DENV serotype 1 isolated in cell culture from a dengue patient, as previously described,12 (link) was 10-fold serial-diluted using Minimum Essential Media (MEM, Gibco, Thermo Fisher Scientific, Waltham, MA). Three consecutive dilutions (“high titer” = H, “medium titer” = M, and “low titer” = L) were selected based on preliminary testing on RDTs. The dilution “L” was the last dilution with detactable RNA purified from RDT.
Zika virus (H/PF/2013, EVA 001v-EVA1545)– and CHIKV (H20235/STMARTIN/2013, EVA 001v-EVA1540)–inactivated strains provided by the European Virus Archive collection (https://www.european-virus-archive.com/) were 10-fold serial-diluted using Minimum Essential Media (MEM, Gibco, Thermo Fisher Scientific). Following RDT manufacturer instructions for NS1 testing, 100 µL of each virus dilution was loaded on the NS1 cassette of RDTs in triplicate and left for 2 hours for the specimen to dry. Each RDT was then opened, and a 15-mm strip piece was cut out from the sample pad, as described.12 (link)For DENV1, each dilution was loaded onto 39 RDTs, three RDTs were immediately processed (D0), and the rest were divided into three groups and placed at three different temperatures: 4°C, −80°C, and 35°C. After different storage times, 2 days (D2), 1 week (W1), 1 month (M1), and 2 months (M2), three RDTs were taken out from each storage condition and then processed.
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