Verotoxin Detection Method

Abstract

The present invention is a method for detecting Verotoxin. Mass spectrometry is used to more accurately detect Verotoxin. The method for detecting Verotoxin comprises: preparing a sample and a molecule that binds to Verotoxin; performing an operation of purifying the Verotoxin in the sample by utilizing the binding of the molecule to Verotoxin; and subjecting the sample obtained by the above operation to a first mass spectrometry analysis.

Description

Technical Field

[0001] The present invention relates to a method for detecting verotoxin. Background Art

[0002] The cause of food poisoning caused by enterohemorrhagic Escherichia coli (EHEC) is verotoxin produced by EHEC. There are two types of verotoxin, type 1 and type 2, which are distinguished by serology, and there are multiple subtypes of type 1 and type 2. Verotoxin detection is important for the identification of foods that cause food poisoning, diagnosis, or prediction of symptoms.

[0003] In non-patent document 1, the gene of Verotoxin was amplified and detected by polymerase chain reaction (PCR). In this method, it is possible to determine whether the gene of Verotoxin exists in the bacteria contained in the sample, but it is not possible to distinguish whether Verotoxin is expressed in the bacteria. In non-patent document 2, type 1 and type 2 of Verotoxin were detected by immunochromatography, but it is not possible to distinguish the subtypes. In non-patent document 3, it is determined whether Verotoxin is contained in the sample by whether there is a peak near the m / z corresponding to Verotoxin in mass spectrometry. However, if the peak corresponding to the impurity exists near the m / z corresponding to Verotoxin, it is a false positive.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: Sedo et al., “Enterohemorrhagic Escherichia coli (EHEC) Detection and Diagnosis Manual”, (Japan), National Institute of Infectious Diseases, September 25, 2019

[0007] Non-patent document 2: NH Foods Ltd., "NH Immunochromatography VT 1 / 2 <<Instructions for Use>> 1st edition (NH Immunochromatography VT 1 / 2 <<Instructions for Use>> 1st edition)", (Japan), NH Foods Ltd., December 2009

[0008] Non-patent document 3: Fagerquist CK, Zaragoza WJ, Sultan O, Woo N, Quinones B, Cooley MB, Mandrell RE. "Top-down proteomic identification of Shiga toxin 2subtypes from Shiga toxin-producing Escherichia coli by matrix-assisted laserdesorption ionization-tandem time of flight mass spectrometry" Applied and environmental microbiology, (USA), American Society for Microbiology, May 2014, Volume 80, Issue 9, pp.2928-2940 Summary of the invention

[0009] Problem that the invention aims to solve

[0010] Mass spectrometry is preferably used to more accurately detect Verotoxin.

[0011] Solutions for solving problems

[0012] A first aspect of the present invention relates to a method for detecting Verotoxin, comprising: preparing a sample and a molecule that binds to Verotoxin; performing an operation of purifying the Verotoxin in the sample by utilizing the binding of the molecule to Verotoxin; and subjecting the sample obtained by the operation to a first mass spectrometry analysis.

[0013] Effects of the Invention

[0014] According to the present invention, it is possible to more accurately detect Verotoxin using mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a flowchart showing the flow of a method for detecting virulence toxin according to one embodiment.

[0016] Figure 2 This is a table showing the amino acid sequence of each subtype of the B subunit of Verotoxin.

[0017] Figure 3 This is a mass spectrum of the sample obtained by the purification operation using the anti-Verotoxin 2 antibody in Example 1.

[0018] Figure 4 This is a mass spectrum of the sample obtained by the purification operation using the anti-Verotoxin 1 antibody in Example 1.

[0019] Figure 5 This is a mass spectrum of the sample obtained by the purification operation using the anti-Verotoxin 2 antibody in Example 2.

[0020] Figure 6 This is a mass spectrum of the sample obtained by the purification operation using the anti-Verotoxin 1 antibody in Example 2. DETAILED DESCRIPTION

[0021] Hereinafter, a mode for carrying out the present invention will be described with reference to the drawings.

