A rapid confocal microscopy-based HP antimicrobial susceptibility testing reagent and its application in Helicobacter pylori detection.

Abstract

This invention relates to the field of biomedical and medical testing reagent technology, specifically disclosing a rapid confocal H. pylori (HP) susceptibility testing reagent and its application in the detection of HP. The reagent comprises a live bacteria fluorescent probe working solution, a dead bacteria fluorescent probe working solution, and an HP activity maintenance solution. The probe working solution contains a composite stabilizer composed of bovine serum albumin, Tween-20, and ascorbic acid in a specific concentration ratio, effectively improving dye stability and staining signal-to-noise ratio. The activity maintenance solution contains a complex reducing system of sodium thioglycolate and L-cysteine, which can maintain HP activity for a short period under normal aerobic conditions. This method reduces the detection time from several days in traditional methods to approximately one hour, is simple to operate, and provides accurate results, offering an efficient tool for precise medication of H. pylori infection in clinical practice.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and medical testing reagents, and in particular to an HP drug susceptibility testing reagent based on rapid confocal microscopy and its application in the detection of Helicobacter pylori. Background Technology

[0002] Helicobacter pylori (HP) is a spiral-shaped, microaerophilic bacterium with extremely demanding growth requirements. First successfully isolated from gastric mucosal biopsy tissue of a patient with chronic active gastritis in 1983, it is currently the only known microorganism capable of surviving in the human stomach. On October 27, 2017, the International Agency for Research on Cancer (IARC) of the World Health Organization published a preliminary list of carcinogens, classifying Helicobacter pylori (infection) as a Group 1 carcinogen. Studies indicate that Helicobacter pylori lives in the pylorus of the human stomach and is one of the most common bacterial pathogens. More than half of the world's population has been infected with Helicobacter pylori, and in some countries, nearly 90% of the population has been infected. Infection typically occurs in childhood, reaching 50% in children under 5 years old. This bacterial infection initially causes chronic gastritis, leading to gastric ulcers and gastric atrophy, and in severe cases, gastric cancer. Statistics show that people who are first infected with Helicobacter pylori at a younger age have a higher incidence of atrophic gastritis and gastric cancer, and there is a parallel relationship between Helicobacter pylori infection and gastric cancer mortality rates. Helicobacter pylori resides in the gastric mucosa and is responsible for 67%–80% of gastric ulcers and 95% of duodenal ulcers. Common symptoms of chronic gastritis and peptic ulcers include postprandial upper abdominal fullness, discomfort, or pain, often accompanied by other adverse symptoms such as belching, bloating, acid reflux, and loss of appetite. Some patients may also experience recurrent episodes of severe abdominal pain and minor upper gastrointestinal bleeding. Early detection of Helicobacter pylori infection and timely and effective eradication of the bacteria with antibiotics are of great significance for the prevention and control of gastric cancer.

[0003] Currently, the treatment of *Helicobacter pylori* (HP) infection mainly employs antibiotic-based combination therapy, such as triple therapy with a proton pump inhibitor (PPI) and two antibiotics, or quadruple therapy with a PPI, bismuth preparation, and two antibiotics. However, with the widespread clinical use of antibiotics, HP resistance has become increasingly prominent. The resistance rates of HP to commonly used antibiotics such as amoxicillin, clarithromycin, and metronidazole are showing a continuously rising trend in different regions. For example, in some areas, the resistance rate of HP to clarithromycin has exceeded 50%, and the resistance rate to metronidazole is even as high as 80%. The emergence of drug resistance significantly reduces the effectiveness of HP infection treatment, not only prolonging the treatment period and increasing patient suffering and financial burden, but also potentially leading to recurrence or even worsening of the condition, increasing the risk of developing serious gastrointestinal diseases. Therefore, accurately detecting the sensitivity of HP to different antibiotics, providing clinicians with a basis for selecting appropriate antibiotics, is crucial for improving the success rate of HP infection treatment and reducing the emergence of drug-resistant strains.

[0004] Existing methods for detecting Helicobacter pylori (HP) drug susceptibility mainly include traditional bacterial culture methods and molecular biological detection methods. Traditional bacterial culture methods are the gold standard for detecting HP drug susceptibility. This involves isolating and culturing HP from patient samples, then performing drug susceptibility tests on culture media containing different antibiotics, and observing bacterial growth to determine its resistance. However, this method has drawbacks such as a long detection cycle (usually 3-7 days), complex operation, and high requirements for laboratory conditions and operators. Furthermore, due to the difficulty in HP culture, the positive rate is low, easily leading to false negatives. For example, the nucleic acid mass spectrometry-based drug resistance gene detection method disclosed in CN117431304A, while having high throughput and speed, detects the drug resistance genotype rather than the actual drug susceptibility phenotype of the bacteria, posing a risk of inconsistency between genotype and phenotype. It also cannot reflect the real-time effect of antibiotics on the physiological state of bacteria and places high demands on equipment and technical personnel. Molecular biological detection methods such as polymerase chain reaction (PCR) and whole genome sequencing (WGS) have advantages such as fast detection speed, high sensitivity and specificity, making them suitable for research on drug resistance mechanisms and analysis of complex drug resistance situations. They mainly detect drug resistance genes of *Helicobacter pylori* (HP) and cannot directly reflect the actual drug sensitivity of bacteria. They also have the problems of false positives and false negatives. At the same time, the detection cost is high and the technical requirements are high, making it difficult to widely use in primary healthcare institutions. They are mainly used for scientific research and detection of some patients with high drug resistance risk.

