Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

Abstract

BackgroundEarly and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.MethodsA single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).ResultsAcute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.ConclusionThe RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

Lyophilized visually readable loop-mediated isothermal reverse transcriptase nucleic acid amplification test for detection Ebola Zaire RNA

“…RT-LAMP technology uses loop-forming oligonucleotides in combination with a strand-displacing polymerase to allow rapid and robust target amplification under isothermal conditions, thus avoiding the need for a thermocycler (Notomi et al, 2000). It has been effective in the amplification of nucleic acid from a variety of human pathogens, including HIV, Dengue, H7N9 Influenza, Hantaan, and Ebola (Curtis et al, 2008; Teoh et al, 2013; Liu et al, 2014; Kurosaki et al, 2007; Hu et al, 2015). However, a significant limitation of the assay is the need for cold storage of reaction components, which is often infeasible in resources limited settings (Njiru, 2012).…”

Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India

“…Dengue is a mosquito borne flaviviral infection, affecting the tropical and subtropical regions of the world and is one of the major emerging global public health problems. There are four antigenically distinct dengue virus serotypes DEN-1, DEN-2, DEN-3 and DEN-4 and each serotype contains phylogenetically distinct genotypes (Teoh et al, 2013). The Dengue virus (DENV) infection induces a lifelong protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection by the other three serotypes.…”

Instrument-Free Point-of-Care Molecular Detection of Zika Virus

“…The smartphone can be programmed not only for image processing and data analysis and reporting but also for communications and GPS recording, which can be useful in tracking the migration of the vector. Other extensions of our work can include multiplexing for concurrent detection of mosquito-borne pathogens such as dengue 42 and chikungunya. 43 …”