Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1–4

Abstract

BackgroundDengue virus (DENV), the most widely prevalent arbovirus, continues to be a threat to human health in the tropics and subtropics. Early and rapid detection of DENV infection during the acute phase of illness is crucial for proper clinical patient management and preventing the spread of infection. The aim of the current study was to develop a specific, sensitive, and robust reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection and differentiation of DENV1-4 serotypes.ResultsThe method detection primers, which were designed to target the different DENV serotypes, were identified by inspection of multiple sequence alignments of the non-structural protein (NS) 2A of DENV1, NS4B of DENV2, NS4A of DENV3 and the 3′ untranslated region of the NS protein of DENV4. No cross-reactions of the four serotypes were observed during the tests. The detection limits of the DENV1-4-specific RT-LAMP assays were approximately 10-copy templates per reaction. The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time PCR. The diagnostic rate was 100 % for clinical strains of DENV, and 98.9 % of the DENV-infected patients whose samples were tested were detected by RT-LAMP. Importantly, no false-positives were detected with the new equipment and methodology that was used to avoid aerosol contamination of the samples.ConclusionThe RT-LAMP method used in our study is specific, sensitive, and suitable for further investigation as a useful alternative to the current methods used for clinical diagnosis of DENV1-4, especially in hospitals and laboratories that lack sophisticated diagnostic systems.

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

“…The sequences of primers used for RT-LAMP on DENV-4 detection, which has been described by Hu et al, 52 are shown in Table 1.…”

Future Perspective of Nucleic Acid-based Detection of Dengue Virus and its Serotypes

“…The genome amplification step is monitored by the visible colour change of HNB (hydroxylnaphthol blue) from violet to sky blue as the LAMP reaction proceeds [10,11]. This method can detect approximately 10-copy templates of dengue virus [12]. However, subtyping of each sample requires the use of either a set of four tubes, each of which has the primer mixture for a particular serotype [9], or of one tube that has a single reaction mixture incorporating all primer sets [11].…”

Serotyping dengue virus with isothermal amplification and a portable sequencer

“…We amplified parts of the 3′UTR of the virus, as opposed to a previously published method 22 . We then identified their sequences with the MinION sequencer.…”