Development of Synthetic Molecular Standards for Dengue Virus

Dengue fever is an acute illness caused by any one of 4 serotypes (1-4) of genetically related dengue viruses (DENV), with an estimated 390 million cases reported annually. Currently, quantitative RT-PCR (qRT-PCR) is the preferred method for the detection and quantification of DENV in clinical diagnostics and epidemiological surveillance. The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known viral genome concentration.

Native DENV RNA can be used as a standard for these assays; however, the full-length dengue viral RNA is on the Commerce Control List and requires a permit from the US Department of Commerce for international shipment. To make DENV RNA standards more accessible, ATCC has developed 4 synthetic molecular standards that represent DENV serotypes 1-4. Each standard contains short fragments from the capsid, membrane, and envelope genes of the DENV genome, as well as target regions encompassing the primer sequences from numerous published RT-PCR assays, including the DENV-1-4 Real-Time RT-PCR Assay developed by the CDC1. The synthetic RNA standards were quantified by Droplet Digital PCR (ddPCR) in order to package precise copies of RNA. Moreover, given the inherent labile nature of RNA, a stabilization matrix was added to the quantitated RNA preparation. As compared to native RNA, these synthetic standards are easier to use as controls for qRT-PCR assays, exhibit less variability, have a longer shelf life, eliminate the need to culture viruses and can be used under BSL-1 conditions. Further, this synthetic quantitative RNA approach can be extended to other pathogenic viruses, which are unculturable or need to be grown in a high-containment facility.

In the following proof-of-concept study, we amplified the synthetic molecular standards with the published primers from the CDC assay and Waggoner et al, and used the generated standard curves to quantify viral RNA extracted from various DENV strains.