Rapid Detection of Helicobacter pylori Virulence and Typing Using Quantum Dot Labeling Technology

Summary

Among the numerous diagnostic methods for Helicobacter pylori, quantum dot immunofluorescence can rapidly type and detect Helicobacter pylori, providing a basis for clinical treatment decisions. This article provides a detailed introduction to the application value of quantum dot technology in the typing and detection of Helicobacter pylori.

Abstract

We present a method based on quantum dot labeling technology for rapidly detecting the virulence of Helicobacter pylori and performing typing, aiming to overcome the limitations of conventional diagnostic methods and provide a basis for personalized clinical treatment. A kit was designed using the double-antigen sandwich method. This involved a series of meticulous steps, including establishing buffer reaction systems and conducting activation and coupling processes for antigen-antibody reactions during kit preparation. Additionally, there were specific procedures for sample collection, processing, and testing operations to determine the infection status and virulence type of Helicobacter pylori. By establishing data models for different results (negative, type II, and type I), the effectiveness of the quantum dot technology in typing detection was verified. In the case of negative samples, there were weak or no significant signal values in relevant detection areas. For type II samples, a specific pattern of positive and negative signal values for different antibodies was observed, and for type I samples, a distinct set of positive signal values corresponding to key antibodies was noted. Moreover, its application in studies related to chronic gastritis and gastric cancer demonstrated its value in serving as a screening indicator. The quantum dot-based method has advantages like improved sensitivity and specificity compared to traditional diagnostic methods. However, it also faces challenges such as potential impacts from sample storage time and relatively high costs. Nevertheless, with continuous technological innovations, it is expected to play a more significant role in the biomedical field for Helicobacter pylori detection and related disease diagnosis in the future.

Introduction

Helicobacter pylori (H. pylori) is a Gram-negative bacterium found between the pyloric mucosa and submucosa of the gastric antrum^1,2. In 1994, the World Health Organization/International Agency for Research on Cancer (WHO/IARC) classified it as a Class I carcinogen. According to whether they express vacuolar cytotoxin A (VacA) and cytotoxin-related gene A (CagA) toxins, they are divided into two types: type I H. pylori infection virulent strain: more virulent and more harmful to patients. Closely related to the incidence of gastric and duodenal ulcers and gastric cancer, Type II H. pylori infection involves low-virulence strains with weak pathogenicity, generally manifesting as mild indigestion^3,4. Researchers have found that the infection rate of type I is higher than that of type II, with the infection rates of type I and type II being 72.4% and 27.6%, respectively⁵. Rapid detection of H. pylori types, especially the distinction between type I and type II, is crucial for clinical treatment and disease prevention.

Chronic gastritis caused by H. pylori infection can lead to severe digestive system diseases, such as atrophic gastritis, peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma in infected patients^6,7,8. The 2014 Global Burden of Disease Study predicts that starting in 2010, approximately 3.5 deaths per 100,000 people will be caused by peptic ulcer disease each year⁹. Research reports found that nearly 800,000 new cases of gastric cancer worldwide in 2018 were related to H. pylori infection¹⁰. In recent years, studies have found that H. pylori infection is also closely related to neurological diseases, cardiovascular diseases, diabetes, etc. H. pylori infection has become a global public health problem. Therefore, accurate detection of H. pylori infection is important for the detection of related diseases. Treatment is crucial, making accurate detection of H. pylori a research hotspot.

Since the infection rate of H. pylori is high in China, timely and accurate detection of H. pylori infection is helpful for early detection and treatment and has positive significance for the prevention and treatment of gastric cancer. There are various methods for detecting H. pylori infection, including invasive methods such as rapid urease test (RUT), stained microscopy of gastric mucosal tissue sections, endoscopy, and C13 or C14 urea breath test. (UBT), H. pylori stool antigen test (SAT) and other non-invasive methods^11,12,13. These detection methods have certain limitations. For example, the histological examination may give false negative results due to the quality and size of the biopsy sample selected at the biopsy site; the breath test may be affected by the patient's recent use of antibiotics or proton pump inhibitors (PPI) and the influence of various influencing factors^11,14,15. In view of the limitations of traditional methods, such as time-consuming and insufficient sensitivity and specificity, the development of rapid diagnostic methods based on quantum dot immunofluorescence (QD-IF) has shown great application potential in the field of bioassays^16,17. QD-IF analysis introduces quantum dot fluorescent probes to achieve specific detection of molecular targets through specific binding to molecular targets^18,19.

