Designing a locally produced DENV-1 nucleic acid diagnostic for low resource regions with endemic disease

Abstract

Using recombinant techniques, to support local production and availability of active diagnostic ingedients (ADIs) in regions with endemic or neglected disease, a reverse transcriptase (RT) fusion construct is reported (Δ3R5-TGP-RT), having the same modular architecture (R5-fluorescent protein-functional enzyme) as reported previously by us. In tandem with the R5-mCherry-Bst this RT construct completes the enzyme set required for RT-LAMP mediated detection of Dengue. The introduction of the thermophilic green fluorescence protein TGP, allowed a heat purification step at 95 °C to be introduced into the workflow, simplifying purification. A 3 terminal amino acid (SSK) truncation of the naturally occurring silaffin R5 binding peptide (Δ3R5) enabled improved protein purification and loading on silica and good storage stabilisation (≥3 months). L-arginine triggered elution of enzyme from silica, immediately prior to use, enabled a soluble enzyme to be delivered directly into the RT-LAMP reactions, producing a clear solution and compatibility with turbidity detection of PPi released complexes that form insoluble Mg₂P₂O₇. A unique primer set was designed for the detection of Dengue Serotype 1 RNA, using a primer set (CAB 3), targeting the RNA-dependent RNA polymerase (RdRp), part of the NS5 region. This was chosen to minimise primer dimer formation, thereby reducing false positives. Amplification was achieved with high sensitivity (10 copies), with turbidity analysis able to distinguish positive and negative reactions. The approach developed has the potential to widen the application of LAMP diagnostics for neglected diseases such as Dengue by removing the availability of biological reagents as a limiting factor.