Quantitative Polymerase Chain Reaction (qPCR)-Based Rapid Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Summary

The protocol presents a noninvasive method for the rapid diagnosis of Helicobacter pylori stomach infections through the string test and determines its antibiotic resistance to clarithromycin and levofloxacin using quantitative polymerase chain reaction (qPCR).

Abstract

Helicobacter pylori is a major human pathogen that infects approximately half of the global population and is becoming a serious health threat due to its increasing antibiotic resistance. It is the causative agent of chronic active gastritis, peptic ulcer disease, and gastric cancer and has been classified as a Group I Carcinogen by the International Agency for Research on Cancer. Therefore, the rapid and accurate diagnosis of H. pylori and the determination of its antibiotic resistance are important for the efficient eradication of this bacterial pathogen. Currently, H. pylori diagnosis methods mainly include the urea breath test (UBT), the antigen test, the serum antibody test, gastroscopy, the rapid urease test (RUT), and bacterial culture. Among them, the first three detection methods are noninvasive, meaning they are easy tests to conduct. However, bacteria cannot be retrieved through these techniques; thus, drug resistance testing cannot be performed. The last three are invasive examinations, but they are costly, require high skills, and have the potential to cause damage to patients. Therefore, a noninvasive, rapid, and simultaneous method for H. pylori detection and drug resistance testing is very important for efficiently eradicating H. pylori in clinical practice. This protocol aims to present a specific procedure involving the string test in combination with quantitative polymerase chain reaction (qPCR) for the rapid detection of H. pylori infection and antibiotic resistance. Unlike bacterial cultures, this method allows for easy, rapid, noninvasive diagnosis of H. pylori infection status and drug resistance. Specifically, we used qPCR to detect rea for H. pylori infection and mutations in the 23S rRNA and gyrA genes, which encode resistance against clarithromycin and levofloxacin, respectively. Compared to routinely used culturing techniques, this protocol provides a noninvasive, low-cost, and time-saving technique to detect H. pylori infection and determine its antibiotic resistance using qPCR.

Introduction

H. pylori is a spiral-shaped, highly motile, gram-negative bacterium that mainly lives in the pylorus region of the stomach¹. It is a common pathogen that infects nearly 50% of the global population². Most people with H. pylori infection have no clinical manifestations, and most develop different diseases after several years of infection, including chronic gastritis, peptic ulcers, gastric ulcers, and gastric cancer³. In several studies based on different populations, the efficacy of eliminating H. pylori for preventing stomach cancer and precancerous lesions has be....

Protocol

The present study was conducted in conformity with ethical considerations established by the ethical committee of Guangdong Provincial People's Hospital, Southern Medical University, Guangzhou, China (Approval Number: KY-Q-2022-384-02). Patients in the age range of 18-60 years old were included in this study. Patients taking antibiotics, antibacterial Chinese medicines, drugs such as proton pump inhibitors (PPI), or H₂ receptor antagonists, etc., within 2 weeks prior to testing were not included in this study. Those patients who had received treatment against H.pylori in the past 3 months were also excluded from this study. Those with serious h....

Results

Detection of H. pylori infection and antibiotic resistance in stomach fluid by qPCR
We performed qPCR for the detection of H. pylori infection by amplifying the ureA gene and determined its antibiotic resistance profile by targeting point mutations in the 23S rRNA gene and gyrA gene (Table 1). The quality control CT values in all three groups of the qPCR experiments were within the recommended range, indicating that the samples were all in a norm.......

Discussion

H. pylori detection can be performed using both invasive and noninvasive methods¹³. Commonly used invasive techniques such as histopathology, the rapid urease test, polymerase chain reaction (PCR), and bacterial culturing require endoscopy and biopsy.Serological tests, urea breath tests, and enzyme-linked immunosorbent assays (ELISA) are recommended among the noninvasive procedures¹⁴. While noninvasive methods are easy to perform, economical, and more comfortable f.......

Materials

Name	Company	Catalog Number	Comments
23S rRNA and gyrA gene point mutations detection kit (PCR-Fluorescence Probing)	Hongmed Infagen		Detection of Helicobacter pylori resistance to clarithromycin and levofloxacin
ABI 7500 fluorescence quantitative PCR machine	Thermo Fisher Scientific	SEDA 20163220767	Fluorescent quantitative PCR amplification
ABI 7500 software	Thermo Fisher Scientific		Data Analysis
BSC-1500IIA2-X	BIOBASE	SEDA 20143222263	Biosafety cabinet
DNA extraction kit	Daan Gene
E-Centrifuge	WEALTEC		Centrifuge the residual liquid off the wall of the tube.
H. Pylori DNA detection kit (PCR-Fluorescence Probing)	Hongmed Infagen		Testing for H. pylori infection
Stream SP96 automated nucleic acid extractor	Daan Gene	SEDA 20140104	For DNA extraction
String test kit	Hongmed Infagen		It contains a capsule attached to a string, scissors, cotton swab, and sample preservation tube
Ultra-low temperature freezers (DW-YL450)	MELING	SEDA 20172220091	-20 °C for storing reagents
Vortex-5	Kylin-bell		For mixing reagent