[0005] Fluorescent dye-based detection methods offer a new approach to rapid drug susceptibility testing. For example, CN103175768B discloses a fluorescent staining kit for rapidly detecting the liveness or deadness of biological cells, using anthocyanin dye and propidium iodide (PI) to distinguish between live and dead bacteria. Furthermore, CN120253795B discloses a rapid detection kit for microorganisms and cells based on confocal multiplex fluorescent labeling, explicitly using DMAO and EthD-III as fluorescent probes for live and dead bacteria, and applying them to bacterial detection. However, these technologies are all designed for general bacteria and cannot be directly used to assess the drug resistance of *Helicobacter pylori* (HP).

[0006] In summary, existing methods for detecting Helicobacter pylori (HP) susceptibility have limitations and cannot meet the clinical need for rapid, accurate, and convenient HP susceptibility testing. Therefore, developing a novel and efficient HP susceptibility testing reagent and method is of significant practical importance. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a rapid confocal HP drug susceptibility detection reagent and its application in Helicobacter pylori detection. The reagent system is complete and highly stable, and can complete the activity identification and drug susceptibility assessment of Helicobacter pylori in a short time, effectively overcoming the problems of long cycle and high cost of traditional culture methods and molecular biology detection. It uses fluorescence staining to detect HP drug susceptibility, which has good stability and can effectively maintain bacterial activity, thereby improving the accuracy of detection.

[0008] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0009] Firstly, the purpose of this invention is to improve an HP drug susceptibility detection reagent based on rapid confocal microscopy, comprising:

[0010] The working solution of the live bacteria fluorescent probe includes the live bacteria fluorescent probe DMAO and a first composite stabilizer system, which includes bovine serum albumin, nonionic surfactant and antioxidant.

[0011] The working solution of the dead bacteria fluorescent probe contains the dead bacteria fluorescent probe EthD-III and a second composite stabilizer system, which contains bovine serum albumin, nonionic surfactant and antioxidant.

[0012] In the first composite stabilizer system and the second composite stabilizer system, the nonionic surfactant is Tween-20 and the antioxidant is ascorbic acid.

[0013] As a preferred technical solution, the concentration of the antioxidant in the working solution of the live bacteria fluorescent probe is higher than its concentration in the working solution of the dead bacteria fluorescent probe; and the concentration of the nonionic surfactant in the working solution of the dead bacteria fluorescent probe is higher than its concentration in the working solution of the live bacteria fluorescent probe.

[0014] As a preferred technical solution, in the working solution of the live bacteria fluorescent probe, the nonionic surfactant is 0.005%-0.02% (v / v) of Tween-20, and the antioxidant is 1-2 mM of ascorbic acid; in the working solution of the dead bacteria fluorescent probe, the nonionic surfactant is 0.02%-0.05% (v / v) of Tween-20, and the antioxidant is 0.5-1 mM of ascorbic acid.

[0015] As a preferred technical solution, the concentration of bovine serum albumin in both the live bacteria fluorescent probe working solution and the dead bacteria fluorescent probe working solution is 0.05%-0.2% (w / v).

[0016] As a preferred technical solution, the working concentration of the live bacteria fluorescent probe DMAO is 0.1-0.5 μg / mL; and the working concentration of the dead bacteria fluorescent probe EthD-III is 0.2-1.0 μg / mL.

[0017] Furthermore, the drug sensitivity testing reagent also includes an HP activity maintenance solution, which contains a basic isotonic solution, a microaerobic environment mimic, an HP-specific growth factor, a growth aid, and a pH stabilizer.

[0018] As a preferred technical solution, the microaerobic environment simulator is 0.01%-0.05% (w / v) sodium thioglycolate; the HP-specific growth factor is 1%-10% (v / v) sterile horse serum; the activity maintenance solution further contains 0.5%-2% (w / v) L-cysteine ​​as a growth aid; and the pH stabilizer is 5-20 mM HEPES buffer.

[0019] As a preferred technical solution, the basic isotonic solution is an aqueous solution containing 0.85% (w / v) sodium chloride and 5% (w / v) glucose.

[0020] Furthermore, the drug sensitivity test reagent also contains at least one or more antibiotic solutions, wherein the antibiotics are selected from at least one or more of amoxicillin, clarithromycin, levofloxacin hydrochloride, metronidazole, furazolidone, and tetracycline.

[0021] Secondly, the present invention further provides the application of the HP drug susceptibility detection reagent based on rapid confocal microscopy provided in the first aspect above in the preparation of medical devices for drug susceptibility detection of Helicobacter pylori.

