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

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.