JP4677525B2 - Detection reagent and detection method for vacuolated toxin of Helicobacter pylori - Google Patents

Description

  The present invention relates to a detection reagent and a detection method for Helicobacter pylori vacuolated toxin. More specifically, the present invention relates to the field of diagnosis of gastritis, gastric ulcer or gastric cancer using a detection reagent for Helicobacter pylori vacuolated toxin VacA.

  Helicobacter pylori, a gram-negative bacilli, inhabits the gastric mucosa of more than half of the world's population. The majority of the infected patients remain asymptomatic, but persistent infection with this strain sometimes causes peptic ulcers from gastritis, and it has been pointed out that it is associated with intestinal metaplasia, gastric cancer, MALT lymphoma, and the like. VacA, a vacuolating toxin produced by Helicobacter pylori, is a toxin that forms vacuoles in the cytoplasm of target cells and kills the cells, and has been implicated in the pathogenicity of this bacterium (non- Patent Document 1). To date, in order to understand the initial action of VacA on host cells, VacA receptor has been purified from human kidney cancer-derived cell line G401 cell or human gastric cancer-derived cell line AZ-521 cell, and VacA receptor 2 It was clarified that it is a kind of receptor tyrosine phosphatase (RPTP), RPTPα and RPTPβ (Non-patent Document 2). Furthermore, it has been clarified that mice that knock out RPTPβ of these two types of RPTP do not develop gastritis or ulcers observed by VacA administration (Non-patent Document 3). It has been reported that for binding of VacA, the O-link sugar chain bound to Thr of the QTTQP sequence located at residues 747-751 of RPTPβ, and the terminal sialic acid of the sugar chain are important ( Non-patent document 4).

As a means for detecting Helicobacter pylori, conventional methods include a method for measuring the activity of urease produced by Helicobacter pylori (Non-patent Document 5), or immunization using an antibody that specifically binds to an antigen such as urease. It depends on the measurement method (Non-patent document 6). For example, a urea breath test is often used as a method for measuring urease activity. This is a method for measuring ¹³ C in exhaled air by utilizing the fact that urea labeled with ¹³ C is converted into ammonia and carbon dioxide by Helicobacter pylori.

  The method is simple and excellent for simply detecting the presence of Helicobacter pylori, but the nature of Helicobacter pylori cannot be determined. Whether Helicobacter pylori produces toxins varies depending on the strain, and the grade of malignancy differs for each strain. Not all Helicobacter pylori infected individuals develop gastric ulcers, gastric cancer, etc. On the other hand, eradication from all infected persons is not meaningful from the viewpoint of preventive medicine. Therefore, there is a demand for treatment corresponding to the degree of malignancy of Helicobacter pylori.

As a conventional method for specifically detecting VacA, there is an ELISA method using an antibody against VacA. Although this method is simple and highly sensitive, it requires a lot of steps such as washing, and it takes time to prepare an antibody, so that it is expensive as a general-purpose test. .
Cover, TL, Blake, SR, Nat. Rev. Microbiol., 2005, 3, p.320-332 Wada, A., Yamasaki, E., Hirayama, T., J. Biochem., 2004, 136, p.741-746 Fujikawa, A., Shirasaka, D., Yamamoto, S., Ota, H., Yahiro, K., Fukada, M., Shintani, T., Wada, A., Aoyama, N., Hirayama, T., Fukamachi, H., Noda, M., Nat. Genet., 2003, 33, p.375-381 Yahiro K., Wada A., Yamasaki E., Nakayama M., Nishi Y., Hisatsune J., Morinaga N., Sap J., Noda M., Moss J., Hirayama T., J. Biol. Chem. , 2004, 279, p.51013-51021 Motoaki Kato, Satoshi Hokari, Toshiro Sugiyama, Masahiro Asaka: Diagnosis and sanitization of H.pyrori infection, Clinicians, 2001, 27, p.28-33 Toshiro Sugiyama: Current Status and Problems of H.pyrori Serological Diagnosis, Prog. Med., 1995, 15, p.515-520

  An object of the present invention is to provide a detection reagent and a detection method for the bacterium capable of being treated according to the malignancy of Helicobacter pylori.

  In view of the above problems, the present inventors are able to detect Helicobacter pylori that correlates with malignancy by focusing on VacA among the pathogenic factors of Helicobacter pylori and binding to VacA. As a result, the present invention has been completed.

