Rapid detection of Yellow fever Virus with Recombinase Aided Amplification(1)

Objectives: Dengue caused by infection with the dengue virus (DENV) is endemic in the tropical andsubtropical regions of the world and of greatest public health con cern, With more large outbreaks in ruralareas, the purpose of this study was to develop a point-of-care test using recombinase-aidedamplification and lateral-flow dipsticks for rapidly detecting DENy in low-re source settings.Methods: The primers for the recombinase-aided amplilfication (RAA) assay were designed based on 3U'iR of the DENy genome and screened. The RAA temperature, time and the concentration of primerswere then optimized, as well as the lateral-flow dipstick assay (LFD) time, Finally, the diagnosticperformance of the reverse transcription (RT-RAA-LFD assay was evaluated using blood samples from247 patients who were clinically suspected to be infected with DENV.Results: The RAA primer pair Fl/R2 was the optimal combination for detecting DENy, The RT-RAA Wasperformed in an incubator block at 37 'c for 20 minutes, and the amplicons were visible in the flowdipsticks from a naked eye within 3 minutes, The de tection limit of the developed RT-RAA-LFD assay was10 copies/uL with high specificity for DENv, Compared with commercal reverse transcriptionquantitative PCR assay, the kappa value of RT-RAA-LFD in the 247 clinical samples was 0.957.Conclusions. in this study, a rapid and visual point-of-care test based on RT-RAA and LFD assay wasde veloped.lt was found to be suitable for reliable detection ofDENVin low-resource settings with limitedlaboratory capabilities and optimal storage conditions.

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