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Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp laem-singh virus (LSNV) as a model
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Document Title
Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp laem-singh virus (LSNV) as a model
Author
Arunrut N., Suebsing R., Withyachumnarnkul B., Kiatpathomchai W.
Name from Authors Collection
Affiliations
Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand; Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Surat Thani Campus, Surat Thani, Thailand
Type
Article
Source Title
PLoS ONE
ISSN
19326203
Year
2014
Volume
9
Issue
9
Open Access
Gold, Green
Publisher
Public Library of Science
DOI
10.1371/journal.pone.0108047
Abstract
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. © 2014 Arunrut et al.
License
CC BY
Rights
Author
Publication Source
Scopus