Rao Xuelian, Zheng Liangkai, Wei Kaipeng, Li Meiqing, Jiang Meng, Qiu Jianlong, Zhou Yulin, Ke Rongqin, Lin Chen
School of Medicine, Huaqiao University, Xiamen, Fujian, China.
Department of Pathology, Women and Children's Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, China.
Microbiol Spectr. 2023 Feb 21;11(2):e0389622. doi: 10.1128/spectrum.03896-22.
RNA plays a vital role in the physiological and pathological processes of cells and tissues. However, RNA hybridization applications in clinical diagnostics are still limited to a few examples. In this study, we developed a novel hybridization assay for human papillomavirus (HPV) E6/E7 mRNA by taking advantage of specific padlock probing and rolling circle amplification, combined with chromogenic readout. We designed padlock probes for 14 types of high-risk HPV and demonstrated that E6/E7 mRNA could be visualized as discrete dot-like signals using bright-field microscopy. Overall, the results are consistent with the clinical diagnostics lab's hematoxylin and eosin (H&E) staining and p16 immunohistochemistry test results. Our work thus shows the potential applications of RNA hybridization for clinical diagnostics using chromogenic single-molecule detection, offering an alternative technical option to the current commercially available kit based on branched DNA technology. detection of viral mRNA expression in tissue samples is of great value for pathological diagnosis to access viral infection status. Unfortunately, conventional RNA hybridization assays lack sensitivity and specificity for clinical diagnostic purposes. Currently, the commercially available branched DNA technology-based single-molecule RNA detection method offers satisfactory results. Here, we present our padlock probe- and rolling circle amplification-based RNA hybridization assay for detecting HPV E6/E7 mRNA expression in formalin-fixed paraffin-embedded tissue sections, providing an alternative yet robust method for viral RNA visualization that is also applicable to different types of diseases.
RNA在细胞和组织的生理及病理过程中发挥着至关重要的作用。然而，RNA杂交在临床诊断中的应用仍仅限于少数例子。在本研究中，我们利用特异性锁式探针和滚环扩增技术，并结合显色读出，开发了一种用于检测人乳头瘤病毒（HPV）E6/E7 mRNA的新型杂交检测方法。我们设计了针对14种高危型HPV的锁式探针，并证明使用明场显微镜可将E6/E7 mRNA可视化为离散的点状信号。总体而言，结果与临床诊断实验室的苏木精和伊红（H&E）染色及p16免疫组织化学检测结果一致。因此，我们的工作展示了基于显色单分子检测的RNA杂交在临床诊断中的潜在应用，为当前基于分支DNA技术的市售试剂盒提供了另一种技术选择。检测组织样本中的病毒mRNA表达对于获取病毒感染状态的病理诊断具有重要价值。不幸的是，传统的RNA杂交检测方法对于临床诊断目的缺乏敏感性和特异性。目前，基于分支DNA技术的市售单分子RNA检测方法能提供令人满意的结果。在此，我们展示了基于锁式探针和滚环扩增的RNA杂交检测方法，用于检测福尔马林固定石蜡包埋组织切片中的HPV E6/E7 mRNA表达，为病毒RNA可视化提供了一种可靠的替代方法，该方法也适用于不同类型的疾病。