[0022] Thirdly, the present invention further provides a method for detecting Helicobacter pylori using the HP antimicrobial susceptibility testing reagent based on rapid confocal imaging provided in the first aspect, specifically including the following steps:

[0023] (1) Sample preparation: The same gastric mucosa brush sample was coated into at least one initial control well and multiple antibiotic test wells of a multi-well glass slide;

[0024] (2) Initial staining and detection: HP activity maintenance solution was added to all sample wells for inoculation and culture, and then removed. Subsequently, the working solution of the dead bacteria fluorescent probe was added to the initial control wells for staining in the dark for 5-10 minutes and then removed. The working solution of the live bacteria fluorescent probe was added to the initial control wells for staining in the dark for 1-3 minutes and then removed. After that, the initial control wells were scanned and counted under a confocal microscope, and the number of initial live bacteria and dead bacteria was recorded as background data.

[0025] (3) Antibiotic treatment and staining detection: 15-30 μL of different types of antibiotic solutions were added to each of the multiple antibiotic test wells and the reaction was carried out at 37°C for 10-15 minutes. After the reaction, the staining process and confocal microscope scanning counting process described in step (2) were repeated for each antibiotic test well to obtain the number of live and dead bacteria after each antibiotic treatment.

[0026] (4) Result interpretation: The number or ratio of live bacteria and dead bacteria after adding antibiotics to each test well is compared with the background data before adding antibiotics to the initial control well. The sensitivity of Helicobacter pylori to the corresponding antibiotics is evaluated based on the decrease in the number of live bacteria or the increase in the number of dead bacteria.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] First, the HP antimicrobial susceptibility testing reagent based on rapid confocal microscopy provided by this invention and its application in Helicobacter pylori detection shortens the antimicrobial susceptibility cycle from "days" to "hours". Traditional culture methods require 3-7 days, while this invention, through culture-free direct staining and rapid imaging technology, compresses the entire detection cycle to about 1 hour, achieving near real-time clinical result feedback and greatly supporting rapid clinical decision-making.

[0029] Secondly, the method of this invention breaks through the stringent dependence of traditional methods on sample activity and complex pretreatment, greatly improving its clinical applicability. This method can directly detect raw samples such as clinical gastric mucosal brushes, without the need for bacterial isolation, purification, and amplification, fundamentally avoiding the risk of culture failure. More importantly, the unique HP activity maintenance solution of this invention, through the synergistic effect of sodium thioglycolate and L-cysteine ​​in a specific ratio, provides a stable and durable chemically reducing microenvironment for strictly anaerobic Helicobacter pylori under conventional aerobic experimental conditions. Experimental data confirm that this maintenance solution can maintain HP activity above 90% for at least 2 hours, extending the effective sample handling time window from several minutes in traditional methods to 1-2 hours. This breakthrough not only significantly reduces the risk of false negatives caused by delayed sample delivery or slightly slow operation, but also allows the entire detection process to be carried out comfortably on a regular laboratory bench, eliminating the absolute dependence on expensive and bulky microaerobic workstations or gas generating bags, and greatly reducing equipment barriers and operational complexity.

[0030] Third, regarding the core aspects of rapid detection, this invention designs a precise and stable dedicated reagent system to ensure the accuracy and reproducibility of the results. Specifically, the composite stabilizer formulations were optimized for the different physicochemical properties of the live bacteria probe (DMAO) and the dead bacteria probe (EthD-III). This differentiated design effectively prevents dye quenching and non-specific aggregation, significantly reducing background noise while enhancing fluorescence signal intensity, thus achieving a high signal-to-noise ratio staining effect. After the reagents were stored at 4°C for 30 days, the fluorescence intensity retention rate still exceeded 97%, ensuring the stability and reliability of the detection results. This staining system, combined with the aforementioned activity maintenance solution, constitutes the core technical guarantee for rapid and accurate counting of live / dead bacteria.

[0031] In summary, this invention provides a complete solution from in vitro sample preservation and efficient, specific staining to rapid quantitative analysis. It not only significantly shortens the detection time but also solves two major problems in rapid HP antimicrobial susceptibility testing—the difficulty in maintaining bacterial activity and the instability of staining results—through innovative key reagent formulations. This enables rapid, accurate, and easy-to-operate antimicrobial susceptibility testing that can be performed in a routine laboratory environment, possessing extremely high clinical translational value and market application prospects. Furthermore, this method has been validated with clinical samples, and its antimicrobial susceptibility testing results are highly consistent with the gold standard of the classic culture method, demonstrating its accuracy and reliability in complex real-world samples. Attached Figure Description

[0032] Figure 1 The image shows the fluorescence staining effect of the HP drug sensitivity test reagent provided in Example 2 of this invention on a gastric mucosal cell brush sample (before adding antibiotics, 40× fluorescence microscope).

[0033] Figure 2 The image shows the fluorescence staining effect of the HP drug sensitivity test reagent provided in Example 2 of this invention on a gastric mucosal cell brush sample (after adding antibiotics, 40× fluorescence microscope). Detailed Implementation

[0034] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0035] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In some other embodiments, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0036] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratio" referred to in the following examples refers to a ratio of parts by mass.

