Wang, Jin-Xing Yang, Xiao -Yuan Yuan, Wen-Bao Qi, Ming Liao and Wu Ai, 2014. Rapid Detection of H4 Subtype Avian Influenza Viruses by Reverse Transcription Loop-Mediated Isothermal Amplification Assay. Journal of Animal and Veterinary Advances, 13: 191-196.