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<p>Messenger RNA (mRNA) from high-risk genotypes of human papillomavirus (hrHPV) is a more specific biomarker for cervical cancer risk than hrHPV DNA due to its ability to differentiate clinically significant infections from transient ones. Detecting mRNA from exfoliated cervical cells, however, is a complex, expensive, and equipment-intensive process, making it infeasible in resource-limited settings. Here we describe a sensitive, specific, and minimally instrumented method to detect HPV16, HPV18, and HPV45 mRNA in exfoliated cervical cells. First, reverse transcription recombinase polymerase amplification (RT-RPA) exo assays were designed to amplify and detect HPV16, HPV18, and HPV45 E7 mRNA ≥100 copies per reaction in real time using a portable fluorimeter, which is on par with the only FDA-approved hrHPV mRNA test. Next, an extraction-free sample preparation method using only an enzymatic reaction was developed to lyse cells and degrade cellular DNA in cultured HPV16, HPV18, and HPV45-positive cells. The RT-RPA assays specifically amplified mRNA from this crude lysate across a clinically relevant range of concentrations. Eleven cervicovaginal samples were tested at the point of care using these methods and showed 100% agreement between RT-RPA and RT-qPCR results, including two samples that were positive for HPV16 mRNA. Additionally, nine negative samples spiked with HPV16, HPV18, and HPV45-positive cells successfully detected each respective HPV type. In total, this prototype assay has potential to make sensitive and specific hrHPV mRNA testing more accessible in resource-limited settings.</p>

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Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年8月19日

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  • Emilie Newsham Novak,
  • Ariel Ma,
  • Kathryn A. Kundrod,
  • Megan M. Chang,
  • Cannon J. Hansen,
  • Jane Richards Montealegre,
  • Michael E. Scheurer,
  • Philip E. Castle,
  • Ming Guo,
  • Mila P. Salcedo,
  • Kathleen Schmeler,
  • Rebecca Richards-Kortum

摘要

Messenger RNA (mRNA) from high-risk genotypes of human papillomavirus (hrHPV) is a more specific biomarker for cervical cancer risk than hrHPV DNA due to its ability to differentiate clinically significant infections from transient ones. Detecting mRNA from exfoliated cervical cells, however, is a complex, expensive, and equipment-intensive process, making it infeasible in resource-limited settings. Here we describe a sensitive, specific, and minimally instrumented method to detect HPV16, HPV18, and HPV45 mRNA in exfoliated cervical cells. First, reverse transcription recombinase polymerase amplification (RT-RPA) exo assays were designed to amplify and detect HPV16, HPV18, and HPV45 E7 mRNA ≥100 copies per reaction in real time using a portable fluorimeter, which is on par with the only FDA-approved hrHPV mRNA test. Next, an extraction-free sample preparation method using only an enzymatic reaction was developed to lyse cells and degrade cellular DNA in cultured HPV16, HPV18, and HPV45-positive cells. The RT-RPA assays specifically amplified mRNA from this crude lysate across a clinically relevant range of concentrations. Eleven cervicovaginal samples were tested at the point of care using these methods and showed 100% agreement between RT-RPA and RT-qPCR results, including two samples that were positive for HPV16 mRNA. Additionally, nine negative samples spiked with HPV16, HPV18, and HPV45-positive cells successfully detected each respective HPV type. In total, this prototype assay has potential to make sensitive and specific hrHPV mRNA testing more accessible in resource-limited settings.