Fish Pathology
Online ISSN : 1881-7335
Print ISSN : 0388-788X
ISSN-L : 0388-788X
Research Articles
Use of Acridine Orange to Visually Improve the Loop-mediated Isothermal Amplification for Detection of Infectious Spleen and Kidney Necrosis Virus
Kuttichantran SubramaniamMohamed ShariffAbdul Rahman OmarMohd Hair-BejoBee Lee Ong
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2014 Volume 49 Issue 4 Pages 173-180

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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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© 2014 The Japanese Society of Fish Pathology
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