Journal Article

A novel method for semi-quantitative detection of HPV16 and HPV18 mRNA with a low-cost, open-source fluorimeter

Chang, Megan M., Scheurer, Michael E., Richards-Kortum, Rebecca R., Kundrod, Kathryn A., Stier, Elizabeth A., Smith, Chelsey A., Castle, Philip E., Schmeler, Kathleen M., Coole, Jackson B., Montealegre, Jane R., Novak, Emilie Newsham, Chiao, Elizabeth, Natoli, Mary E.

2025

Overview

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.

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: Analytical Chemistry / Assaying / Biochemistry / Biomarkers / Cancer / Cervical cancer / Characterization and Evaluation of Materials / Chemistry / Chemistry and Materials Science / Complexity / cost effectiveness / Deoxyribonucleic acid / detection limit / Diagnosis / DNA / Female / fluorescence / Fluorimetry / Fluorometers / Fluorometry - economics / Fluorometry - instrumentation / Fluorometry - methods / Food Science / Genotypes / Health aspects / Human papillomavirus / Human papillomavirus 16 - genetics / Human papillomavirus 16 - isolation & purification / Human papillomavirus 18 - genetics / Human papillomavirus 18 - isolation & purification / Humans / infrastructure / Laboratory Medicine / Limit of Detection / Low cost / Measurement / Messenger RNA / Methods / Monitoring/Environmental Analysis / mortality / Nucleic Acid Amplification Techniques - economics / Nucleic Acid Amplification Techniques - methods / Paper in Forefront / Papillomavirus infections / Papillomavirus Infections - diagnosis / Papillomavirus Infections - virology / patients / Portability / Real time / Recombinase / recombinase polymerase amplification / Reverse transcription / Ribonucleic acid / RNA / RNA, Messenger - analysis / RNA, Messenger - genetics / RNA, Viral - analysis / RNA, Viral - genetics / Sample preparation / uterine cervical neoplasms / Uterine Cervical Neoplasms - diagnosis / Uterine Cervical Neoplasms - virology