  That is, the present invention is as follows.

[1] A detection reagent for Helicobacter pylori vacuolated toxin VacA comprising at least one member selected from the group consisting of gangliosides and chemically modified lyso forms thereof.
[2] The detection reagent according to [1], wherein the ganglioside is composed of a group consisting of GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b.
[3] The detection reagent according to [1], wherein the ganglioside is GM1.
[4] The detection reagent according to [1], wherein the lyso form is a lyso form of GM1.
[5] The detection reagent according to [1], wherein the chemical modification is an isotope label, a protein label, an antibody label, an antibody epitope tag label, or a fluorescent label.
[6] The detection reagent according to any one of [1] to [5], which is carried on the surface of a carrier.
[7] A step of preparing a sample solution by mixing the detection reagent according to any one of [1] to [6] and a sample containing VacA, and a detection means corresponding to the detection reagent used in the preparation step A method for detecting VacA in a sample, comprising a step of measuring a signal from the sample solution by using the method.
[8] A step of preparing a sample solution by mixing the fluorescently labeled detection reagent according to the above [5] and a sample containing VacA, and the intensity of the entire fluorescence signal of the sample solution using a fluorescence detection system A method for detecting VacA in a sample, comprising a step of measuring a change in fluorescence signal over time or a degree of fluorescence polarization.
[9] A step of preparing a sample solution by mixing the detection reagent according to [6] above and a sample containing VacA;
A method for detecting VacA in a sample, comprising: separating VacA bound to a detection reagent from the sample solution together with a carrier; and measuring the separated VacA.
[10] Any one of the above [7] to [9], wherein the sample containing VacA has been subjected to a treatment for physically or chemically crushing part or all of the Helicobacter pylori cells in advance. The detection method according to.
[11] The detection method according to any one of [7] to [10], further including a pretreatment step for extracting and / or purifying VacA in the sample before the step of preparing the sample solution. .
[12] A method for diagnosing a disease associated with Helicobacter pylori, comprising using the detection method according to any one of [7] to [11].
[13] The diagnostic method according to [12], wherein the disease is gastritis, gastric ulcer, or gastric cancer.

  According to the detection reagent for VacA of the present invention, a ganglioside that is a basic structure or a substance that is chemically modified to lyso form thereof is contained, and specifically binds to VacA among proteins produced by Helicobacter pylori.・ Detection based on the pathogenicity of H. pylori is possible. Since the VacA detection reagent has a relatively low molecular weight and can be easily synthesized in large quantities, it can be easily detected by a detection device that uses a change in the molecular weight of a fluorescent substance as an index, for example, fluorescence correlation spectroscopy or fluorescence polarization. It is possible to provide the detection means more rapidly and with higher sensitivity and lower cost than when using. According to the detection method of the present invention using such a detection reagent, it is possible to quickly select pathogen-bearing carriers from those infected with Helicobacter pylori. In addition, according to the diagnostic method of the present invention, by accurately diagnosing pathogenic carriers using the detection reagent and detection method of the present invention, it is possible to accurately determine the necessity and timing of administration of a therapeutic agent and antibiotics. Abuse of substances and the like can be prevented, the occurrence of multidrug-resistant bacteria can be reduced, and as a result, medical costs can be reduced.

  The VacA detection reagent of the present invention (hereinafter sometimes simply referred to as a detection reagent) contains at least one selected from the group consisting of gangliosides and chemical modifications of lyso forms thereof.

  VacA is a toxin produced by Helicobacter pylori and has an action of causing vacuolar degeneration of epithelial cells. Such vacuolar degeneration can be confirmed by observing the morphology of epithelial cells with a microscope or examining the uptake of neutral red into the vacuole. The genomic sequence of Helicobacter pylori is disclosed in Gene Bank Accession Nos. NC — 000921 and NC — 000915, and the base sequence encoding VacA or the encoded amino acid sequence is disclosed in AY — 737319, AB — 190958, AB — 190988, and the like. VacA may be a monomer or a multimer (eg, hexamer), but is preferably a monomer or multimer (eg, hexamer) activated by acid treatment. .

  The ganglioside refers to a glycolipid containing sialic acid and may be a natural product or a chemically synthesized product. A preferred ganglioside is GM1, GM2, GM3, GD1a, GD1b, GD3 or GT1b, more preferably GM1, from the viewpoint of binding to VacA.