[0022] - Implementation Methods -

[0023] In the method for detecting Verotoxin of the present embodiment, after performing an operation of purifying Verotoxin in a sample using a molecule that binds to Verotoxin, mass spectrometry analysis is performed.

[0024] Figure 1 This is a flowchart showing the flow of the method for detecting virooxin according to the present embodiment. In step S1001, a sample and a molecule that binds to virooxin are prepared.

[0025] (About the sample)

[0026] The sample is not particularly limited as long as it is a liquid or solid containing or possibly containing verotoxin. For example, the sample includes a liquid or solid containing or possibly containing enterohemorrhagic Escherichia coli. Samples that may contain verotoxin include, for example, food or beverages that have been ingested or touched by organisms such as humans with symptoms of food poisoning, and vomitus of the organisms. Furthermore, bacterial liquids or bacterial communities obtained by culturing bacteria from these samples, and bacterial liquids or bacterial communities obtained by culturing bacteria from these samples, and Community The lysate obtained by lysing the bacteria is also a sample suitable as the object of the method of this embodiment. By applying the method of this embodiment to a sample that may contain Verotoxin, and detecting Verotoxin or identifying the type or subtype, it is possible to identify, diagnose or predict symptoms of food or beverages that are the cause of food poisoning. The method of this embodiment can also be performed on a sample that detects Verotoxin. For example, using the method of this embodiment, the following operations can be performed: when the type or subtype of the detected Verotoxin is unknown, the type or subtype is identified, or it is useful for the study of Verotoxin, or the Verotoxin detection result obtained by other methods is confirmed.

[0027] When the sample contains multiple microorganisms, it is preferred to extract a single type of microorganism by culturing the microorganism using a plate culture medium, etc., and recovering the colonies obtained by the culture. In this case, as described above, a bacterial solution containing the colonies or a lysate obtained by lysing the colonies can be used as the sample.

[0028] (About molecules that bind to Verotoxin)

[0029] Hereinafter, the molecule that binds to Verotoxin is referred to as Verotoxin binding molecule. For Verotoxin binding molecule, there is no particular limitation as long as the Verotoxin binding molecule can be used to purify the sample when the sample contains Verotoxin by combining with Verotoxin. Here, the purification of the sample refers to: the concentration of at least a portion of the molecules other than Verotoxin in the sample is relatively reduced compared to the concentration of Verotoxin.

[0030] The viroxin binding molecule is preferably an antibody or globotriaosylceramide, which is a receptor for viroxin in cells. In the following embodiments, "antibodies" include immunoglobulins such as IgG and immunoglobulin-like molecules that are not immunoglobulins but contain variable regions that bear antigen specificity in immunoglobulins.

[0031] The molecule that can be combined with the type or subtype of the Verotoxin that is desired to be detected can be used as the Verotoxin binding molecule. Verotoxin is composed of a mode that includes 1 A subunit and 5 B subunits. The A subunit is composed of a mode that includes an A1 subunit and an A2 subunit. The Verotoxin binding molecule can be a molecule combined with the A1 subunit, a molecule combined with the A2 subunit, or a molecule combined with the B subunit.

[0032] Figure 2It is a diagram showing the types and subtypes of Verotoxin, and the amino acid sequences of the B subunits of Verotoxin of each type and subtype. Verotoxin type 1 has subtypes of Stx1a, Stx1c and Stx1d. The B subunit of Stx1a has an amino acid sequence of "TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR" (sequence number 1). The B subunit of Stx1c has an amino acid sequence of "APDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR" (sequence number 2). The B subunit of Stx1d has an amino acid sequence of "APDCVTGKVEYTKYNDDDTFTVKVADKELFTNRWNLQSLLLSAQITGMTVTIKTTACHNGGGFSEVIFR" (sequence number 3).