[0037] Example 1: The purpose of this example is to provide an HP drug susceptibility detection reagent based on rapid confocal microscopy and its preparation method. The reagent composition and preparation steps are as follows:

[0038] (1) Working solution of live bacteria fluorescent probe: Take 100 mL of sterile PBS (phosphate buffer, pH 7.2), add 0.1 g BSA (bovine serum albumin) and stir to dissolve, add 5 μL Tween-20 (final concentration 0.005% v / v) and mix well, add 0.0176 g ascorbic acid (final concentration 1 mM) to dissolve, add 0.03 mg DMAO powder (final concentration 0.3 μg / mL) and stir until completely dissolved, filter through a 0.22 μm filter membrane for sterilization, dispense and store in a sealed container at 4℃ protected from light. The shelf life is up to 3 months.

[0039] (2) Working solution of dead bacteria fluorescent probe: Take 100mL of sterile PBS (pH 7.2), add 0.1g BSA to dissolve, add 20μL Tween-20 (final concentration 0.02% v / v) and mix well, add 0.0088g ascorbic acid (final concentration 0.5mM) to dissolve, add 0.06mg EthD-III powder (final concentration 0.6 μg / mL) and stir until completely dissolved, filter through a 0.22μm filter membrane for sterilization, dispense and store at 4℃ in the dark, shelf life up to 3 months.

[0040] (3) HP activity maintenance solution: Take 100 mL of deionized water, add 0.85 g NaCl and 5 g glucose to dissolve as a basic isotonic solution, then add 0.03 g sodium thioglycolate (0.03% w / v), 5 mL sterile horse serum (5% v / v), 1 g L-cysteine ​​(1% w / v) and 0.238 g HEPES (10 mM), stir evenly, adjust the pH to 7.2 ± 0.1 with dilute hydrochloric acid or sodium hydroxide solution, filter through a 0.22 μm filter membrane for sterilization, and store at 4 °C.

[0041] (4) Antibiotic solution:

[0042] Prepare according to the following method:

[0043] Amoxicillin solution: Accurately weigh 1.6 mg of amoxicillin, add an appropriate amount of 0.85% physiological saline, stir until completely dissolved, and then dilute to 100 mL with 0.85% physiological saline. The resulting concentration is 16 μg / mL.

[0044] Clarithromycin solution: Accurately weigh 1.6 mg of clarithromycin, add 0.2 mL of dimethyl sulfoxide (DMSO), vortex until completely dissolved, then dilute to 100 mL with 0.85% physiological saline and mix thoroughly. The resulting concentration is 16 μg / mL.

[0045] Levofloxacin hydrochloride solution: Accurately weigh 0.8 mg of levofloxacin hydrochloride, add an appropriate amount of 0.85% physiological saline, stir until completely dissolved, and then dilute to 100 mL with 0.85% physiological saline and mix well. The resulting concentration is 8 μg / mL.

[0046] Metronidazole solution: Accurately weigh 1.6 mg of metronidazole, add an appropriate amount of 0.85% physiological saline, stir until completely dissolved, and then dilute to 100 mL with 0.85% physiological saline. The resulting concentration is 16 μg / mL.

[0047] Furazolidone solution: Accurately weigh 1.6 mg of furazolidone, add 0.2 mL of dimethyl sulfoxide (DMSO), vortex until completely dissolved, then dilute to 100 mL with 0.85% physiological saline and mix thoroughly. The resulting concentration is 16 μg / mL.

[0048] Tetracycline solution: Accurately weigh 1.6 mg of tetracycline, add an appropriate amount of 0.85% physiological saline, stir until completely dissolved, and then dilute to 100 mL with 0.85% physiological saline and mix well. The resulting concentration is 16 μg / mL.

[0049] The reagent system prepared in this embodiment exhibits good dispersibility between the live and dead bacterial fluorescent probe working solutions, low background fluorescence, and the HP activity maintenance solution effectively supports the preservation of Helicobacter pylori activity in vitro for a short period. When applied to the HP drug susceptibility testing of clinical samples, the specific method is as follows:

[0050] (1) Sample preparation

[0051] Clinical gastric mucosal brushing samples were taken, gently rinsed with sterile PBS, and then directly spread onto two trisected multi-well slides. Bacterial cells from the same sample were spread into at least one initial control well and six antibiotic test wells on the slides, ensuring a consistent bacterial background across all wells.

[0052] (2) Initial staining and background detection

[0053] Add an appropriate amount of HP activity maintenance medium to all sample wells (including the initial control wells and all antibiotic test wells) for short-term inoculation (37°C, approximately 10 minutes) to restore and maintain bacterial activity. After incubation, remove the HP activity maintenance medium from the wells.

[0054] Subsequently, the following staining procedure was performed only on the initial control wells:

[0055] Add the working solution of the dead bacterial fluorescent probe to completely cover the sample area, and stain at 37°C for 5 minutes in the dark. After staining, remove the working solution of the dead bacterial fluorescent probe.

[0056] Immediately add the live bacteria fluorescent probe working solution, completely covering the sample area, and stain at 37°C for 2 minutes in the dark. After staining, remove the live bacteria fluorescent probe working solution.

[0057] The slide was quickly placed under a fully automated fluorescence confocal microscope, and the initial control wells were automatically scanned and images were acquired. The initial number of viable bacteria (green fluorescence) and the initial number of dead bacteria (red fluorescence) were analyzed using built-in software and recorded. This data will serve as a background control for assessing the antibiotic's effect.

