Towards Fluorescence <i>In Vivo</i> Hybridization (FIVH) Detection of <i>H</i>. <i>pylori</i> in Gastric Mucosa Using Advanced LNA Probes

In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.近年来，学界已开展多项尝试以优化幽门螺杆菌（Helicobacter pylori）感染的诊断方案。荧光原位杂交（Fluorescence in situ hybridization）是当前检测幽门螺杆菌感染的常用技术，但该技术需获取胃部活检组织。因此，开发一种基于FISH的体内检测方法（in vivo FISH-based method，简称FIVH），可直接在人体内检测并可视化该细菌，将大幅缩短分析时长，实现内镜下同步诊断。 在既往研究中，我们采用标准亚磷酰胺化学合成法，设计并合成了一种硫代磷酸酯锁核酸（phosphorothioate locked nucleic acid，简称LNA）/2’-O-甲基RNA（2’ O-methyl RNA，简称2’OMe）探针，并成功在37℃下对贴附及悬浮状态的细菌完成了FISH杂交反应。 本研究对FISH技术进行了简化、优化并适配胃部pH环境，如今杂交步骤仅需30分钟，且除缓冲液外，仅需使用无毒浓度的尿素与探针。值得注意的是，在所有测试条件范围内，即便在低严谨性条件（如低pH环境）下，该FISH方法的灵敏度与特异性均得以保持。 综上，该方法是一种极具应用前景的方案，未来可与共聚焦激光内镜显微镜联用，实现体内幽门螺杆菌的可视化检测。