  The lyso form means a structure containing sphingosine in which a fatty acid is liberated from the ceramide portion of the ganglioside to become an amino group. A preferred lyso form is a lyso form of GM1, GM2, GM3, GD1a, GD1b, GD3 or T1b, and more preferably a lyso form of GM1, from the viewpoint of binding to VacA. For example, GM1 and its lyso form are as shown in FIG.

  The chemical modification of the ganglioside or lyso form thereof refers to a compound that is chemically modified so that the ganglioside or lyso form of the ganglioside or lyso form is suitable for detection of VacA. As such a chemical modification, a label is preferable from the viewpoint of easy detection. From the viewpoint of ease of introduction of chemical modification, it is preferable to use a lyso form having an amino group as a basic structure, but it is not limited thereto.

Preferable examples of the label include isotope labeling, protein labeling, antibody labeling, antibody epitope tag labeling, and fluorescent labeling. Examples of the isotope label include ² H, ³ H, ¹³ C, ¹⁴ C, ³² P, ³⁵ S, and ¹²⁵ I. Examples of protein labels include alkaline phosphatase, peroxidase, glutathione-S-transferase (GST), avidin, streptavidin, maltose binding protein, green fluorescein protein (GFP), and GAL4 protein. Examples of the antibody label include an anti-glutathione-S-transferase antibody, an anti-influenza hemagglutinin antibody, an anti-flag epitope antibody, an anti-maltose binding protein antibody, an anti-green fluorescein protein (GFP) antibody, and an anti-GAL4 protein antibody. . Examples of the antibody epitope tag tag include polyhistidine, influenza hemagglutinin epitope, and flag epitope. Examples of fluorescent labels include Alexa, various types of rhodamine (rhodamine 6G, rhodamine green, TMR, TAMRA), Bodipy, Cy5, R6G, FAM, JOE, ROX, EDANS, fluorescein isothiocyanate (FITC), and the like.

  The chemical modification can be introduced by a known method depending on the modifying group to be introduced.

  The detection reagent of the present invention preferably contains a fluorescently labeled chemical modification in view of detection sensitivity and ease of detection.

  In addition, the detection reagent of the present invention is preferably supported on the surface of the carrier from the viewpoint of operability in the detection method.

  Examples of the carrier include sepharose, agarose, polyacrylic acid derivatives, polystyrene, and the like, which are preferably processed into a bead shape, a stick shape, or a flat plate shape, but are not limited thereto. By immobilizing a detection reagent, that is, a VacA binding substance on the surface of these carriers, it becomes easy to separate from the sample solution together with VacA bound to the reagent by an operation such as centrifugation or filtration or washing. It is possible to provide detection means.

  The detection reagent of the present invention only needs to contain at least one chemical modification, but may contain two or more chemical modifications as long as the object of the present invention is achieved.

  The detection reagent of the present invention may be the chemical modification as it is, or may contain a known additive. Examples of the additive include preservatives such as sodium azide, sodium benzoate, sodium hydrogen sulfite, methyl paraben, propyl paraben, diluents such as water, physiological saline, buffer solution, methanol, ethanol, dimethyl sulfoxide, dimethyl Examples include organic solvents such as formamide and glycerol, but are not limited thereto.

  The content of the chemically modified product can be appropriately set within a range in which a desired detection effect can be obtained, but is usually 0.01 to 100% by weight, preferably 0.1 to 99.9% by weight. %, More preferably 0.5 to 99.5% by weight.

  Since the detection reagent of the present invention specifically binds to VacA among proteins produced by Helicobacter pylori, it can be suitably used for a detection method using the pathogenicity of Helicobacter pylori as an index.

The present invention provides a method for detecting Helicobacter pylori VacA, which comprises using the VacA detection reagent of the present invention. The detection method is as follows:
A step of preparing a sample solution by mixing the detection reagent and a sample containing VacA, and a step of measuring a signal from the sample solution using a detection means corresponding to the detection reagent used in the preparation step.

1) Step of preparing a sample solution by mixing the detection reagent and a sample containing VacA The sample containing VacA may be any sample as long as VacA may exist in the sample.