Quantum dots are semiconductor nanoparticles with a radius smaller than or close to the Bohr exciton radius, generally 1 to 10 nm. Quantum dots are used as fluorescent probes. Fluorescence signals are generated by exciting quantum dots, and quantitative data is obtained by measuring the device. Its advantages include a wide range of excitation wavelengths, narrow emission wavelengths, adjustable fluorescence size, high sensitivity, good optical stability, long fluorescence lifetime, large Stokes shift, and high quantum fluorescence efficiency. Quantum dots overcome the shortcomings of other markers, such as short luminescence time, precise environmental requirements, poor repeatability, poor stability (such as fluorescent dyes), enzyme inactivation, and low sensitivity. Quantum dot fluorescence immunoassay combines the advantages of quantum dot fluorescence immunoassay and biomarker fluorescence immunoassay and has the advantages of simplicity, rapidity, high specificity, and high sensitivity^20,21.

Traditional diagnostic tools for H. pylori detection, including bacterial culture, urea breath test, and serological tests, have their own limitations. Bacterial culture is time-consuming and requires strict laboratory conditions, often resulting in relatively low sensitivity due to the fastidious growth requirements of H. pylori. The urea breath test may yield false-negative results under certain circumstances, like recent use of antibiotics or proton pump inhibitors^22,23,24. Serological tests can only indicate past exposure rather than current infection status in some cases, and they lack the ability to distinguish between different virulence types of the pathogen.

In contrast, Quantum Dot Immunofluorescence (QD-IF) technology has emerged as a promising alternative. QD-IF combines the excellent optical properties of quantum dots, such as high fluorescence intensity, narrow emission spectra, and good photostability, with the specificity of immunological reactions. This enables rapid and sensitive detection of H. pylori antigens or antibodies in biological samples. Moreover, it has the unique advantage of being able to simultaneously identify the virulence type of the infecting strain, which traditional methods struggle to achieve. By filling this gap in the diagnostic process, QD-IF technology holds great potential to revolutionize the way we diagnose and manage H. pylori infections, facilitating more personalized and effective medical interventions.

Overall, there is an urgent clinical need for a non-invasive, effective diagnostic method that can detect H. pylori infection and its virulence types (type I and type II) simultaneously. This article aims to use the QD-IF method to evaluate the presence of H. pylori infection in tissue samples and construct a rapid and accurate method for detecting H. pylori infection types. Rapid detection of H. pylori infection and its virulence types is of great significance for achieving precision medicine, improving treatment effects, reducing the risk of related diseases, and preventing gastric cancer. It provides a scientific theoretical basis for clinical staff to choose accurate H. pylori infection detection methods.

Protocol

This study (KY2025-191-01) was performed adhering to the guidelines of the human research ethics committee of the Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University. This article complies with the application for exemption from informed consent.

NOTE: This study aims to use quantum dot technology to design and develop a kit for diagnosing whether participants' blood species contain Helicobacter pylori urease (Urease) antibodies, vacuolar cytotoxin A (VacA) antibodies, and cytotoxin-related gene A (CagA) ) toxin antibodies to determine whether H. pylori is infected and whether the infected strain is type I or type II. This designed kit uses a double-antigen sandwich method to measure Helicobacter pylori Urease antibodies, CagA antibodies, and VacA antibodies. When the test sample containing the target substance is added to the sample pad, it flows through the quantum pad and membrane via siphon action, forming a solid-phase antigen-antibody complex. After spotting, the detection line and mass are read respectively by the analyzer. The signal value of the control line is used to determine the negative and positive status of the Urase antibody, CagA antibody, and VacA antibody based on the T/C signal values of each of the three antibodies. The design diagram is shown in Figure 1.

Figure 1: Schematic diagram of H. pylori typing test card. The figure shows the structure and composition of the test card. Please click here to view a larger version of this figure.

