(i) Rapid sample processing method. Samples were mixed with TNA-Cifer reagent E (BioCifer Pty. Ltd., Brisbane, Queensland, Australia) at different ratios depending on the sample type. Initial testing identified optimum ratios (sample to TNA-Cifer reagent E) of 5:1 for cell culture medium, 1:1 for blood, 2:1 for plasma, and 5:1 for serum, which were used for spiked samples. Rapidly processed samples were diluted in nuclease-free water at a ratio (processed sample to nuclease-free water) of 1:1 for blood, plasma, and serum and were used undiluted for cell culture medium.
(ii) Serotype-specific DENV RT-RPA-LFD assays. Serotype-specific DENV RT-RPA-LFD assays were performed using the TwistAmp Nfo kit (TwistDX, Cambridge, United Kingdom) and HybriDetect lateral flow strips (Milenia Biotec, Giessen, Germany). In brief, reaction mixes for the respective DENV-1 to -4 tests were prepared separately and incubated at 39°C for 20 min as previously described (24 (link)). Amplicon detection was performed using preactivated lateral flow strips (23 (link)), which were then placed into running buffer (30 (link)) for 5 min, analyzed visually, and photographed or scanned.
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