(M-481) Rapid Test for high risk HPV to Sensitively Detect HPV 16 for Cervical Cancer Screening

Introduction:: Every year 12,000 Americans develop cervical cancer resulting in 4,000 deaths. This cancer is almost entirely preventable with proper screening, yet it is estimated that over a third of the women who died from cervical cancer had never been screened for HPV, the virus causing almost all cervical cancers. While existing tests such as CareHPV are used to provide same-day results in resource-limited settings, they cannot provide same-visit results that would minimize loss to follow-up care, which occurs in around 20% of positive screenings in the US. Rapid testing using isothermal molecular detection methods for high-risk human papillomavirus (hrHPV), such as recombinase polymerase amplification (RPA), could facilitate hrHPV testing and follow-up care as a more accessible cervical cancer screening technology. In previous work our group surveyed and interviewed clinicians who perform cervical cancer screening to establish prioritized user requirements for such a rapid test. We now present the initial work in designing an RPA rapid test for hrHPV to meet these specifications, starting with HPV 16, the most carcinogenic of the 14 hrHPV types.


Materials and Methods:: RPA Basic Kits were purchased from TwistDx and performed as instructed with primers for high-risk HPV at 39oC for 20 minutes. A fluorescent probe for the HPV 16 L1 gene was designed to test its limit of detection based on the work of Gong et al.¹ detecting HPV L1 gene. To make the test amenable to the point of care, the assay was then adapted to include a labelled primer and probe to detect amplicons on a dipstick readout. Amplicon sequence identity was confirmed by restriction digest using Bst EII restriction digest (Ipswich, MA, USA) and analysis of product length on a polyacrylamide gel. A primer set for beta-globin is being added to the primer pool to act as a sample adequacy control.


Results, Conclusions, and Discussions:: The HPV L1 gene was successfully amplified using labelled primers and probes with a limit of detection of 100 copies/50 µL reaction with a probe that generates a fluorescent signal upon binding to the amplicons (n=3, fig 1). HPV 16 was also detected on a lateral flow strip to a limit of detection of 1000 copies/50 µL (n=3, fig 2). The identity of the sequence was verified with a restriction digest. Several beta-globin primer candidates have been evaluated, and specificity testing is currently underway.

Discussion/conclusion: This limit of detection is superior to the 5000 copies /50 μL of CareHPV, a comparator rapid test for HPV DNA. Since the primer set used here has also been shown to amplify the other hrHPV strains, the creation of a probe for HPV 18/45 and hrHPV the other 11 less dangerous hrHPV types would allow for the detection and identification of nearly all 14 hrHPV types. With the sample adequacy control, this would give this test as much diagnostic information as current hrHPV laboratory tests.


Acknowledgements (Optional): : Acknowledgements: This work was funded by the NCI grant number K01CA241073, PI: Rodriguez


References (Optional): : References: Gong, J. Nature Sci Rep.(2021) v.11 pp.12800.