Optimisation of reverse transcriptase loop-mediated isothermal amplification assay for rapid detection of Macrobrachium rosenbergii noda virus and extra small virus in Macrobrachium rosenbergii

The standardisation and optimisation of a one step single tube reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macro brachium rosenbergii noda virus (MrNV) and extra small virus (XSV), in giant fresh water prawn, M. rosenbergii. Time, temperature and quantity of each reagent were optimised for the detection of the two viruses. This method was more sensitive than the conventional reverse transcriptase polymerase chain reaction (RT-PCR) for detecting the two viruses. The RT-LAMP reaction is highly suited for disease diagnosis in developing countries. Amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of a whitish precipitate of magnesium pyrophosphate as a by-product. The cost of RI-LAMP for one reaction is nearly 4 times less than that of RT-PCR. (C) 2010 Elsevier B.V. All rights reserved.