[0033] Verotoxin type 2 has subtypes of Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f and Stx2g. The B subunit of Stx2a has an amino acid sequence of "ADCAKGKIEFSKYNEDDTFTVKVDGKEYWTSRWNLQPLLQSAQLTGMTVTIKSSTCESGSGFAEVQFNND" (sequence number 4). The B subunit of Stx2b has an amino acid sequence of "ADCAKGKIEFSKYNENDTFTVKVAGKEYWTNRWNLQPLLQSAQLTGMTVTIKSNTCASGSGFAEVQFN" (sequence number 5). The B subunit of Stx2c has an amino acid sequence of "ADCAKGKIEFSKYNENDTFTVKVAGKEYWTSRWNLQPLLQSAQLTGMTVTIKSSTCESGSGFAEVQFNND" (sequence number 6). The B subunit of Stx2d has an amino acid sequence of “ADCAKGKIEFSKYNENDTFTVKVAGKEYWTSRWNLQPLLQSAQLTGMTVTIKSSTCASGSGFAEVQFNND” (sequence number 7). The B subunit of Stx2e has an amino acid sequence of “ADCAKGKIEFSKYNEDNTFTVKVSGREYWTNRWNLQPLLQSAQLTGMTVTIISNTCSSGSGFAQVKFN” (sequence number 8). The B subunit of Stx2f has an amino acid sequence of “ADCAVGKIEFSKYNEDDTFTVKVSGREYWTNRWNLQPLLQSAQLTGMTVTIISNTCSSGSGFAQVKFN” (sequence number 9). The B subunit of Stx2g has an amino acid sequence of “ADCAKGKIEFSKYNGDNTFTVKVDGKEYWTNRWNLQPLLQSAQLTGMTVTIKSNTCESGSGFAEVQFNND” (sequence number 10).

[0034] Hereinafter, the situation that Verotoxin is combined with a specific type or subtype and is not combined with other types or subtypes is referred to as "selective binding". The Verotoxin binding molecule can be an antibody that selectively combines with Verotoxin type 1, an antibody that selectively combines with Verotoxin type 2, or an antibody that can combine with Verotoxin type 1 and type 2. The Verotoxin binding molecule can be an antibody that can combine with at least one of Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f and Stx2g. It is expected that the Verotoxin binding molecule uses a site common to each type or subtype of Verotoxin as an epitope, but is not particularly limited thereto.

[0035] Verotoxin binding molecule can be a monoclonal antibody or a polyclonal antibody. In addition, in the present embodiment, as Verotoxin binding molecule, a variety of molecules can be used. Verotoxin binding molecule can comprise antigen-specific or structurally different multiple antibodies, in which case, in the purification operation described later, the sample will be exposed to multiple antibodies.

[0036] Back to Figure 1 After step S1001 is completed, step S1003 is started. In step S1003, an operation for purifying Verotoxin is performed. Hereinafter, this operation is referred to as a purification operation. By the purification operation, when Verotoxin is contained in the sample, the Verotoxin will be purified.

[0037] For the purification of Verotoxin by purification operation, as long as the combination of Verotoxin binding molecule and Verotoxin is utilized to purify, its method is not particularly limited.In purification operation, the reaction that Verotoxin binding molecule is combined with Verotoxin is carried out.For example, the sample is contacted with the solution comprising Verotoxin binding molecule.The purification of Verotoxin by purification operation is preferably carried out by utilizing ultrafiltration to separate the complex obtained by combining Verotoxin binding molecule with Verotoxin.Below, if it is abbreviated as complex, it refers to the complex of Verotoxin binding molecule and Verotoxin.The purification of Verotoxin by purification operation can be carried out by using the affinity purification of Verotoxin binding molecule.The example of affinity purification can include: immunoprecipitation, pull-down assay and affinity chromatography using post, pipette end, micro flow path or spin column (Spin Column) etc.When Verotoxin binding molecule is combined with Verotoxin, Verotoxin can be the state separated into subunits, or it can be the unseparated state. Prior to the operation of binding the Verotoxin-binding molecule to the Verotoxin, a process of separating the Verotoxin into its subunits may be performed.

