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Development of a monoclonal antibody-based flow-through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

Authors

  • R Patil,

    1. Fish Pathology and Biotechnology Laboratory, Department of Aquaculture, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Mangalore, Karnataka, India
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  • K M Shankar,

    Corresponding author
    • Fish Pathology and Biotechnology Laboratory, Department of Aquaculture, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Mangalore, Karnataka, India
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  • B T N Kumar,

    1. Fish Pathology and Biotechnology Laboratory, Department of Aquaculture, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Mangalore, Karnataka, India
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  • A Kulkarni,

    1. Fish Pathology and Biotechnology Laboratory, Department of Aquaculture, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Mangalore, Karnataka, India
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  • P Patil,

    1. Frontier Science Research Center, University of Miyazaki Miyazaki-ken, Miyazaki-shi Kiyotake-chou, Kihara, Japan
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  • N Moger

    1. Institute of Agribiotechnology, University of Agricultural Sciences, Dharwad, Karnataka, India
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Correspondence K M Shankar, Fish Pathology and Biotechnology Laboratory, Department of Aquaculture, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Mangalore 575002, Karnataka, India (e-mail: kalkulishankar@gmail.com)

Abstract

A flow-through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL−1 crude WSSV protein, respectively. The FTA could be completed in 8–10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1-step polymerase chain reaction (PCR) and in between that of the 1- and 2-step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post-larvae, WSSV was first detected 14, 16 and 18 h post-infection (hpi) by FTA, immunodot and one-step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one-step PCR, respectively. The FTA was more sensitive (25/27) than one-step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4–8 °C. The FTA is available as a rapid test kit called ‘RapiDot’ for the early detection of WSSV under field conditions.

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