  The method of mixing the detection reagent and the sample containing VacA may be performed under conditions that allow the reagent and VacA to come into contact with each other in a solution suitable for the detection reagent to be used. For example, in a solvent in which the detection reagent and VacA can be dissolved, the detection reagent and VacA can be mixed for several minutes to one day at a temperature of about 0 to 40 ° C. The mixing includes not only a mechanical mixing means but also a stationary means. The prepared sample solution is subjected to the following measurement process.

2) A step of measuring a signal from the sample solution using a detection means corresponding to the detection reagent used in the preparation step The detection means corresponds to the detection reagent used and can be performed by a known method. . For example, when an isotope-labeled detection reagent is used, a signal can be measured using a radiation detection apparatus corresponding to the isotope. When an enzyme protein-labeled detection reagent is used, a signal emitted by adding an enzyme substrate and decomposing the substrate can be measured. When a detection reagent labeled with a protein or antibody is used, the degree of staining can be measured by immunostaining using a secondary antibody and / or antigen that recognizes the protein or antibody. When a detection reagent labeled with an antibody epitope tag is used, the degree of staining can be measured by immunostaining using an antibody that recognizes the tag. When a fluorescently labeled detection reagent is used, the intensity of fluorescence can be measured with a fluorescence detector or the like by irradiating excitation light corresponding to the fluorescent label.

  In the present invention, when the sample containing VacA also contains Helicobacter pylori cells for the purpose of increasing the sensitivity of detection or facilitating the measurement process, It is preferable to apply a treatment for physically or chemically crushing a part or all of it. Examples of such treatment include freezing and thawing of the sample, ultrasonic treatment of the sample, lysis of bacterial cells to which a surfactant or the like is added in the sample, treatment with acid or alkali, heat treatment, etc. Is not to be done.

  In the present invention, it is preferable to further include a pretreatment step for extracting or purifying VacA in the sample and both for the purpose of increasing the sensitivity of detection or facilitating the measurement step. . As such a pretreatment step, a normal method for extracting and purifying proteins in a solution can be used without limitation. For example, a method of performing a treatment such as centrifugation, ammonium sulfate precipitation, dialysis and the like after the crushing step of the microbial cells can be mentioned, but the method is not limited thereto.

  The signal obtained by the measurement step is compared with a signal in a sample not containing VacA, and in some cases, compared with a signal in a sample containing VacA at a certain concentration, and VacA contained in the sample to be detected is qualitatively determined. Or it can detect quantitatively. In addition, when there is a possibility that the measurement target object contains VacA and other substances, both can be determined based on the difference in molecular weight of the measurement target object. Alternatively, by using a detection reagent containing multiple chemical modifications such as gangliosides with different affinities, both can be distinguished by comparing the differences in binding strength of VacA and other substances to multiple chemical modifications. It is.

In the present invention, it is preferable to use a fluorescently labeled detection reagent from the viewpoint of ease of detection and sensitivity. In this case, the detection method of the present invention is based on the step of preparing a sample solution by mixing with a sample containing VacA, and the intensity of the entire fluorescence signal of the sample solution using the fluorescence detection system, and the time course of the fluorescence signal. Measuring the degree of change or fluorescence polarization.

1) The preparation process of the sample solution is as described above.

2 ′) Step of measuring the intensity of the entire fluorescence signal of the sample solution, the change of the fluorescence signal over time or the degree of fluorescence polarization using a fluorescence detection system In the present invention, as a suitable fluorescence detection system, fluorescence polarization Detection system based on the detection method, detection system based on fluorescence correlation spectroscopy, and the like. Fluorescence polarization is a method that measures changes in the molecular size of fluorescent substances by irradiating polarized excitation light into a solution and examining the depolarization caused by the rotation of the molecules (Kakehi, K., Oda , Y., and Kinoshita, M. Anal. Biochem., 2001, 297, 2, p.111-116). Fluorescence correlation spectroscopy is a method that measures changes in the molecular size of fluorescent substances by examining the degree of fluctuation of fluorescence intensity over time by the movement of molecules in a minute space (Eigen, M. and Rigler, R Proc. Natl. Acad. Sci. USA, 1994, 91, p.5740-5747).

