Development of an isothermal point of care method for detection of human papilloma virus (HPV)

Current commercial methods of detection of high-risk human papilloma virus (hrHPV) rely mostly on the polymerase chain reaction (PCR) that use either 14 specific primer pairs or a single consensus primer pair (e.g., GP5+/GP6+). However, PCR requires the use of a thermal cycler (PCR machine) and electricity and therefore is not suitable for a low-cost Point of Care Test (PoCT). Moreover, PCR primers are often not suitable for isothermal amplification since at lower temperature they may anneal non-specifically leading to background amplification. We have developed an isothermal point of care method for the rapid detection of hrHPV that works at a constant temperature (< 42 o C) using recombinase polymerase amplification (RPA). A novel consensus primer pair was designed able to amplify all 14 hrHPV types. To enable the detection of the amplified double-stranded RPA products, one primer was labelled with a 5’-phosphate, and the other primer was labelled at the 5’-end with 6-carboxyfluorescein (FAM). Then, the double-stranded labelled RPA product was digested with Lambda exonuclease, which preferentially digests 5’-phosphorylated DNA ends whereas the 5’-FAM ends are protected, generating single-stranded 5’-FAM-labelled RPA products. These products can be detected by paper-based lateral flow assays (LFA) using biotinylated detection probes that detect all 14 hrHPV types at the same time (universal consensus detection probe). Lateral flow assay conditions were developed that allow these probes to detect all 14 hrHPV types. Using this novel isothermal rapid hrHPV detection method, amplification of all 14 hrHPV types at constant temperature (currently set at 37 o C) within short time (< 1h) was achieved. • An isothermal point of care method for the rapid detection of 14 high-risk human papilloma virus (hrHPV) is developed using recombinase polymerase amplification (RPA) and modified consensus primers • One primer is labelled at the 5’-end with a phosphate, and the other primer is labelled with 6-carboxyfluorescein (FAM) • The amplified double-stranded RPA products are digested with Lambda exonuclease that preferentially digests 5’-phosphorylated DNA ends whereas the 5’-FAM ends are protected • The resulting single-stranded 5’-FAM-labelled amplified DNA can be detected by paper-based lateral flow assays (LFA) using biotinylated detection probes