Gastric Mucosa Quantitative Polymerase Chain Reaction Analysis for Detecting Helicobacter pylori and Antibiotic Resistance

Summary

A rapid and accurate method for H. pylori detection and drug resistance testing is very significant for efficiently eradicating H. pylori in clinical practice. This protocol aims to present a specific methodology involving gastric mucosa quantitative polymerase chain reaction (qPCR) for the rapid detection of H. pylori and antibiotic resistance.

Abstract

Helicobacter pylori is a main pathogen that infects nearly half of the global population and is threatening public health due to its increasing antibiotic resistance. Besides, Helicobacter pylori is also responsible for chronic gastritis, gastric and duodenal ulcers, gastric carcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, it is essential to perform a timely and accurate diagnosis of H. pylori and the determination of its antibiotic resistance. Nowadays, existing methods of H. pylori diagnosis mainly include the rapid urease test (RUT), the urea breath test (UBT), the serum antibody test, the antigen test, gastroscopy, and bacterial culture. However, bacteria could not be cultured through the first five detection methods, not to mention the detection of drug resistance. The bacterial culture is time-consuming, and antibiotic sensitivity tests cannot be carried out rapidly and routinely. In clinical settings, the swift and precise identification of H. pylori and its susceptibility to antibiotics is crucial for its effective elimination. The objective of this protocol is to outline a targeted approach utilizing quantitative polymerase chain reaction (qPCR) on gastric mucosal samples to expedite the diagnosis of H. pylori and assess its resistance to antimicrobial agents. qPCR was exploited to detect the ureA gene for H. pylori infection and mutations in the 23S rRNA and gyrA genes associated with resistance to clarithromycin and quinolones, respectively. Currently, there remain challenges in gastric mucosa qPCR due to the lack of standard operating procedures. Therefore, it is essential to share methodologies with experimental details to ensure accurate communication of experimental procedures, contributing to gold-standard protocols that enable greater transparency. Overall, this protocol offers an economical and expeditious alternative to conventional methods for assessing H.pylori infection and its resistance to antibiotics through the application of quantitative polymerase chain reaction (qPCR) technology.

Introduction

H. pylori is a gram-negative bacteria that can survive in the presence of a low oxygen level. The organism belongs to several distinct genetic populations and shows high genetic diversity. Although the organism is usually spirally shaped, it could be changed to a rod¹. It is a main pathogen that infects nearly 50% of the global population². Mostly, infection occurs when patients remain healthy carriers during childhood, and symptoms manifest later in adulthood. It has been reported to be relative to chronic gastritis, gastric and duodenal ulcers, gastric carcinoma, and gastric mucosa-associated lymphoid tissue (....

Protocol

The existing 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: KY2024-1115-01). Patients aged from 18 to 60 were enrolled in this study. For this study, participants were excluded if they had recently taken antibiotics, antibacterial herbal remedies, proton pump inhibitors (PPIs), or H₂ receptor antagonists within the 2 weeks prior to testing. Additionally, individuals who had undergone anti-H. pylori therapy within the last 3 months, or those with significant cardiac, hepatic, or renal issu....

Discussion

Traditional testing such as RUT, UBT, histology, culturing as well as serology are exploited for the detection of H. pylori. Each diagnostic approach offers distinct advantages and faces specific challenges depending on the clinical context¹⁷. The cultivation of H. pylori from gastric mucosal biopsies is often considered the benchmark for diagnosis. Nonetheless, this method is labor-intensive, and its accuracy is constrained by technical challenges, the conditions required for in.......

Materials

Name	Company	Catalog Number	Comments
Bath Incubator	ALLSHENG	MK2000-2	Provide a constant temperature environment
Biosafety cabinet	Haier	HR1500-B2
Centrifuge	Thermo Fisher Scientific	THERMO ST16R	Centrifuge the residual liquid off the wall of the tube.
Chelex-100	sigma	C7901	Resin
Gastroscope biopsy forceps	Boston Scientific Corporation		Sampling of the gastric mucosa
Helicobacter pylori 23S rRNA gene and gyrA gene mutation detection kit	Jiangsu Mole Bioscience	CFDA 20223400137	qPCR detection kit for H. pylori and drug-resistance genes (clarithromycin and quinolones)
Nucleic acid extraction reagent	Jiangsu Mole Bioscience	SEDA 20150076	For DNA extraction
SDS Software	Applied Biosystems	7300/7500	Data analysis
Thermocylcer	Thermo Fisher Scientific	ABI 7500	For qPCR detection of H. pylori and drug-resistance genes (clarithromycin and quinolones)
Vortex mixer	JOANLAB	VM-5005	For mixing reagent