  In the fluorescence detection system, it is possible to qualitatively or quantitatively measure the intensity of the entire fluorescence signal, the change of the fluorescence signal over time, or the degree of fluorescence polarization. For example, in the case of fluorescence polarization, since the degree of depolarization varies depending on the mass of the molecule bound to the fluorescence detection reagent, not only the quantification of VacA but also the difference in the bound molecular weight can be detected. Therefore, it can be examined whether the bound VacA is a monomer or a multimer such as a hexamer.

  The signal obtained by the measurement step is compared with a signal in a sample not containing VacA, and in some cases, compared with a signal in a sample containing VacA at a certain concentration, and VacA contained in the sample to be detected is qualitatively determined. Or it can detect quantitatively.

In the present invention, it is preferable to use a detection reagent supported on a carrier from the viewpoint of ease of detection and sensitivity. In this case, the detection method of the present invention comprises a step of preparing a sample solution by mixing a detection reagent supported on a carrier and a sample containing VacA.
Separating the VacA bound to the detection reagent from the sample solution together with a carrier, and measuring the separated VacA.

  The preparation process of the sample solution is as described above.

  The separation step can be performed by centrifuging or filtering VacA bound to a detection reagent together with a carrier. By further washing the separated VacA, the separation of VacA from the sample solution becomes better.

  The step of measuring VacA is as described above.

  The present invention provides a method for diagnosing a disease associated with Helicobacter pylori by using the detection method.

  The disease to be diagnosed is not particularly limited as long as it is a disease involving Helicobacter pylori, but is preferably gastritis, gastric ulcer, or gastric cancer, and gastritis that is particularly suggested to be associated with VacA. Or a gastric ulcer is more preferable.

  The animals to be diagnosed are humans and non-human mammals, and examples of non-human mammals include monkeys, cows, horses, pigs, sheep, rabbits, dogs, cats, mice, rats, hamsters, and guinea pigs. It is done.

  In this diagnostic method, the sample containing VacA is preferably a biological sample derived from the animal, particularly a sample derived from the stomach inhabiting Helicobacter pylori, such as gastric juice, gastric mucosa, gastric biopsy sample, and the like. Preferably exemplified.

  In this diagnostic method, the signal obtained by the measurement step is compared with the signal in the sample not containing VacA, and in some cases, compared with the signal in the sample containing VacA at a certain concentration, and included in the sample to be detected. VacA can be detected qualitatively, preferably quantitatively. If VacA is detected, the subject is carrying a pathogenic Helicobacter pylori and is appropriately administered a drug aimed at eradication or reduction of symptoms before or after the onset of the disease be able to. Further, it can be used as an index for diagnosis of whether the prognosis of treatment is successful, for example, whether sterilization is successful or not.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention in detail, this invention is not restrict | limited at all by these Examples.

Example 1: Binding of VacA to a ganglioside bound to a carrier Lyso-GM1, which is one of gangliosides, was bound to a resin, and whether or not VacA bound to this resin was determined by binding VacA to the ganglioside. The binding ability was evaluated. Lyso-GM1-Sepharose or control Sepharose was incubated at 4 ° C. with a dialyzed sample of Helicobacter pylori culture supernatant precipitated with ammonium sulfate. After centrifugation (20 min, 15000 g), the precipitated Sepharose was washed. The protein bound to Lyso-GM1-Sepharose or control Sepharose was analyzed by SDS-PAGE, and the protein specifically bound to Lyso-GM1 was analyzed. In addition, Western blotting using an anti-VacA antibody prepared by immunizing rabbits with VacA protein by a conventional method was used to confirm whether VacA was specifically bound to Lyso-GM1. As a result, it was revealed that the protein that specifically binds to Lyso-GM1-Sepharose is VacA.

Example 2: Synthesis of Rhodamine labeled Lyso-GM1
Lyso-GM1 (manufactured by Takara Bio Inc.) 2.5 mM and rhodamine-NHS (N-hydroxysuccinimide) (manufactured by Molecular Probes Inc.) 2.5 mM were reacted in DMF containing 0.1% triethylamine for 16 hours at room temperature. . After distilling off DMF under reduced pressure, the reaction solution was separated by a reverse phase HPLC (Gilson HPLC system, Tosoh TSKgel ODS-120A column 4.6 × 150 mm) with an acetonitrile (0.05% trifluoroacetic acid) concentration gradient. A fraction eluted at 66% acetonitrile with light absorption at 570 nm was obtained. NMR measurement was performed, and the molecular structure of the target product represented by the following chemical formula was confirmed.