1. Kit preparation

1. Establishment of QDs-2-(n-morphine) ethanesulfonic acid (MES) buffer reaction system
   1. Equilibrate the original QDs solution to room temperature (RT). Then, aspirate 1000 µL and add an equal volume of 2x MES solution (the final concentration of MES was 10 mmol/L).
   2. Mix, shake, and sonicate for 30 s to 3 min.
2. Activation
   1. Add 20 mg/mL 1-Ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC)/ N-Hydroxysuccinimide (NHS) into the QDs-MES buffer system at a ratio of 10:1. Mix, shake, and sonicate it for 3 min.
   2. Place the prepared solution in a 37 °C water bath for 15 min and centrifuge at 13,000 × g for 20 min.
   3. Remove the supernatant from the centrifuged solution and re-dissolve it with 1x MES solution. Blow the solution repeatedly with a pipette, shake it to mix it evenly, and sonicate it for 3 min.
   4. Place the above solution in a 37 °C water bath for 15 min and centrifuge at 13,000 × g for 20 min.
3. Coupling
   1. Add 0.03 mg of Urease antigen, CagA antigen, VacA antigen, and goat anti-mouse IgG to 1000 µL of the activation solution, pipette the tip repeatedly, shake, and sonicate for 3 min.
   2. Place the above solution in a 37 °C water bath and react for 3 h.
4. Closure and storage
   1. Add 100 µL of blocking solution (Supplementary Table 1) and incubate for 1 h.
   2. Centrifuge the blocked solution at 13,000 × g for 15 min.
   3. Remove the supernatant from the centrifuged solution, add 750 µL of reconstitution solution (Supplementary Table 1), repeatedly pipette, shake, and sonicate for 5 min, then store at 4 °C.

2. Preparation of coated plates

1. Preparation of quantum pads
   1. Dilute the quantum dot-labeled H. pylori antigen complex 1:5 with quantum dot dilution buffer to prepare the required amount according to batch production.
   2. Take the quantum pad and evenly spray a certain amount of quantum dot-labeled H. pylori antigen complex onto the pad using a film sprayer and dry at 37 °C for 30 min.
2. Membrane preparation
   1. Use the printing buffer to dilute the control line and test line coating materials and prepare the required amount according to the batch production volume.
   2. Stick the nitrocellulose membrane on the polyvinyl chloride (PVC) plate, use a film-scratch gold sprayer to evenly coat the quality control line and test line materials on the membrane, and dry at 57 °C for 24 h.

3. Assembly

1. Paste the quantum and sample pads onto the film-coated PVC plate in sequence.

4. Cutting into strips

1. Use a strip cutter to cut the assembled large board into test strips.

5. Inner packaging

1. Complete the packaging according to the kit composition requirements.
   NOTE: The components of the kit are as follows: (i) Test card: It consists of a test strip shell and a test strip. The test strip consists of a PVC plate, a sample pad, a quantum pad (an antigen complex labeled with quantum dots; the complex contains Urease antigen, CagA antigen, VacA antigen, and mouse IgG), a nitrocellulose membrane (the detection area is fixed with VacA antigen, CagA antigen and Urease antigen (recombinant antigen eukaryotic expression), and the quality control area is coated with goat anti-mouse IgG (goat poly antibody)), and absorbent paper. Different packaging specifications have different test card quantities. One test card is required for one person's reagent. (ii) SD card: 1 piece/box. Test card and secure digital (SD) card components from different batches of test kits must not be mixed.

6. Sample processing

NOTE: For sample collection, refer to the National Clinical Laboratory Operation Procedures.

1. Collect blood in a separation gel tube. Take care to avoid hemolysis.
2. Test the separated serum sample within 4 h at room temperature (RT). If it cannot be tested in time, store the sample at 2-8 °C for 3 days. For long-term (within 1 year), seal and store it below -20 °C.
3. Restore the samples to RT and thoroughly mix them before use.
   NOTE: Frozen and thawed samples must be completely thawed before use, and the number of freeze-thaw cycles should not exceed five. Repeated freeze-thawed samples may cause the analyte to deteriorate and should be avoided. Severe chyle and hemolysis samples will also affect the test and should be avoided.