[0058] (3) Antibiotic treatment and final detection

[0059] In each of the six antibiotic test wells, add 20 μL of amoxicillin, clarithromycin, levofloxacin hydrochloride, metronidazole, furazolidone, and tetracycline solution, respectively, according to clinical needs, ensuring the liquid covers the sample area. Place the slide at 37°C to allow the antibiotics to react with the bacteria for 10 minutes.

[0060] After the reaction is complete, repeat the complete staining procedure described in step (2) for each antibiotic test well:

[0061] Add the working solution of the dead bacteria fluorescent probe, stain in the dark for 5 minutes, and then remove the working solution of the dead bacteria fluorescent probe.

[0062] Add the working solution of the live bacteria fluorescent probe, stain in the dark for 2 minutes, and then remove the working solution of the live bacteria fluorescent probe.

[0063] Immediately scan and count the bacteria under a fully automated fluorescence confocal microscope to obtain the number of terminal viable and dead bacteria after treatment with each antibiotic.

[0064] (4) Result interpretation

[0065] The number of terminal viable / dead bacteria (or the calculated viability rate) obtained after adding antibiotics to each test well is compared with the baseline data before adding antibiotics to the initial control well.

[0066] Sensitivity determination: If the number of live bacteria decreases significantly and / or the number of dead bacteria increases significantly after the treatment of a certain antibiotic test well, the clinical isolate of Helicobacter pylori is determined to be sensitive to this antibiotic.

[0067] Drug resistance determination: If the number of live / dead bacteria after the treatment of a certain antibiotic test well does not change significantly compared with the baseline data, the clinical isolate of Helicobacter pylori is determined to be resistant to this antibiotic.

[0068] The above process allows for the assessment of the sensitivity of multiple antibiotics to be completed within approximately one hour after sampling, providing a reference for timely adjustments to the clinical medication regimen.

[0069] Example 2: Please refer to Example 1. The purpose of this example is to provide an HP antimicrobial susceptibility testing reagent based on rapid confocal microscopy and its preparation method. Its characteristic is that the concentration of stabilizer in the probe working solution varies to optimize the staining effect under specific conditions. The reagent composition and preparation steps are as follows:

[0070] (1) Working solution of live bacteria fluorescent probe: Take 200mL of sterile PBS (pH 7.2), add 0.4g BSA to dissolve, add 40μL Tween-20 (final concentration 0.02% v / v) and mix well, add 0.0704g ascorbic acid (final concentration 2mM) to dissolve, add 0.1mg DMAO (final concentration 0.5 μg / mL) and stir until completely dissolved, filter to sterilize, dispense and store at 4℃ protected from light.

[0071] (2) Working solution of dead bacteria fluorescent probe: Take 200mL of sterile PBS (pH 7.2), add 0.4g BSA to dissolve, add 100μL Tween-20 (final concentration 0.05% v / v) and mix well, add 0.0352g ascorbic acid (final concentration 1mM) to dissolve, add 0.2mg EthD-III (final concentration 1.0 μg / mL) and stir until completely dissolved, filter to sterilize, dispense into containers, and store at 4℃ protected from light.

[0072] (3) HP activity maintenance solution: Take 200mL of deionized water, add 1.7g NaCl and 10g glucose to dissolve, add 0.1g sodium thioglycolate (0.05% w / v), 20mL sterile horse serum (10% v / v), 4g L-cysteine ​​(2% w / v) and 0.953g HEPES (20mM), stir evenly and adjust the pH to 7.2 ± 0.1, filter to sterilize and store at 4℃.

[0073] (4) Antibiotic solution, prepared in the same way as in Example 1.

[0074] The method for detecting HP susceptibility in clinical HP samples using HP susceptibility testing reagents is described in Example 1. As an example, before antibiotic administration, the HP fluorescence staining effect of a gastric mucosal cell brush sample (40× fluorescence microscope) is shown below. Figure 1 As shown; HP fluorescence staining effect of gastric mucosal cell brush sample after antibiotic treatment (40× fluorescence microscope). Figure 2 As shown, the criteria for interpreting this result can be any of the following:

[0075] Qualitative judgment criteria: Before the addition of antibiotics, green fluorescence (live HP bacteria) is significantly dominant in the field of view; after the addition of antibiotics, if the number of red fluorescence (dead HP bacteria) in the field of view increases significantly and becomes dominant, the antibiotic is considered sensitive; if the ratio of red to green fluorescence does not change significantly compared with before the addition of antibiotics, the antibiotic is considered resistant.

[0076] Alternatively, a quantitative judgment criterion can be used: Calculate the reduction rate of viable bacteria in each antibiotic test well relative to the initial control well using confocal microscopy scanning counting. If the reduction rate is ≥ 90%, the antibiotic is considered sensitive; if the reduction rate is < 90%, it is considered resistant. It should be noted that the aforementioned 90% threshold can be optimized and adjusted based on extensive clinical validation data to balance sensitivity and specificity.