Example 3: Detection of VacA by fluorescence polarization method using rhodamine-labeled Lyso-GM1 Purified VacA was diluted to various concentrations, and 150 μl of PBS solution mixed with rhodamine-labeled Lyso-GM1 having a final concentration of 100 nmol / l was measured for fluorescence This was dispensed into a 96-well plate (manufactured by NUNKU). The fluorescence polarization of these solutions was measured with a multi-functional plate reader / Fusion Alpha FP manufactured by PerkinElmer, Inc. using a polarization excitation light filter 535 nm and a polarization fluorescence filter 580 nm (FIG. 2). As a result, an increase in the degree of fluorescence polarization was observed from the VacA concentration of 0.1 nmol / l, and an increase of 200 mP from the reference value was observed at 10 nmol / l of VacA.

FIG. 1 shows the structure of ganglioside GM1 and its lyso form. FIG. 2 is a graph showing the fluorescence polarization signal of VacA using rhodamine-labeled Lyso-GM1.

Claims (14)

Evacuation of Helicobacter pylori comprising at least one selected from the group consisting of gangliosides composed of the group consisting of GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b and chemical modifications of those lyso forms Detection reagent for toxin VacA.   The detection reagent according to claim 1, wherein the ganglioside is GM1.   The detection reagent according to claim 1, wherein the lyso form is a lyso form of GM1.   The detection reagent according to claim 1, wherein the chemical modification is an isotope label, a protein label, an antibody label, an antibody epitope tag label or a fluorescent label. The detection reagent of any one of Claims 1-4 currently carry | supported by the support | carrier surface. A sample solution is prepared by mixing the detection reagent according to any one of claims 1 to 5 and a sample containing VacA, and using the detection means corresponding to the detection reagent used in the preparation step, the sample solution A method for detecting VacA in a sample, comprising the step of measuring a signal from the sample. Step of mixing a sample containing a fluorescent labeled detection reagent and VacA of claim 4 to prepare a sample solution, and said using a fluorescence detection system sample solution fluorescence signals overall strength of the fluorescence signal A method for detecting VacA in a sample, comprising a step of measuring a change with time or a degree of fluorescence polarization. A step of preparing a sample solution by mixing the detection reagent according to claim 5 and a sample containing VacA;
A method for detecting VacA in a sample, comprising: separating VacA bound to a detection reagent from the sample solution together with a carrier; and measuring the separated VacA. A step of preparing a sample solution by mixing the detection reagent according to any one of claims 1 to 5 and a sample containing VacA;
A step of measuring a signal from the sample solution using a detection means corresponding to the detection reagent used in the preparation step; and
Comparing the signal obtained in the measurement step with a signal in a sample not containing VacA, and detecting VacA contained in the sample to be detected.
A method for detecting Helicobacter pylori having pathogenicity, comprising: A step of mixing the fluorescently labeled detection reagent according to claim 4 and a sample containing VacA to prepare a sample solution;
Measuring the intensity of the entire fluorescence signal of the sample solution, the change of the fluorescence signal over time, or the degree of fluorescence polarization using a fluorescence detection system; and
Comparing the signal obtained in the measurement step with a signal in a sample not containing VacA, and detecting VacA contained in the sample to be detected.
A method for detecting Helicobacter pylori having pathogenicity, comprising: A step of preparing a sample solution by mixing the detection reagent according to claim 5 and a sample containing VacA;
Separating VacA bound to a detection reagent from the sample solution together with a carrier;
Measuring the separated VacA using the detection reagent according to any one of claims 1 to 5 using a detection means corresponding to the detection reagent; and
Comparing the signal obtained in the measurement step with a signal in a sample not containing VacA, and detecting VacA contained in the sample to be detected.
A method for detecting Helicobacter pylori having pathogenicity, comprising: The detection method according to any one of claims 6 to 11 , wherein the sample containing VacA has been subjected to a treatment for physically or chemically disrupting a part or all of Helicobacter pylori cells in advance. Before step preparation of the sample solution, further comprising a pretreatment step for the extraction and / or purification of VacA in the sample, detecting how according to any one of claims 6-12. The detection method according to any one of claims 9 to 13, wherein the Helicobacter pylori having pathogenicity is a pathogen of gastritis, gastric ulcer, or gastric cancer.

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