title: Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp Laem-Singh virus (LSNV) as a model.
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.
authors.affiliation: 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.
authors.forename: Narong
authors.initials: N
authors.lastname: Arunrut
authors.affiliation: 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.
authors.forename: Rungkarn
authors.initials: R
authors.lastname: Suebsing
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand; Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand; Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, Surat Thani, Thailand.
authors.forename: Boonsirm
authors.initials: B
authors.lastname: Withyachumnarnkul
authors.affiliation: 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.
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
mesh_terms: D000818:Animals; D003365:Costs and Cost Analysis; D006358:Hot Temperature; D007753:Laboratories; D009391:Nephelometry and Turbidimetry; D033561:Penaeidae / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics* / Q000302:isolation & purification*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics* / Q000295:instrumentation*
chemical_list: D012324:RNA-Dependent RNA Polymerase