水凝胶环介导等温扩增技术在无需核酸提取的情况下用于粪便样本中幽门螺杆菌的超快速诊断。

Hydrogel loop-mediated isothermal amplification for ultra-fast diagnosis of Helicobacter pylori in stool samples without nucleic acid extraction.

机构信息

College of Chemistry and Molecular Engineering, College of Biological Engineering, Shandong Provincial Key Laboratory of Biochemical Engineering, Qingdao Key Laboratory of Rapid Nucleic Acid Detection, Qingdao Rapid Nucleic Acid Detection Engineering Research Center, Qingdao University of Science and Technology, Qingdao, 266042, PR China.

Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao, 226000, PR China.

出版信息

Anal Chim Acta. 2025 Jan 2;1333:343384. doi: 10.1016/j.aca.2024.343384. Epub 2024 Nov 7.

DOI:10.1016/j.aca.2024.343384

PMID:39615902

Abstract

BACKGROUND

The gastrointestinal diseases caused by Helicobacter pylori (H. pylori) infection made the accurate detection of H. pylori infection more important. Non-invasive methods, such as molecular diagnostic methods, had become a promising method for detection of H. pylori. Stool samples combined with loop-mediated isothermal amplification (LAMP), showed potential practicability for real-time detection. However, complex nucleic acid extraction steps were required to remove the large numbers of amplification inhibitors in stool samples before LAMP reaction. And the limited number of H. pylori made the detection with long reaction time and low sensitivity. The problems mentioned above were urgently to be solved.

RESULTS

In this study, we proposed a strategy for ultra-rapid sensitive detection of H. pylori in stool samples by hydrogel LAMP (hLAMP) without extraction. The hydrogel was combined with stool samples after simple thermal cracking, and amplification spaces were formed in its nanopore structures by nano-localization. The LAMP reaction was accelerated by nano space-localization. Besides, this method based on hLAMP could specifically and sensitively detect as low as 100 CFU/mL H. pylori within 40 min from sampling to result due to good anti-inhibition effect on complex samples of hydrogel. The whole process involved sample simple disposal for 10 min and LAMP reaction for 30 min. Furthermore, the excellent anti-inhibition mechanism of hydrogel was discussed, and the mechanism of hydrogel accelerating LAMP was explored.

SIGNIFICANCE

This is the first application of that hydrogel and LAMP systematically combined to detect H. pylori in stool samples. The developed method had been verified in actual clinical applications that the accuracy rate reached 88.9 % compared with routine histopathology. And it also provided a potential idea for the diagnosis and prevention of H. pylori.

摘要

背景

由幽门螺杆菌（H. pylori）感染引起的胃肠道疾病使得准确检测 H. pylori 感染变得更加重要。非侵入性方法，如分子诊断方法，已成为检测 H. pylori 的有前途的方法。粪便样本与环介导等温扩增（LAMP）相结合，显示出实时检测的潜在实用性。然而，在 LAMP 反应之前，需要复杂的核酸提取步骤来去除粪便样本中大量的扩增抑制剂。并且有限数量的 H. pylori 使得检测具有长反应时间和低灵敏度的问题。上述问题亟待解决。

结果

在这项研究中，我们提出了一种无需提取即可通过水凝胶 LAMP（hLAMP）超快速灵敏检测粪便样本中 H. pylori 的策略。水凝胶与粪便样本简单热裂后结合，在其纳米孔结构中通过纳米定位形成扩增空间。纳米空间定位加速了 LAMP 反应。此外，由于水凝胶对复杂样本具有良好的抗抑制作用，这种基于 hLAMP 的方法可以从采样到结果在 40 分钟内特异性和灵敏地检测低至 100 CFU/mL 的 H. pylori。整个过程涉及 10 分钟的样本简单处理和 30 分钟的 LAMP 反应。此外，还讨论了水凝胶的优异抗抑制机制，并探讨了水凝胶加速 LAMP 的机制。