Center for Medical Ethics and Health Policy Staff Publications
Language
English
Publication Date
4-1-2025
Journal
Analytical and Bioanalytical Chemistry
DOI
10.1007/s00216-025-05765-8
PMID
39920386
PMCID
PMC11913951
PubMedCentral® Posted Date
2-7-2025
PubMedCentral® Full Text Version
Post-print
Abstract
Despite global calls to eliminate cervical cancer, rates of cervical cancer incidence and mortality remain high in resource-limited settings, where it is challenging to implement and sustain screening, diagnosis, and treatment programs. The presence of high-risk HPV mRNA in cervical cells is a sensitive and specific biomarker of cervical precancer. Yet, current testing methods are too costly and complex for use in resource-limited settings. Here, we present a novel method for semi-quantitative detection of HPV16 and HPV18 mRNA with minimal infrastructure requirements. The assay relies on isothermal reverse transcription recombinase polymerase amplification (RT-RPA) with real-time fluorescence readout, demonstrated on rugged, portable, and affordable instruments. We demonstrate adapting the assay from DNA detection to RNA detection, characterizing the test with samples of increasing biological complexity, and ultimately establishing a limit of detection of 1000 HPV16 or HPV18 transcripts per reaction with RNA extracted from cell lines. HPV16 and HPV18 mRNA assays were used to test total RNA from 11 patient samples; results for 10 samples (91%) agreed with the gold standard of RT-qPCR. To reduce cost, the assay was demonstrated with multiplexed detection of HPV16 and HPV18 DNA, validated with a reaction volume that was reduced from 50 to 5 µL with DNA and RNA, and performed using a low-cost, portable reader with DNA and RNA. With incorporation of point-of-care-friendly sample preparation and detection of additional genotypes, this test has the potential to expand global access to HPV testing.
Keywords
Human papillomavirus 18, Humans, Human papillomavirus 16, RNA, Messenger, RNA, Viral, Female, Papillomavirus Infections, Fluorometry, Nucleic Acid Amplification Techniques, Uterine Cervical Neoplasms, Limit of Detection, Cervical cancer, HPV testing, MRNA, Point-of-care diagnostics
Published Open-Access
yes
Recommended Citation
Kundrod, Kathryn A; Natoli, Mary E; Smith, Chelsey A; et al., "A Novel Method for Semi-quantitative Detection of HPV16 and HPV18 mRNA With a Low-Cost, Open-Source Fluorimeter" (2025). Center for Medical Ethics and Health Policy Staff Publications. 94.
https://digitalcommons.library.tmc.edu/med_ethics/94