Molecular Diagnostics of Dengue by Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) in Disposable Polyester-Toner Microdevices

Abstract

Dengue is one of the most prevalent infectious tropical diseases in the world, with high incidence in over 100 countries. Rapid and reliable diagnosis of dengue is of great importance for public health. To simplify molecular diagnostics, isothermal amplification techniques have recently emerged as an alternative to conventional methods of deoxyribonucleic acid (DNA) amplification. Here, we developed a one-step method for dengue virus detection from real sample based on RT-LAMP (reverse transcription-loop mediated isothermal amplification) in a disposable microdevice. The reaction was thermally controlled with a thermoblock for 15 min at 72 °C. At the end of the incubation time, we either removed the solution for detection of fragments by gel electrophoresis or added DNA intercalator for visual detection on-chip. Our results demonstrated that it is possible to detect dengue virus through RT-LAMP directly from a serum sample, without previous ribonucleic acid (RNA) extraction. The success of RT-LAMP was confirmed in reactions initiated with 0.8 fg μL −1 of RNA, which represents 200 copies of RNA per μL. RT-LAMP in a polyester-toner (PeT) microdevice is a simple and inexpensive method that allows for rapid detection of dengue virus with high reliability and great potential for point-of-care applications.

Recent advances in loop-mediated isothermal amplification (LAMP) for rapid and efficient detection of pathogens

“… Pathogen Time Sensitivity Detection Method References Zika 20 min 10 −5 PFU Colorimetric ( Calvert et al., 2017 ) Ebola 15–20 min 256 copies Real-time fluorometer ( Oloniniyi et al., 2017 ) HIV 60 min 10 4 RNA copies/mL RT Quencher probe ( Rudolph et al., 2015 ) Influenza 60 min 0.1 RNA copies Phenol-red pH indicator ( Ahnet al. et al., 2019 ) Hepatitis B 15 min 2.2fg/µl Fluorometer ( Quoc et al., 2018 ) Dengue 15 min 0.8 fg/µl Colorimetric (SYBR Green I) ( Mendes et al., 2019 ) HPV 60 min 10 copies Colorimetric (SYBR Green I) ( Mudhigeti et al., 2019 ) H1N1 <40 min 10 copies RNA Real-time turbidimeter, UV ( Kubo et al., 2010 ) Encephalitis 70 min 50 pg RNA Lateral flow dipstick ( Deng et al., 2015 ) MERS-CoV 60 min 4 RNA copies Colorimetric (EvaGreen) ( Lee et al., 2017 ) West Nile virus 17 min 0.1 PFU Real-time turbidimeter ( Parida et al., 2004 ) …”

“…Similarly, dengue, causing seasonal attacks, is still prevalent in large parts of the world, and needs early diagnosis before the multiplication of the virus. Mendes et al. (2019) developed RT-LAMP which can produce results in 15 min with high sensitivity (in femtograms).…”

All-Serotype Dengue Virus Detection through Multilayered Origami-Based Paper/Polymer Microfluidics

“…Despite very limited outreach outside laboratory settings, RT-LAMP assays for the rapid detection and differentiation of DENV serotypes appear to be well established in microfuge tubes − with further adaptation to miniature platforms. , In recent times, this fostered the emergence of miniaturized LAMP-based testing platforms for several infectious diseases albeit with no particular contextualization with DENV, such as bead-mediated dielectrophoresis-based microfluidic immunofluorescence assay, disposable lab-on-a-chip for highly efficient RNA isolation, and paper-based diagnostic kits for resource-constrained settings . Further advancements resulted in a multiplexed electrochemical paper-based analytical device deploying graphene oxide-silicon dioxide nanocomposites for detecting the DENV serotypes as well as origami-based device technologies for detecting different variants of mosquito-borne infections. , These technologies also found their particular use in detecting Hepatitis C virus, assessing the quality of the serum for veterinary disease control, and diagnosing infections in skin and mucous membranes due to methicillin-resistant Staphylococcus aureus .…”