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.

Rapid and visual Chlamydia trachomatis detection using loop-mediated isothermal amplification and hydroxynaphthol blue

“…; Arunrut et al . ). During the LAMP reaction, Mg 2+ is formed to insoluble magnesium pyrophosphate, and therefore the concentration of Mg 2+ in the solution decreases and can be titrated by a metal ion indicator, e.g.…”

“…Loop-mediated isothermal amplification uses Bst DNA polymerase and a set of 4-6 target-specific primers for an autocycling and strand displacement DNA synthesis at a constant temperature of 60-65°C in 30-60 min. This method is low price, high speed and has ease-of-use, comparable sensitivity and specificity to the PCR (Goto et al 2009(Goto et al , 2010Tao et al 2011;Arunrut et al 2014). During the LAMP reaction, Mg 2+ is formed to insoluble magnesium pyrophosphate, and therefore the concentration of Mg 2+ in the solution decreases and can be titrated by a metal ion indicator, e.g.…”

Development and evaluation of real‐time loop mediated isothermal amplification assay for rapid and sensitive detection of Salmonella spp. in chicken meat products

“…To determine the optimal temperature for the real‐time LAMP assay, reactions were performed on a portable turbidimeter (Arunrut et al, ) at 60, 63, and 65 °C for 1 hr. Results of the real‐time reaction monitoring were accomplished by measuring the turbidity every second using the optical density (OD) at 650 nm.…”

“…To determine the optimal temperature for the real-time LAMP assay, reactions were performed on a portable turbidimeter (Arunrut et al, 2014)…”

Loop-Mediated Isothermal Amplification untuk Mendeteksi Gen blaTEM Sebagai Penyandi Extended-Spectrum Beta-Lactamase pada Isolat Enterobacteriaceae

“…Magnesium pirofosfat terbentuk akibat reaksi antara ion magnesium dan ion pirofosfat yang dilepaskan oleh dNTP saat polimerisasi asam nukleat. 19 Magnesium pirofosfat yang terbentuk kemudian akan mengendap dan memiliki warna putih sehingga akan membuat cairan menjadi keruh. Kekeruhan ini tidak mudah untuk diamati oleh mata telanjang dan memerlukan bantuan turbidimeter, yaitu alat yang dapat mengukur turbiditas atau kekeruhan.…”