[0038] In ultrafiltration, macromolecules such as proteins dissolved in water can be separated from microparticles through the pores on the porous ultrafiltration membrane. Due to the deviation of pore size and the difficulty of measurement, the molecular weight cutoff is used instead of the pore size as an indicator to represent the separation performance of the membrane. Each manufacturer of ultrafiltration membrane defines a nominal molecular weight cutoff (Nominal Molecular Weight Limit: NMWL) according to their own different standards. Molecules with a molecular weight of the same degree as the molecular weight cutoff sometimes pass through the ultrafiltration membrane, and sometimes do not. Below, in the retention curve obtained by introducing a variety of standard substances with different molecular weights into the ultrafiltration membrane, the molecular weight with a rejection rate of 90% is used as the molecular weight cutoff.

[0039] In the method using ultrafiltration, after the Verotoxin binding molecule is contacted and combined with Verotoxin, the obtained complex is separated by ultrafiltration. The molecular weight of the A1 subunit of Verotoxin is about 28kDa, the molecular weight of the A2 subunit is about 4kDa, the molecular weight of each B subunit is about 7500~8000Da, and the molecular weight of the IgG of the antibody is about 150kDa. Therefore, the molecular weight of the complex is about 150~230kDa. Therefore, the molecular weight cut-off of the ultrafiltration in the purification operation is preferably 10kDa~200kDa, more preferably 40kDa~150kDa, and further preferably 70kDa~120kDa. Thus, the complex can be retained on the ultrafiltration membrane while the impurities close to the molecular weight of the A subunit or B subunit of Verotoxin are separated in the filtrate. As a result, when the components remaining on the ultrafiltration membrane are subjected to mass spectrometry analysis, the peaks corresponding to impurities located near the m / z of the ions of the A subunit or B subunit of Verotoxin can be reduced. Although m / z is used below as the mass-to-charge ratio, there is no particular limitation as long as it can represent the ratio of the mass to the charge of an ion.

[0040] Use in the method for ultrafiltration, preferably react so that free Verotoxin binding molecules and free Verotoxin are combined in solution.Thus, compared with the situation that the antibody that is fixed on the carrier is combined with Verotoxin, can shorten the reaction time, carry out the purification of Verotoxin rapidly.The rapid purification of Verotoxin is owing to realize the detection of Verotoxin in a short time, therefore especially preferred clinically.In addition, before carrying out the operation that Verotoxin is combined with Verotoxin binding molecules, can also carry out the following operation: by micropore filtration or ultrafiltration etc., remove molecular weight and antibody for equal degree or larger molecule, or remove the molecule that quality is greater than the molecular weight of Verotoxin.

[0041] In the purification operation, after the complex is separated, the operation of separating Verotoxin from Verotoxin binding molecules and the operation of separating Verotoxin into each subunit can also be carried out. These operations can be carried out by, for example, adding an organic solvent in the complex, or adding acids such as sulfuric acid or trifluoroacetic acid (trifluoroacetic acid; TFA). However, when preparing the mass spectrometry sample used by MALDI as described below, Verotoxin can be separated from Verotoxin binding molecules such as antibodies due to the matrix solution, and can be separated into each subunit, so there is no need to carry out these operations.

[0042] It should be noted that in step S1003, microfiltration may be performed using a microporous filtration membrane having equivalent characteristics to the molecular weight of the molecules passed therethrough as a substitute for ultrafiltration or in addition to ultrafiltration.

[0043] After step S1003 is completed, step S1005 is started. In step S1005, a sample for mass spectrometry analysis is prepared.

[0044] (About Preparation of Samples for Mass Spectrometry Analysis)

[0045] The method for preparing the sample for mass spectrometry analysis is not particularly limited as long as it is performed using a method corresponding to the type of ionization in mass spectrometry analysis (step S1007) described later.

