Detection of Helicobacter pylori Infection and Antibiotic Resistance via Stool Quantitative Polymerase Chain Reaction Analysis

Summary

The protocol employs a non-invasive stool sampling combined with a quantitative polymerase chain reaction to offer a convenient and rapid diagnostic method for Helicobacter pylori infection and its resistance to clarithromycin and quinolones.

Abstract

Helicobacter pylori (H. pylori) is widely prevalent worldwide, with approximately 50% of the global population having a history of H. pylori infection. In China, the infection rate ranges from 40% to 70%. H. pylori is primarily associated with gastrointestinal diseases such as chronic gastritis, gastric ulcers, and duodenal ulcers. Currently, the clinical treatment for H. pylori infection involves either triple or quadruple therapy. However, the extensive use of antibiotics has led to the development of antibiotic resistance in H. pylori. Therefore, detecting both H. pylori and its antibiotic resistance is crucial for guiding clinical treatment.

Diagnostic methods for H. pylori include urea breath test (UBT), antigen test, serological antibody test, endoscopy, rapid urease test (RUT), and bacterial culture. While the first three methods are non-invasive, they do not allow for bacterial recovery and, thus, cannot be used for resistance testing. The latter three methods are invasive, expensive, require high technical expertise, and may cause harm to patients.

Therefore, a non-invasive, rapid method for simultaneous detection of H. pylori infection and antibiotic resistance is of utmost clinical importance for the effective eradication of H. pylori. This paper aims to introduce a specific protocol that combines quantitative polymerase chain reaction (qPCR) with TaqMan fluorescent probe technology to rapidly detect H. pylori infection and antibiotic resistance. This method provides a convenient, rapid, and non-invasive way to diagnose H. pylori infection and resistance, unlike traditional bacterial culture and other techniques. qPCR is used to identify the infection and detect mutations in the 23S rRNA and gyrA genes, which are linked to resistance to clarithromycin and quinolones, respectively. Compared to conventional culture techniques, this approach offers a non-invasive, cost-effective, and time-efficient method for detecting Helicobacter pylori infection and determining its antibiotic resistance.

Introduction

Helicobacter pylori is a gram-negative, spiral-shaped bacterium that can persistently infect the human gastric epithelium¹. In 1994, the World Health Organization classified H. pylori as a Group 1 carcinogen for gastric cancer, with ~3% of infected individuals ultimately developing the disease². A recently published systematic review has indicated that a general trend of increased antibiotic resistance rate of H. pylori was observed in the last decade, which has reached alarming levels worldwide, especially clarithromycin resistance³. In China, the latest survey data sho....

Protocol

This study adheres to the ethical guidelines established by the Ethics Committee of Guangdong Provincial People's Hospital, Southern Medical University, Guangzhou, China (Approval No: KY2024-445-01). Detailed information regarding the materials used in this research (reagents, chemicals, equipment, and software) can be found in the Table of Materials.

1. Participant selection

1. Choose participants in the age range of 18 to 60 years.
2. Choose participants who may undergo Helicobacter pylori infection screening, such as patients presenting with symptoms like gastritis, peptic ul....

Results

Application of qPCR for detection of Helicobacter pylori infection and antibiotic resistance in fecal samples
We designed primers and probes based on point mutations in the conserved genes of Helicobacter pylori, as well as in the 23S rRNA gene and the gyrA gene. These primers and probes were labeled with different fluorescent dyes and then used for qPCR detection. The quality control results for the qPCR ex.......

Discussion

In recent years, molecular detection methods have been extensively applied in the field of microbiology, significantly altering the clinical management of several infectious diseases. These methods operate at the genetic level, allowing for not only the confirmation of bacterial presence but also gene typing and antibiotic resistance testing. Real-time fluorescence quantitative PCR (qPCR) is increasingly favored due to its short processing time, high sensitivity and accuracy, and low risk of cross-contamination. It has b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSC-1500IIA2-X	BIOBASE	SEDA 20143222263	Biosafety cabinet
Disposable fecal collection and storage tube	Mole		Collect fecal specimens
E-Centrifuge	WEALTEC		Centrifuge the residual liquid off the wall of the tube
Helicobacter pylori nucleic acid, clarithromycin, and quinolone resistance mutation detection kit	Mole		Detection of Helicobacter pylori infection and antibiotic resistance; freeze-dried H. pylori reagent (containing primers, probes, Taq polymerase, UNG enzyme, dNTPs, etc.), a positive control for H. pylori (containing Helicobacter pylori and human β-actin target genes), and a negative control for H. pylori (containing human β-actin target genes)
Mole 96M automated nucleic acid extractor	Mole		For DNA extraction
Nucleic acid extraction kit	Mole		To extract nucleic acid; contains lysis buffer (guanidine salt, tris hydroxymethyl aminomethane, Tween-20, sodium chloride), Wash Buffer 1 (sodium chloride), Wash Buffer 2 (tris hydroxymethyl aminomethane, Tween-20), Wash Buffer 3 (magnetic beads, Tween-20), Wash Buffer 4 (nuclease-free water), and Elution Buffer (tris hydroxymethyl aminomethane), along with a magnetic rack
SLAN Fully automatic medical PCR analysis system	HONGSHI		Data Analysis
SLAN-96S Real-Time PCR machine	HONGSHI		Fluorescent quantitative PCR amplification
Ultra-low temperature freezers (DW-YL450)	MELING	SEDA 20172220091	-20 °C for storing reagents
Vortex-5	Kylin-bell		For mixing reagent