7. Procedure for the test

1. Take out the reagent kit and quality control materials to equilibrate to RT.
2. Sample preparation: Centrifuge the whole blood samples at 1467 g for 10 min.
3. Verify that the SD card lot number matches that of the reagent kit. Insert the SD card into the instrument for automatic calibration.
4. Power on the instrument and the operating computer. Launch the designated software and select the Test module. Choose Initialization Settings to initialize the instrument and wait for successful completion.
5. Access the instrument's reagent area and remove the test card slot. Open the reagent kit and select the required number of test cards.
6. Open the foil pouches of the test cards and place them in the slot according to the indicated orientation. Reinsert the card slot into the instrument's reagent area.
7. Quality Control (QC) Testing
   1. Within the Test module, select Manual Entry. Click Information Entry and input the specific QC material information. Click Generate. Place the QC materials in the designated sample rack, ensuring correct order and orientation.
   2. Click Start Test to initiate QC testing. The instrument's sample needle will aspirate 80 µL of the sample and dispense it vertically into the sample well. The instrument will start timing automatically after sample dispensing. After 15 min, the instrument will perform detection, read the measurement values and output the results.
8. Sample Testing: Within the Test module, select Bidirectional LIS. Place the samples to be tested in the designated sample rack, ensuring proper order and orientation. Click Start Test. The instrument will similarly aspirate 80 µL of sample from each and process them identically to the QC process.
9. Once testing is completed, remove the used test cards from the instrument and dispose of them as medical waste.

8. Quality control

1. H. pylori negative: Check and ensure that the results of Helicobacter pylori negative reference products, namely Urease antibody, CagA antibody, and VacA antibody, are all negative.
2. H. pylori type II infection: Check and ensure that the Helicobacter pylori type II reference product, that is, the Urease antibody, is positive, and the CagA antibody and VacA antibody results are all negative.
3. H. pylori type I infection: Check and ensure that the Helicobacter pylori type I reference product, that is, Urease antibody is positive, CagA antibody, and/or VacA antibody are positive.

9. Results analysis

1. Type II infection with weak or non-toxigenic strains: When the Urease antibody reading is positive, and the CagA antibody and VacA antibody readings are negative, interpret it as an infection with a weak or non-toxigenic H. pylori strain.
2. Type I infection with highly pathogenic toxin-producing strains: When the Urease antibody reading is positive, and either the CagA antibody or the VacA antibody reading is positive, or both are positive, interpret it as an infection with a highly pathogenic H. pylori toxin-producing strain.
3. Negative: When the readings of the Urease antibody, CagA antibody, and VacA antibody are all negative, interpret it as no infection with H. pylori strains.
4. Invalid: When the instrument judges the result as invalid, check for incorrect operation or reagent kit failure and try the procedure again.

10. Calibration of the fluorescence analyzer

1. First, turn on the analyzer and let it warm up for about 15 min to stabilize its performance. Then, insert the calibration standard provided by the manufacturer into the sample holder.
2. Next, access the calibration function on the analyzer's interface and initiate the process. The analyzer will automatically detect the fluorescence of the standard and adjust its internal parameters accordingly to match the preset reference values.
3. After that, remove the calibration standard and insert a blank sample to verify that the readings are within the acceptable range.
4. Regularly repeating these steps as per the recommended schedule helps maintain the accuracy of the analyzer for reliable measurements.

Results

Figure 2 shows the working principle of the quantum dot fluorescence immunoanalyzer. It mainly consists of an excitation light source, a receiving light source, a photoelectric conversion circuit module, a signal processing circuit, an AD value conversion circuit, and a signal processing calculation module; the excitation light source generates fluorescent markers. It absorbs monochromatic light with a similar wavelength, and the monochromatic light shines on the fixed scanning point of the ...

Discussion

Current diagnostic methods for Helicobacter pylori are mainly divided into two categories: invasive and non-invasive²⁶. Methods such as rapid urease test (RUT), histology, molecular biology, and bacterial culture have limitations in invasiveness, sensitivity, specificity, time constraints, cost, equipment requirements, sample handling, and real-time monitoring. More common are rapid in vitro diagnostics using methods such as urea breath testing, fecal antigen, and serological ant...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-(3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride(EDC)	Sigma	25952-53-8	5 g/bottle
2-(n-morphine) ethanesulfonic acid (MES)	Sigma	145224-94-8	100 g/bottle
Centrifuge	Sigma	3K15
Fluorescence Immuno Analyzer	Guangzhou Labsim Biotech Co.,Ltd	Guangdong Province Medical Device Registration No. 2017221743
Helicobacter pylori Typing Test Kit	Chongqing iSIA BIO-Technology Co., Ltd	National medical device registration No. 20203400330	QDs Immunofluorescence Method
Sodium salt of N-hydroxythiosuccinimide (NHS)	Sigma	106627-54-7	1 g/bottle