[0077] Example 3: Please refer to Example 1. The purpose of this example is to provide an HP drug susceptibility detection reagent based on rapid confocal microscopy and its preparation method. Its feature is that it provides another stable intermediate concentration formulation. The reagent composition and preparation steps are as follows:

[0078] (1) Working solution of live bacteria fluorescent probe: Take 150 mL of sterile PBS (pH 7.2), add 0.15 g of BSA to dissolve, add 22.5 μL of Tween-20 (final concentration 0.015% v / v) and mix well, add 0.0396 g of ascorbic acid (final concentration 1.5 mM) to dissolve, add 0.045 mg of DMAO (final concentration 0.3 μg / mL) and stir until completely dissolved, filter to sterilize and store at 4℃ protected from light.

[0079] (2) Working solution of dead bacteria fluorescent probe: Take 150mL of sterile PBS (pH 7.2), add 0.15g BSA to dissolve, add 45μL Tween-20 (final concentration 0.03% v / v) and mix well, add 0.0198g ascorbic acid (final concentration 0.75mM) to dissolve, add 0.09mg EthD-III (final concentration 0.6 μg / mL) and stir until completely dissolved, filter to sterilize and store at 4℃ protected from light.

[0080] (3) HP activity maintenance solution: Take 150 mL of deionized water, add 1.275 g NaCl and 7.5 g glucose to dissolve, add 0.045 g sodium thioglycolate (0.03% w / v), 7.5 mL sterile horse serum (5% v / v), 1.5 g L-cysteine ​​(1% w / v) and 0.357 g HEPES (10 mM), adjust the pH to 7.2 ± 0.1, filter to sterilize, and store at 4℃.

[0081] (4) Antibiotic solution: Its preparation method is the same as in Example 1.

[0082] The method for detecting HP susceptibility in clinical HP samples using HP susceptibility testing reagents is described in Examples 1 and 2.

[0083] Experimental Example 1

[0084] The purpose of this experimental example is to compare the traditional HP drug susceptibility detection method with the new method provided in Example 2 of this invention, as follows:

[0085] 1. Research Methods

[0086] Gastric mucosal brushing samples were selected from 135 patients with positive C14 urea breath tests. All samples were subjected to parallel comparative tests using both the traditional disk diffusion method (KB method) and the rapid confocal-based drug susceptibility testing reagents and methods provided in this invention.

[0087] Paper disc diffusion method (KB method): Paper discs containing six antibiotics—amoxicillin, clarithromycin, levofloxacin, furazolidone, tetracycline, and metronidazole—were placed on culture media uniformly coated with the corresponding *H. pylori* strains. The media were then incubated at 37°C under microaerophilic conditions for 3 days. The diameter (mm) of the inhibition zone of each of the six inhibitory discs was measured. Drug susceptibility was confirmed according to the latest CLSI M100 standard. No resistance to any antibiotic in a single sample was considered uniformly susceptible; resistance to only one antibiotic was considered monodrug-resistant; resistance to two antibiotics was considered dual-drug-resistant, and so on.

[0088] The rapid detection method of this invention: using the HP drug sensitivity test reagent prepared in Example 2, the same gastric mucosa brush sample is directly coated onto one initial control well and six antibiotic test wells of a multi-well glass slide. The specific test method is carried out as in Example 1, and the sensitivity judgment is based on a reduction of ≥90% in the number of viable bacteria.

[0089] 2. Results and Analysis

[0090] The test results were processed using relevant statistical software. The clinical concordance rate was calculated as follows: using the disk diffusion method as a reference standard, the interpretation results of the present invention were compared with those of the disk diffusion method. The percentage of cases where both results were consistent (both sensitive and both resistant) out of the total number of cases was the clinical concordance rate. Simultaneously, the Kappa test was used to assess the degree of consistency between the two methods (Kappa value > 0.75 indicates good consistency).

[0091] Table 1. Comparison of HP susceptibility test results between the two methods (n=135)

[0092]

[0093] As shown in Table 1, for all six antibiotics, the clinical concordance rate of the method described in this invention was higher than 92% (range: 92.59% - 99.26%) compared to the disk diffusion method. Meanwhile, all Kappa values ​​were greater than 0.75 (range: 0.755 - 0.885), indicating a highly consistent to almost perfectly consistent statistical correlation between the two methods.

[0094] The above clinical data show that the rapid confocal HP susceptibility testing reagent and method provided by this invention are equivalent to the traditional, time-consuming disk diffusion method in terms of detection accuracy. However, the method of this invention significantly reduces the detection time from 72 hours to approximately 1 hour, and is simpler to operate, without relying on bacterial culture. This breakthrough advantage enables clinicians to receive near real-time susceptibility results, which is of great significance for achieving precise treatment of HP infection, avoiding empirical drug failure, and curbing the spread of drug resistance.

[0095] Experimental Example 2

[0096] The purpose of this experiment is to observe the effects of the composite stabilizer system and its specific ratio on preventing dye quenching / aggregation, reducing dyeing background, and improving the signal-to-noise ratio, as detailed below:

[0097] 2.1 Experimental Design and Grouping:

[0098] This experiment tested the working solutions of live bacteria fluorescent probes and dead bacteria fluorescent probes, respectively. In addition to comparing the formulation of this invention with conventional solvents, a control group with adjusted proportions of key components was added. The groups are shown in Table 2.

