|Combination of multiplex reverse transcription recombinase polymerase amplification assay and capillary electrophoresis provides high sensitive and high-throughput simultaneous detection of avian influenza virus subtypes
HAN YOU LIN
|Article; avian influenza virus; capillary electrophoresis; controlled study; data analysis; disease control; gene amplification; gene targeting; HA gene; limit of detection; multiplex reverse transcription polymerase chain reaction; nonhuman; NP gene; Taiwan; virus detection; virus gene; waterfowl; animal; avian influenza; capillary electrophoresis; high throughput screening; Influenza A virus; isolation and purification; multiplex polymerase chain reaction; procedures; reverse transcription polymerase chain reaction; veterinary medicine; virology; viral protein; Animals; Anseriformes; Electrophoresis, Capillary; Genes, Viral; High-Throughput Screening Assays; Influenza A virus; Influenza in Birds; Multiplex Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Taiwan; Viral Proteins
|Journal of Veterinary Science
The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 184.108.40.206 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 220.127.116.11 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry. ? 2020 The Korean Society of Veterinary Science.
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