Rapid detection of hepatitis C virus using recombinase polymerase amplification

Figures

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  Fig 1. RT-RPA target sequence location and overview of assay. (A) HCV RPA assay was designed to target the 5’ untranslated region (UTR) gene which has the highest homology across all genotypes. We plot locations of mismatches in red for an alignment of the reference sequences of the six genotypes of HCV, with regions of high homology colored grey or white. We show the locations of the RPA primers and probe on 5’ UTR, as well as the published PCR primers and probe [26]. (B) Schematic of the experimental protocol for HCV RNA detection using either RT-RPA or RT-PCR. Viral RNA is extracted from human serum using a column-based solid phase extraction kit, RNA is reverse transcribed into complementary DNA (cDNA), and HCV cDNA is detected with either PCR (1–2 hr runtime) or RPA (15 min runtime). (C) A simple experimental setup for reverse transcription and RPA includes basic laboratory consumables, a pipette, and a benchtop fluorometer with an integrated heating block (Axxin T16).

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  Fig 2. Detection limit of HCV RNA standards. The limit of detection of one-step RT-RPA was measured using HCV RNA synthetic standards. Real-time fluorescence measured from reaction tubes is plotted over 17-minutes for experimental data of 1000, 100, 10, and 1 copy per reaction. All reactions with input copies of RNA greater than or equal to 10 copies per reaction amplified in 15-minutes or less, while replicates with 1 copy per reaction and no template controls (NTCs) did not amplify. The inset table lists data for all input RNA concentrations tested. All concentrations were run with 4 replicates.

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  Fig 3. Viral load distribution of HCV clinical samples. Viral loads of clinical samples used in this study were measured using RT-qPCR on freshly extracted RNA from serum samples (GiCasRes biorepository). The measurements are stratified by genotype on the x-axis, and the black horizontal bar represents the median. We evaluated a total of 58 HCV-positive samples representing genotypes 1–4, and 7 samples lacking a paired genotype test.