[0099] Table 2. Experimental grouping of fluorescent probe working solutions

[0100]

[0101] 2.2 Test Methods and Results:

[0102] Prepare the fluorescent probe working solutions according to Table 2 above, and perform the following tests:

[0103] (1) Stability test: Each working solution was stored at 4°C in the dark. The fluorescence intensity (FI) was detected by a fluorescence spectrophotometer on day 0 and day 30, and the fluorescence intensity retention rate (%) on day 30 was calculated accordingly.

[0104] (2) Performance testing: The working solutions of each group were used to stain the known concentration of *HP* dead (heat-inactivated) bacterial suspensions, and the mean fluorescence intensity (MFI, representing staining efficiency) and signal-to-noise ratio (SNR) were quantitatively analyzed by flow cytometry. For the working solutions of dead bacterial fluorescent probes, the hydrodynamic diameter (Dh) was measured by dynamic light scattering (DLS) to evaluate the degree of dye aggregation.

[0105] The experimental results are shown in Table 3:

[0106] Table 3 Performance test results of fluorescent probe working solution

[0107]

[0108] Note: The staining performance and background of the live bacteria fluorescent probe working solution DMAO have been indirectly reflected by the signal-to-noise ratio after staining with its corresponding dead bacteria fluorescent probe working solution; therefore, MFI and SNR were not measured again. The particle size of the live bacteria fluorescent probe working solution DMAO is not a key evaluation indicator in this experiment, therefore it was not measured.

[0109] The experimental results above show that the working solution formulation containing a specific proportion of composite stabilizer has significant advantages in maintaining the long-term stability of fluorescent probes, improving staining efficiency, controlling background interference, and preventing dye aggregation.

[0110] For the working solution of the live bacteria fluorescent probe, the experimental group using the optimized ratio exhibited the highest fluorescence stability. Comparative experiments showed that the concentration of the surfactant (Tween-20) had a significant impact on stability. When the concentration was below the optimal level, stability decreased significantly; while when the concentration was slightly above the optimal level, stability remained at a high level (control group L3). The concentration of the antioxidant (ascorbic acid) had a clear optimal range: too low a concentration led to insufficient protection and decreased stability (control group L4); while when the concentration exceeded the optimal level (control group L5), a noticeable decrease in stability occurred, indicating that the concentration effect of ascorbic acid is not a simple linear relationship, and its protective effect has an optimal range.

[0111] For the working solution of the dead bacteria fluorescent probe, the experimental group with the optimal ratio achieved the best performance in all indicators. The effect of surfactant concentration was particularly prominent: when the concentration was below the optimal level, dye molecule aggregation was significantly aggravated, the average particle size increased significantly, and the staining efficiency and signal-to-noise ratio also decreased simultaneously, indicating that sufficient surfactant is crucial for maintaining the dispersion of dye and ensuring its effective binding with the target. When the concentration was increased to above the optimal level (control group D3), it still had a good effect on inhibiting aggregation, but no further improvement in staining efficiency and signal-to-noise ratio was observed. The change in antioxidant concentration showed a specific effect relationship: lower concentrations led to a decrease in stability and staining efficiency (control group D4); while when the concentration was increased to above the optimal level (control group D5), although good dispersibility was still maintained, the fluorescence intensity retention rate, average fluorescence intensity, and signal-to-noise ratio all showed a slight but clear decrease, and the average particle size also increased slightly.

[0112] In summary, this experiment, through systematic gradient comparison, confirms that the proposed composite stabilizer formulation and its specific ratio can effectively solve problems such as dye quenching, aggregation, and background interference. It achieves an optimized balance between the long-term storage stability of the probe and its immediate staining performance, providing a reliable guarantee for obtaining detection results with a high signal-to-noise ratio.

[0113] Experimental Example 3

[0114] The purpose of this experiment is to observe the technical effects of sodium thioglycolate and L-cysteine ​​on maintaining HP activity over a long period of time in the HP activity maintenance solution provided by this invention. The specific experiment is as follows:

[0115] 3.1 Experimental Design and Grouping:

[0116] Based on the basic components of the HP activity maintenance solution from Example 2 (0.85% physiological saline + 5% glucose + 10% horse serum + 20mM HEPES, pH 7.0), the following experimental and control groups were set up. The reducing agent system composition of each experimental and control group is shown in Table 4:

[0117] Table 4 HP Activity Maintenance Solution Test Grouping

[0118]

[0119] 3.2 Test Methods and Results:

[0120] HP standard strains were mixed with equal volumes of the different maintenance solutions described above and placed in an aerobic environment at 37°C. Samples were taken at 0, 1, and 2 hours, stained with the optimized live bacteria fluorescent probe working solution of this invention, and accurately counted by flow cytometry.

[0121] Table 5. Changes in HP viable bacterial concentration over time under different maintenance solutions (viable bacterial concentration 10^6 CFU / m³, mean ± SD)

[0122]

[0123] The above experimental results show that this experiment systematically evaluated the effects of different reducing agent formulations on maintaining the activity of Helicobacter pylori (HP) by simulating an aerobic stress environment.

