Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus

Abstract

During dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. To facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that detects all 4 serotypes of dengue virus (DENV). We used a quencher to reduce nonspecific amplification. The assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at 63 °C. Results can be visualized using UV fluorescence, handheld readers, or lateral flow immunochromatographic tests. We report a sensitivity of 86.3% (95% confidence interval [CI], 72.7-94.8%) and specificity of 93.0% (95% CI, 83.0-98.1%) using a panel of clinical specimens characterized by DENV quantitative reverse transcription-polymerase chain reaction. This pan-serotype DENV RT-LAMP can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need.

A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses

“…Isothermal NAATs eliminate the need for power hungry thermal cyclers, significantly lowering the complexity of hardware required for these tests. RT-LAMP detection of dengue15161718, chikungunya19, and other RNA viruses has been demonstrated previously, and at least one recent report has demonstrated RT-LAMP detection of Zika virus13. In most cases, these reports detect amplification with non-specific indicators of total DNA synthesis, such as monitoring turbidity20, observing the color-change indicator hydroxynaphtol blue (HNB)17, performing a post-reaction analysis involving addition of an indicator dye ( e.g .…”

New Tools for Dengue Diagnostics

“…The study showed that pan-serotype RT-LAMP could discriminate dengue with a sensitivity of 86%. This study also visualized the amplification of target gene on lateral flow assay [49]. In one study Lau et al employed hydroxynaphthol blue (HNB) dye for the colorimetric visualization of RT-LAMP-amplified product through the naked eye, thus, making the assay simpler as it does not require specific tool to interpret the LAMP product.…”

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions

“…This process can be further accelerated by the addition of loop primers also targeted to the exposed single-stranded region of the loop (9). This technique has found widespread application primarily in the diagnosis and monitoring of infectious diseases such as malaria (10,11), West Nile virus (12,13) and dengue (14).…”

“…The potential of quantitative LAMP for monitoring and control operations remains to be fully exploited. Until this point, most qLAMP assays have focused on ascertaining the concentrations of pathogens within specific infection contexts (e.g 14,21,29,30),. although studies have considered microorganismal eDNA(14), pathogenic viruses in water (31) and forensic applications (32).…”

“…Until this point, most qLAMP assays have focused on ascertaining the concentrations of pathogens within specific infection contexts (e.g 14,21,29,30),. although studies have considered microorganismal eDNA(14), pathogenic viruses in water (31) and forensic applications (32). Our study reveals another application -ascertaining the approximate frequency of individuals infected with a symbiotic bacterium in disease-related releases.…”