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Title: Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp laem-singh virus (LSNV) as a model
Authors: Narong Arunrut
Rungkarn Suebsing
Boonsirm Withyachumnarnkul
Wansika Kiatpathomchai
Mahidol University
Thailand National Center for Genetic Engineering and Biotechnology
Faculty of Science and Industrial Technology
Keywords: Agricultural and Biological Sciences;Biochemistry, Genetics and Molecular Biology;Medicine
Issue Date: 25-Sep-2014
Citation: PLoS ONE. Vol.9, No.9 (2014)
Abstract: © 2014 Arunrut et al. Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP) using Laem-Singh virus (LSNV) as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase (RdRp) gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED) light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR) photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65°C for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p<0.05). This platform can be easily adapted for detection of other pathogens under field laboratory settings.
ISSN: 19326203
Appears in Collections:Scopus 2011-2015

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