[0124] In the absence of any reducing agent (control group - M9), *Helicobacter pylori* (HP) activity was rapidly and acceleratedly lost in an aerobic environment, highlighting the crucial importance of external chemical reduction protection for the survival of such strict anaerobes under routine operating conditions. Compared to the control groups (M1, M2) using only a single reducing agent, the experimental group - M (a combination of both) not only showed an overwhelming advantage in total viable cell rate after 2 hours, but more importantly, its activity maintenance curve was the flattest throughout the experiment. This indicates that the combined system is not a simple additive effect, but rather provides a more durable and stable reducing microenvironment, effectively delaying the accelerated decline in activity caused by reducing agent consumption or oxygen damage accumulation. The results of the concentration gradient control groups (M3-M6) show that the effect of this combined system is highly sensitive to the concentrations of both components. Deviations in the concentrations of either sodium thioglycolate or L-cysteine ​​from their optimal values ​​resulted in a noticeable decrease in protective efficacy, especially in the ability to maintain activity in the later stages. Although a moderate increase in the concentration of individual components (such as M4 and M6) can bring about some improvement, its protective efficacy curve still fails to reach the stable level of the experimental group -M. Moreover, considering the rationality of cost and potential side effects, the current optimized ratio achieves the best balance between protective effect and system stability.

[0125] When L-cysteine ​​was replaced with equal amounts of glutathione (control group - M7) or ascorbic acid (control group - M8), the protective effect was significantly weakened. The ascorbic acid group, in particular, showed a sharp collapse in late-stage protective capacity. This strongly suggests that, in addition to its role as a reducing agent, L-cysteine's unique molecular properties or metabolic functions may provide specific support for HP survival under stress, making it an indispensable key component in this highly efficient complex system.

[0126] In summary, the experimental data clearly demonstrate that the designed low-concentration sodium thioglycolate and L-cysteine ​​complex reduction system can provide a highly efficient, stable, and durable reducing microenvironment for Helicobacter pylori for at least 2 hours through synergistic effects, significantly delaying its death process under aerobic exposure. This effect depends on precise composition and ratio. This characteristic is a key technical guarantee for ensuring the reliable completion of subsequent rapid drug susceptibility testing procedures in routine laboratory environments.

[0127] The embodiments provide a detailed description of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A rapid confocal Hippolytetrafluoroethylene (HP) susceptibility testing reagent, characterized in that, It includes: The working solution of the live bacteria fluorescent probe includes the live bacteria fluorescent probe DMAO and a first composite stabilizer system, which includes bovine serum albumin, nonionic surfactant and antioxidant. The working solution of the dead bacteria fluorescent probe contains the dead bacteria fluorescent probe EthD-III and a second composite stabilizer system, which contains bovine serum albumin, nonionic surfactant and antioxidant. In both the first and second composite stabilizer systems, the nonionic surfactant is Tween-20, and the antioxidant is ascorbic acid. Specifically, in the working solution of the live bacteria fluorescent probe, the nonionic surfactant is 0.02% (v / v) of Tween-20, and the antioxidant is 2 mM ascorbic acid. In the working solution of the dead bacteria fluorescent probe, the nonionic surfactant is 0.05% (v / v) of Tween-20, and the antioxidant is 1 mM ascorbic acid. The concentration of bovine serum albumin in both the live and dead bacteria fluorescent probe working solutions is 0.05%-0.2% (w / v). The working concentration of the live bacteria fluorescent probe DMAO is 0.1-0.5 μg / mL, and the working concentration of the dead bacteria fluorescent probe EthD-III is 0.2-1.0 μg / mL.

2. The HP drug susceptibility testing reagent based on rapid confocal microscopy according to claim 1, characterized in that, The drug sensitivity testing reagent also includes an HP activity maintenance solution, which contains a basic isotonic solution, a microaerobic environment mimic, an HP-specific growth factor, a growth aid, and a pH stabilizer.

3. The HP drug susceptibility testing reagent based on rapid confocal microscopy according to claim 2, characterized in that, The microaerobic environment mimic is 0.01%-0.05% (w / v) sodium thioglycolate; the HP-specific growth factor is 1%-10% (v / v) sterile horse serum; the activity maintenance solution also contains 0.5%-2% (w / v) L-cysteine ​​as a growth aid; and the pH stabilizer is 5-20 mM HEPES buffer.

4. The HP drug susceptibility testing reagent based on rapid confocal microscopy according to claim 2, characterized in that, The basic isotonic solution is an aqueous solution containing 0.85% (w / v) sodium chloride and 5% (w / v) glucose.

5. The HP drug susceptibility testing reagent based on rapid confocal microscopy according to claim 2, characterized in that, The drug sensitivity test reagent further comprises at least one or more antibiotic solutions, wherein the antibiotics are selected from at least one or more of amoxicillin, clarithromycin, levofloxacin hydrochloride, metronidazole, furazolidone, and tetracycline.

6. The use of the HP drug susceptibility detection reagent based on rapid confocal imaging as described in any one of claims 1-5 in the preparation of a medical device for drug susceptibility detection of Helicobacter pylori.

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