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Use of acridine orange to visually improve the loop-mediated isothermal amplification for detection of infectious spleen and kidney necrosis virus


Citation

Subramaniam, Kuttichantran and Mohamed Din, Mohamed Shariff and Omar, Abdul Rahman and Bejo, Mohd Hair and Ong, Bee Lee (2014) Use of acridine orange to visually improve the loop-mediated isothermal amplification for detection of infectious spleen and kidney necrosis virus. Fish Pathology, 49 (4). pp. 173-180. ISSN 0388-788X; ESSN: 1881-7335

Abstract

The advancement in nucleic acid amplification has improved the diagnostic methods for many diseases. In the current study, an improved technique for the detection of infectious spleen and kidney necrosis virus (ISKNV) based on loop-mediated isothermal amplification (LAMP) is described. The addition of acridine orange at the end of the reaction causes distinct color change which can be recognized by the naked eye. In addition, a new set of primers was designed based on the ISKNV major capsid protein (MCP) gene sequences. The primers are highly specific for ISKNV and there was no cross amplification with red sea bream iridovirus (RSIV), white spot syndrome virus (WSSV), Aeromonas hydrophila or Vibrio parahaemolyticus. The detection limit of LAMP assay was 1.4 × 104 copies of the virus DNA and the optimum temperature and time for assay was 65°C and 60 min respectively, and this protocol could successfully detect the virus from asymptomatically infected fish. This improved LAMP assay is a simple and inexpensive diagnostic tool for the detection of ISKNV without the need of specialized equipment.


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Additional Metadata

Item Type: Article
Divisions: Faculty of Veterinary Medicine
Institute of Bioscience
DOI Number: https://doi.org/10.3147/jsfp.49.173
Publisher: Japanese Society of Fish Pathology
Keywords: Infectious spleen and kidney necrosis virus; Megalocytivirus; Major capsid protein gene; Loop-mediated isothermal amplification; Acridine orange; LAMP
Depositing User: Nurul Ainie Mokhtar
Date Deposited: 11 Feb 2016 08:55
Last Modified: 11 Feb 2016 08:55
Altmetrics: http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.3147/jsfp.49.173
URI: http://psasir.upm.edu.my/id/eprint/35934
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