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.
Highlights
Rapid 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
Development of a combined reverse-transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) unit For simultaneous detection of dengue virus serotypes 1-4 (DENV1-4), Japanese encephalitis virus (JEV), and West Nile virus (WNV), we developed a flexible RT-LAMP unit composed of six rows and each row had four 0.2 ml MicroAmp reaction tubes with cap (Applied Biosystems, Foster City, CA)
Specificity and sensitivity of the RT-LAMP unit Under the amplification condition, DENV1-4, JEV, and WNV were successfully amplified in the RT-LAMP unit and observed as ladder-like patterns on the gel (Figure 1)
Summary
Rapid 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. JEV is transmitted among birds, pigs, and other domestic animals and human beings by Culex species mosquitoes. Infection with WNV causes a self-limited febrile illness. WNV epidemic in North America is a major public health concern [3] In most circumstance, these viruses appear transiently in acute sera after onset of the diseases and transmit quickly by mosquito bites. These viruses appear transiently in acute sera after onset of the diseases and transmit quickly by mosquito bites This has necessitated the development of rapid detection methods for these viruses in acute sera of the patients before admission to hospital and in vector mosquitoes for the prediction and prevention of large-scale epidemics
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