J Vet Sci.  2020 Mar;21(2):e24. 10.4142/jvs.2020.21.e24.

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

• Tsai SK
• Chen CC
• Lin HJ
• Lin HY
• Chen TT
• Wang LC

• KMID: 2506182
• DOI: http://doi.org/10.4142/jvs.2020.21.e24

Abstract

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 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 2.3.4.4 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.

Keyword

Capillary electrophoresis; influenza virus; reverse transcription

Figure

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  Fig. 1. Detection limit comparison between traditional plate agarose gel electrophoresis (A) and capillary electrophoresis (B). In vitro transcribed RNA from each targeted viral gene was quantified and used for multiplex reverse transcription recombinase polymerase amplification reaction templates. M, 100 bp ladder marker; LM, lower alignment marker (size = 20 bp); UM, upper alignment marker (size = 1,000 bp); 1, 108 copy numbers; 2, 107 copy numbers; 3, 106 copy numbers; 4, 105 copy numbers; 5, 104 copy numbers; 6, 103 copy numbers; 7, 102 copy numbers; 8, 101 copy numbers; 9, 100 copy numbers; IBV, infectious bronchitis virus; NDV, Newcastle disease virus. Other common avian respiratory viruses (IBV and NDV) and type A influenza viruses (clade 2.2.1 low pathogenic H5N2, human H1N1 and human H3N2) were used as control.

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  Fig. 2. The representative capillary electrophoresis results of the field samples (Blue line, tested field sample; Red line, positive control which was from the in vitro transcribed RNA standards). (A) All negative. (B) NP and H6 positive. (C) NP, H6 and H5 positive. (D) NP, H6 and H7 positive. (E) All of the four genes (NP, H6, H5, and H7) were positive. LM, lower alignment marker (20 bp); UM, upper alignment marker (1,000 bp); Peak 1, H7 HA gene (137 bp); Peak 2, H5 HA gene (173 bp); Peak 3, H6 HA gene (199 bp); Peak 4, NP gene (217 bp); NP, nucleoprotein.

Reference

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