Rapid detection of hepatitis C virus using recombinase polymerase amplification

Abstract

Over 71 million people are infected with hepatitis C virus (HCV) worldwide, and approximately 400,000 global deaths result from complications of untreated chronic HCV. Pan-genomic direct-acting antivirals (DAAs) have recently become widely available and feature high cure rates in less than 12 weeks of treatment. The rollout of DAAs is reliant on diagnostic tests for HCV RNA to identify eligible patients with viremic HCV infections. Current PCR-based HCV RNA assays are restricted to well-resourced central laboratories, and there remains a prevailing clinical need for expanded access to decentralized HCV RNA testing to provide rapid chronic HCV diagnosis and linkage to DAAs in outpatient clinics. This paper reports a rapid, highly accurate, and minimally instrumented assay for HCV RNA detection using reverse transcription recombinase polymerase amplification (RT-RPA). The assay detects all HCV genotypes with a limit of detection of 25 copies per reaction for genotype 1, the most prevalent in the United States and worldwide. The clinical sensitivity and specificity of the RT-RPA assay were both 100% when evaluated using 78 diverse clinical serum specimens. The accuracy, short runtime, and low heating demands of RT-RPA may enable implementation in a point-of-care HCV test to expand global access to effective treatment via rapid chronic HCV diagnosis.

Biosensor‐based multiple cross displacement amplification platform for visual and rapid identification of hepatitis C virus

“…27 Chia et al applied an assay based on RT-recombinase polymerase amplification and identified an LoD of 25 copies per test. 28 It was obvious that the novel assay in this study was more sensitive than the approaches previously reported.…”

Rapid detection of goose astrovirus genotypes 2 using real-timereverse transcription recombinase polymerase amplification

“…The optimal probe-primer combination from two probes and 10 primers was selected to amplify the ORF2 gene, which were used as the target sequence because they were reported to be highly conserved in several previously developed methods based on nucleic acid ampli cation, but a few sequence variations still appeared in the ORF2 genes of GoAstV-2 strains. The RPA assay was reported to be able to tolerate 5-9 target mismatches with little in uence on the results of the assay [31,32]. The probe-primer set used in this study had a strong uorescence signal and no cross-reaction with other related pathogens, demonstrating good ampli cation e ciency and high speci city of the assay.…”

DNA-directed formation of plasmonic core–satellite nanostructures for quantification of hepatitis C viral RNA

“…Enzyme-assisted isothermal methods include nucleic acid sequence amplication (NASBA), 11,12 loop-mediated isothermal amplication (LAMP), [13][14][15] rolling circle amplication (RCA), 16,17 and recombinase polymerase amplication (RPA). 18,19 However, dependence on enzyme catalysis introduces supply, cost, and technical hurdles that need to be addressed for decentralized adoption. Enzyme-free techniques present a promising solution, instead utilizing the inherent self-assembly of nucleic acidssuch as Watson-Crick base pairing and nucleoside modicationsto construct nanostructures in a bottom-up approach.…”