Novak Emilie Newsham, Ma Ariel, Kundrod Kathryn A, Chang Megan M, Hansen Cannon J, Montealegre Jane Richards, Scheurer Michael E, Castle Philip E, Guo Ming, Salcedo Mila P, Schmeler Kathleen, Richards-Kortum Rebecca
Department of Bioengineering, Rice University, Houston, TX, USA.
Divisions of Cancer Prevention and Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD, USA.
Sci Rep. 2025 Aug 19;15(1):30450. doi: 10.1038/s41598-025-13583-2.
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.
由于能够区分具有临床意义的感染和短暂感染，来自人乳头瘤病毒（hrHPV）高危基因型的信使核糖核酸（mRNA）是比hrHPV DNA更具特异性的宫颈癌风险生物标志物。然而，检测脱落宫颈细胞中的mRNA是一个复杂、昂贵且设备要求高的过程，在资源有限的环境中并不可行。在此，我们描述了一种灵敏、特异且所需仪器最少的方法，用于检测脱落宫颈细胞中的HPV16、HPV18和HPV45 mRNA。首先，设计了逆转录重组酶聚合酶扩增（RT-RPA）外切酶分析，以使用便携式荧光计实时扩增和检测每个反应中≥100拷贝的HPV16、HPV18和HPV45 E7 mRNA，这与唯一获得美国食品药品监督管理局（FDA）批准的hrHPV mRNA检测相当。接下来，开发了一种仅使用酶促反应的免提取样品制备方法，以裂解培养的HPV16、HPV18和HPV45阳性细胞中的细胞并降解细胞DNA。RT-RPA分析在临床相关浓度范围内从这种粗裂解物中特异性扩增mRNA。使用这些方法在护理点对11份宫颈阴道样本进行了检测，结果显示RT-RPA与RT-qPCR结果之间的一致性为100%，包括两份HPV16 mRNA阳性样本。此外，9份加样了HPV16、HPV18和HPV45阳性细胞的阴性样本成功检测到了各自的HPV类型。总体而言，这种原型检测方法有潜力在资源有限的环境中使灵敏且特异的hrHPV mRNA检测更易于实现。
