Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification

Department of Aquaculture, College of Fisheries, Kerala Agricultural University, Cochin, India.
Journal of Fish Diseases (Impact Factor: 2.06). 06/2006; 29(5):275-83. DOI: 10.1111/j.1365-2761.2006.00718.x
Source: PubMed


A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product.

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    • "a large amount of target sequences under isothermal conditions between 63 and 65°C with - out the requirement for a thermal cycler within 60 min . Amplification of DNA can be detected by observing the presence of a white precipitate of magnesium pyrophos - phate as a by - product without the use of agarose gel electrophoresis ( Mori et al . 2001 ; Pillai et al . 2006 ) . Besides observing the presence of a white precipitate of magnesium pyrophosphate , there are several ways to ver - ify the LAMP reaction . The addition of ethidium bromide ( EtBr ) will yield a pink colour on the positive LAMP reaction product and the solution will remain the original orange colour if there are no reaction products "
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    • "Following reverse transcription (RT) of RNA into cDNA, the LAMP method can also be used to detect RNA (Whiting and Champoux, 1998). RT-LAMP methods have been developed to detect shrimp RNA viruses including yellow head virus (YHV) (Mekata et al., 2006), Macrobrachium rosenbergii nodavirus (MrNV) (Pillai et al., 2006) and Taura syndrome virus (TSV) (Kiatpathomchai et al., 2007). In these methods, DNA amplified is detected by agarose gel electrophoresis, ethidium bromide staining and UV transillumination. "
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    • "In previous reports, the RT-LAMP reaction can be accelerated by adding specific loop primers (Nagamine et al., 2002; Blomstrom et al., 2008). However, the presence of specific loop primers can result in unstable reactions according to some other studies (Pillai et al., 2006; Teng et al., 2007). In this study, the loop primers were not designed, and no RT-LAMP products were detected <45 min. "
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