Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

Abstract

BackgroundRapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics.ResultsWe developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4), Japanese encephalitis virus (JEV), and West Nile virus (WNV). The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR.ConclusionsThe RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

“…Since many of these diseases are co-endemic, it is desirable to leverage resources and integrate diagnostic platforms wherever possible. Multiplexing of the LAMP reaction has been demonstrated for the detection of Babesia parasites in cattle [37] malaria and heartworm in mosquitoes [69] and human arboviruses [81]. More recently a real-time, multiplex LAMP technique was described that enables detection of up to 4 distinct LAMP targets in a single reaction [65].…”

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

“…The RT-LAMP assay is being increasingly used by various investigators for rapid detection and typing of emerging viruses (Chan and Fox, 1999;Mori et al, 2001;Parida et al, 2005). These earlier reports, however, evaluated their RT-LAMP assays for the detection of DENV infection with a small clinical sample size (<100) and using the C-prM gene (Lu et al, 2012) or serotype-specific regions of the 3 untranslated region (UTR) (Parida et al, 2005;Li et al, 2011;Sahni et al, 2013). The C-prM gene, however, was relatively less conserved among all the four DENV serotypes (inter-serotype) in comparison to the 3 UTR (Teoh et al, 2013).…”

Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice

“…LAMP of Salmonella-specific sequences. A LAMP reaction of Salmonella-specific sequences requires the identification of DNA primers that recognize a region of the Salmonella genome that is sufficiently conserved-such that a single set of LAMP primers amplifies the region in a large subset of pathogenic Salmonella serovars-yet divergent enough that internal sequences within the amplicon can readily discriminate closely related serovars (60)(61)(62). To this end, we employed PrimerExplorer v4 software to design a primer set for the amplification of a region in the recF gene, a well-conserved gene encoding the RecF gap repair protein (63,64).…”