期刊
JOURNAL OF VIROLOGICAL METHODS
卷 187, 期 2, 页码 384-389出版社
ELSEVIER
DOI: 10.1016/j.jviromet.2012.11.005
关键词
GCRV; Detection; Isothermal amplification; RT-LAMP; Grass carp
资金
- National Key Technology RD Program [2012BAD17B01]
- Major agricultural project of major science and technology special project of Science and Technology Department of Zhejiang Province [2010C02001]
- Science and Technology Research Project of Huzhou [2011GG11]
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a gene amplification method that can amplify the RNA template by isothermal incubation. This paper reports a rapid and sensitive RT-LAMP method which was developed for the detection of grass carp reovirus (GCRV). The present study concluded the optimal conditions for the LAMP reaction of which the Mg2+ concentrations in the reaction mixtures, the incubation temperature, and the reaction time are at 8 mM, 64 degrees C, and 30 min, respectively. The analytical sensitivity of the RI-LAMP method was revealed as low as 7 copies of viral templates and 100-fold more sensitive than the published RT-PCR method. A visual inspection of in-tube LAMP products stained with a DNA fluorescent dye demonstrated that the positive and negative reactions exhibit distinct and different colors in daylight, which means that gel electrophoresis is not necessary to judge the positive or negative results. As the application of the method is rapid, easy, and no complicated instrument required, the GCRV-RT-LAMP method established in this study has great potential for the detection of GCRV in both the laboratory and the farm. (C) 2012 Elsevier B.V. All rights reserved.
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