Broad-spectrum dengue virus detection using the commercial RealStar dengue RT-PCR kit 3.0 (Altona) and an in-house combined real-time RT-PCR assay

In endemic areas, the genetic diversity among co-circulating dengue virus (DENV) strains is considerable and new, highly divergent strains are identified on a regular basis. It is thus critical to ensure that molecular diagnostic tools effectively detect virus genomes even in case of important genetic variation. Here, we tested both the pan-DENV detection capacity and the limit of detection of two real-time RT-PCR assays: (i) the commercial RealStar Altona 3.0 system and (ii) a laboratory developed test (LDT) combining two RT-PCR systems in a single reaction tube (DenAllDUO). We used a panel of DENV strains representative of the genetic diversity within DENV species, combined with three in vitro transcribed RNAs as surrogates for unavailable strains corresponding to recently discovered strains with substantial genetic divergence: DENV serotype 1 (DENV-1) Brun2014, DENV-2 QML22 and DENV-4 DKE121. Both systems (i) targeted the genome 3' untranslated region, (ii) displayed a broad detection spectrum, encompassing most of DENV species diversity, and (iii) detected the three aforementioned divergent strains. DenAllDUO detected all the strains tested, whereas the RealStar system failed to detect strains from DENV-4 genotype III. Altogether, our findings support the value of these two RT-PCR systems as part of the Dengue diagnostic arsenal.