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Immunology and Infection

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

Published: July 28th, 2023

DOI:

10.3791/65689

1Laboratory Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, 2Division of Microbiology and Immunology, School of Biomedical Sciences, The University of Western Australia, 3Center for Precision Health, School of Medical and Health Sciences, Edith Cowan University, 4Medical Technology School of Xuzhou Medical University, 5Marshall Laboratory of Biomedical Engineering, School of Medicine, Shenzhen University, 6The Marshall Centre for Infectious Diseases Research and Training, The University of Western Australia, 7Marshall International Digestive Diseases Hospital, Zhengzhou University, 8Marshall Medical Research Center, Fifth Affiliated Hospital of Zhengzhou University
* These authors contributed equally

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).

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.

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....

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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 st.......

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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.......

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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.......

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This work was supported by the Sanming Project of Medicine in Shenzhen (Grant No. SZSM201510050) and the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2022A1515220023). Research Foundation for Advanced Talents of Guandong Provincial People's Hospital (No. KJ012021097), and the National Natural Science Foundation of China (81871734, 82072380, 82272423). The funders had no role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

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NameCompanyCatalog NumberComments
23S rRNA and gyrA gene point mutations detection kit (PCR-Fluorescence Probing)Hongmed InfagenDetection of Helicobacter pylori resistance to clarithromycin and levofloxacin
ABI 7500 fluorescence quantitative PCR machineThermo Fisher ScientificSEDA 20163220767Fluorescent quantitative PCR amplification
ABI 7500 softwareThermo Fisher ScientificData Analysis
BSC-1500IIA2-XBIOBASESEDA 20143222263Biosafety cabinet
DNA extraction kitDaan Gene 
E-CentrifugeWEALTECCentrifuge the residual liquid off the wall of the tube.
H. Pylori DNA detection kit (PCR-Fluorescence Probing) Hongmed InfagenTesting for H. pylori infection
Stream SP96 automated nucleic acid extractorDaan GeneSEDA 20140104For DNA extraction 
String test kitHongmed InfagenIt contains a capsule attached to a string, scissors, cotton swab, and sample preservation tube 
Ultra-low temperature freezers (DW-YL450) MELINGSEDA 20172220091-20 °C for storing reagents 
Vortex-5Kylin-bellFor mixing reagent 

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