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. 2023 Mar 23:14:1094600.
doi: 10.3389/fmicb.2023.1094600. eCollection 2023.

A novel loop-mediated isothermal amplification-lateral flow dipstick method for Helicobacter pylori detection

Wenwen Liu  1 , Gang Lu  2   3 , Yu Wang  2   3 , Zhenghong Chen  4 , Yunyun Gao  2 , Zhipeng Yin  2 , Yi Wu  2 , Xiaoqian Lv  2 , Pengbo Guo  2   3 , Yinghui Zhao  2   3
Affiliations

A novel loop-mediated isothermal amplification-lateral flow dipstick method for Helicobacter pylori detection

Wenwen Liu et al. Front Microbiol. .

Abstract

Introduction: To eradicate Helicobacter pylori (H. pylori) and reduce the risk of gastric cancer, a sensitive, specific, convenient, and simple detection method is needed. This study aimed to establish a novel loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for H. pylori detection.

Methods: LAMP primer design software was used to design primers for the conserved sites of the H. pylori ureB gene. UreB-FIP-labeled biotin was used for LAMP amplification, and FAM-labeled probes were specifically hybridized with LAMP amplification products, which were then detected by LFD. In addition, a clinical study was conducted to assess LAMP-LFD in 20 fecal samples.

Results: The results of the optimization indicated that H. pylori could be specifically detected by LFD without cross-reaction with other non-H. pylori bacteria when the LAMP was performed at 65°C for 60 min. The lower limit of the detection method was 102 copies/μL, which was 100 times the sensitivity of polymerase chain reaction (PCR). H. pylori-positive fecal samples were detected by LAMP-LFD in 13/20 patients.

Discussion: In conclusion, a new LAMP-LFD assay has been fully established and confirmed for H. pylori detection. The entire process can be completed in approximately 1.5 h, with the advantages of strong specificity, high sensitivity, and simple operation. This study provides a novel potential method for the detection of H. pylori in the clinical settings of primary hospitals and low-resource countries.

Keywords: Helicobacter pylori; lateral flow dipstick; loop-mediated isothermal amplification; novel detection method; ureB gene.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Optimization of the LAMP reaction. (A) Optimization of dNTPs concentration, M: DNA Maker DL5000; lane 1: Negative control; lane 2: 4 mM; lane 3: 6 mM; lane 4: 8 mM; lane 5: 10 mM; lane 6: 12 mM; lane 7: 14 mM. (B) Optimization of Mg+ content, M: DNA Maker DL5000; lane 1: Negative control; lane 2: 0 μL; lane 3: 0.5 μL; lane4: 1.0 μL; lane 5: 1.5 μL; lane 6: 2.0 μL; lane 7: 2.5 μL; lane 8: 3.0 μL. (C) Optimization of enzyme content, M: DNA Marker DL5000; lane 1: Negative control; lane 2: 0.5 μL; lane 3: 1.0 μL; lane 4: 1.5 μL; lane 5: 2.0 μL. (D) Optimization of primer concentration ratio (internal primer: external primer), M: DNA Marker DL5000; lane 1: Negative control; lane 2: 1:1; lane 3: 2:1; lane 4: 4:1; lane 5: 8:1; lane 6: 10:1. (E) Optimization of reaction temperature, M: DNA Maker DL5000; lane 1: Negative control; lane 2: 50°C; lane 3: 55°C; lane 4: 60°C; lane 5: 65°C; lane 6: 70°C. (F) Optimization of reaction time, M: DNA Maker DL5000; lane 1: Negative control; lane 2: 15 min; lane 3: 30 min; lane 4: 45 min; lane 5: 60 min; lane 6: 75 min. The lanes marked red are the optimal conditions.
Figure 2
Figure 2
Establishment of LAMP-LFD for H. pylori, lane 1: Negative control; lane 2: sample detection.
Figure 3
Figure 3
Specific analysis of LAMP-LFD for H. pylori. (A) Detection of agarose gel electrophoresis; (B) Detection by LAMP-LFD; M: DNA Maker DL5000; lane 1: Negative control; lane 2: H. pylori; lane 3: Escherichia coli; lane 4: Staphylococcus aureus; lane 5: Pseudomonas aeruginosa.
Figure 4
Figure 4
Detection of sensitivity. (A) Sensitivity of PCR; (B) Sensitivity of LAMP agarose gel electrophoresis; (C) Sensitivity of LAMP-LFD; M: DNA Marker DL5000; lane 1: Negative control; lane 2: 108copies/μL; lane 3: 107 copies/μL; lane 4: 106 copies/μL; lane 5: 105 copies/μL; lane 6: 104 copies/μL; lane 7: 103 copies/μL; lane 8: 102 copies/μL; lane 9: 101 copies/μL.
Figure 5
Figure 5
Test of repeatability. (A) Test of LAMP agarose gel electrophoresis repeatability; (B) Repeatability detection by LAMP-LFD; M: DNA Marker DL5000; lane 1: Negative control; lane 2: H. pylori DNA; lane 3: Negative control; lane 4: H. pylori DNA; lane 5: Negative control; lane 6: H. pylori DNA.
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