Background: The family Flaviviridae contains a large number of viral agents capable of causing encephalitis and jaundice. Individual members such as dengue (DENV), yellow-fever virus (YFV) and West Nile virus (WNV) cause significant morbidity and mortality worldwide with up to 100 million new dengue infections alone per year worldwide. Using 3base™ technology we developed rapid real-time PCR assays that are able to detect the presence of any flavivirus, any dengue serotype and any alphavirus using a single primer and probe set for each.
Material/methods: The assay was validated using whole virus samples (Zeptometrix), viral RNAs (Vircell) and proficiency panels (QCMD). To simulate clinical samples mock specimens consisting of serum, urine and saliva were prepared using whole virus particles. The assay was then tested using matched serum/plasma, urine and saliva obtained from patients during a dengue outbreak on the South Pacific Island of Vanuatu. RNA extraction was performed on a GS-mini automated extraction platform (Genetic Signatures). PCR was performed on a MIC real-time PCR instrument (BMS).
Results: We observed sensitivity for all targets to be less than 10 copies of target when introduced into the RT-PCR reaction. Results obtained using the 2016 QCMD DENV, WNV and Zika challenges showed 100% concordance with expected results. No inhibition was observed confirming the assay was compatible with a full range of clinical samples. For Vanuatu primers and probes to geographically relevant species including DENV 1-4, Zika and chikungunya were used to type positive samples. Full results of the Vanuatu study will be presented in detail, including serotyping and geographical distribution data.
Conclusions: As a result of the genome complexity reduction achieved using 3base™ technology we were able to design a single primer/probe set that detected all flavivirus and alphavirus species. Positive samples are then reflexed to determine the exact flavivirus/alphavirus responsible for the disease. The assay can be fully automated and provides healthcare workers with a fast and accurate diagnosis of flavivirus/alphavirus infections.