Department of Obstetrics and Gynecology, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing 210004, P.R. China.
Centre for Diseases Prevention and Control of Eastern Theater, Nanjing 210018, P.R. China.
J Microbiol Biotechnol. 2023 Aug 28;33(8):1091-1100. doi: 10.4014/jmb.2304.04009. Epub 2023 May 19.
Human papillomavirus (HPV) types 16 and 18 are the major causes of cervical lesions and are associated with 71% of cervical cancer cases globally. However, public health infrastructures to support cervical cancer screening may be unavailable to women in low-resource areas. Therefore, sensitive, convenient, and cost-efficient diagnostic methods are required for the detection of HPV16/18. Here, we designed two novel methods, real-time ERA and ERA-LFD, based on enzymatic recombinase amplification (ERA) for quick point-of-care identification of the HPV E6/E7 genes. The entire detection process could be completed within 25 min at a constant low temperature (35-43°C), and the results of the combined methods could be present as the amplification curves or the bands presented on dipsticks and directly interpreted with the naked eye. The ERA assays evaluated using standard plasmids carrying the E6/E7 genes and clinical samples exhibited excellent specificity, as no cross-reaction with other common HPV types was observed. The detection limits of our ERA assays were 10 and 10 copies/μl for HPV16 and 18 respectively, which were comparable to those of the real-time PCR assay. Assessment of the clinical performance of the ERA assays using 114 cervical tissue samples demonstrated that they are highly consistent with real-time PCR, the gold standard for HPV detection. This study demonstrated that ERA-based assays possess excellent sensitivity, specificity, and repeatability for HPV16 and HPV18 detection with great potential to become robust diagnostic tools in local hospitals and field studies.
人乳头瘤病毒（HPV）16 型和 18 型是导致宫颈病变的主要原因，与全球 71%的宫颈癌病例有关。然而，资源匮乏地区的妇女可能无法获得支持宫颈癌筛查的公共卫生基础设施。因此，需要一种敏感、方便、具有成本效益的诊断方法来检测 HPV16/18。在这里，我们设计了两种新的方法，实时 ERA 和 ERA-LFD，基于酶促重组酶扩增（ERA），用于快速即时识别 HPV E6/E7 基因。整个检测过程可以在恒定的低温（35-43°C）下在 25 分钟内完成，并且联合方法的结果可以呈现为扩增曲线或棒上的条带，并直接用肉眼解释。使用携带 E6/E7 基因的标准质粒和临床样本评估的 ERA 分析显示出优异的特异性，因为没有观察到与其他常见 HPV 类型的交叉反应。我们的 ERA 分析的检测限分别为 HPV16 和 18 的 10 和 10 拷贝/μl，与实时 PCR 分析相当。使用 114 个宫颈组织样本评估 ERA 分析的临床性能表明，它们与 HPV 检测的金标准实时 PCR 高度一致。这项研究表明，基于 ERA 的分析在 HPV16 和 HPV18 的检测中具有出色的灵敏度、特异性和可重复性，具有成为当地医院和现场研究中强大诊断工具的巨大潜力。