[0046] Hereinafter, an example of performing matrix-assisted laser desorption / ionization (MALDI) in mass spectrometry will be described. A solution containing or possibly containing verotoxin obtained by purification is prepared. The purified solution is desalted using a solid phase extraction chip or the like, and a solution containing a matrix (hereinafter referred to as a matrix solution) is added and dripped onto a sample plate for MALDI and dried, thereby obtaining crystals containing a sample and a matrix. The crystals are used as a sample for mass spectrometry. The above solution obtained by purification may be placed on a sample plate for MALDI and then the matrix solution may be added. The type of matrix is ​​not particularly limited, and CHCA (α-cyano-4-hydroxycinnamic acid), sinapinic acid, or DHB (2,5-dihydroxybenzoic acid) may be used as appropriate. The solvent of the matrix solution may be a solvent obtained by adding 0 to 3% by volume of trifluoroacetic acid (TFA) to an aqueous solution containing several tens of% by volume of an organic solvent such as acetonitrile.

[0047] In addition, the sample for mass spectrometry analysis may be prepared by appropriately using additives for improving sensitivity and the like.

[0048] After step S1005 is completed, step S1007 is started. In step S1007, mass spectrometry analysis is performed on the sample obtained in the purification operation. When the sample obtained in the purification operation contains verotoxin, the purified fraction containing verotoxin is subjected to mass spectrometry analysis.

[0049] The method of mass spectrometry is just not particularly limited as long as the ion with the m / z corresponding to the subunit of the Verotoxin to be detected can be mass separated and detected.Mass spectrometry can carry out mass spectrometry of any type such as quadrupole type, ion trap type or time-of-flight type.When detecting the ion of 1 valence, from the viewpoint of each subunit of high-quality Verotoxin more than several kDa detected with good accuracy, preferably time-of-flight type mass spectrometry.In the present embodiment, Verotoxin can be detected by mass spectrometry of one stage.Mass spectrometry can be carried out by comprising more than one mass spectrometer in any mass spectrometer such as quadrupole type, ion trap type or time-of-flight type.Chromatography such as liquid chromatography can also be carried out before mass spectrometry.

[0050] The method of ionization in mass spectrometry is not particularly limited, and MALDI or electrospray ionization (ESI) can be performed. From the viewpoint of easily generating monovalent ions and obtaining easily analyzable data, MALDI is preferred. In the case of MALDI, the mass spectrometry sample prepared as described above is irradiated with a laser beam to ionize the sample.

[0051] In mass spectrometry, it is preferred to obtain data for obtaining a mass spectrum by scanning the m / z of ions to be mass-separated. The data obtained in mass spectrometry is referred to as mass spectrometry data. The mass spectrometry data is stored in a storage medium that can be referenced by a processing device such as an electronic computer.

[0052] After step S1007 is completed, step S1009 is started. In step S1009, the mass spectrometry data is analyzed. The analysis of the mass spectrometry data can be performed by a processing device such as an electronic computer. It is preferred to produce mass spectrometry data corresponding to the mass spectrogram based on the mass spectrometry data. For example, in the case of time-of-flight mass spectrometry, the flight time in the mass spectrometry data is correlated with the intensity of the detection signal of the ion detected at each flight time. The flight time can be converted into m / z based on the calibration data obtained in advance, and the mass spectrometry data in which m / z and intensity are correlated are obtained.

[0053] Based on the m / z of the ions of the A1 subunit, A2 subunit, or B subunit of each type or subtype of verotoxin, it can be determined whether an ion having an m / z within the allowable range of accuracy based on mass spectrometry has been detected. When an ion having an m / z within the allowable range is detected, it can be considered that the corresponding type or subtype of verotoxin has been detected. The m / z of the ions of each subunit of each type or subtype can be determined using a previously measured value or a molecular weight of each type or subtype (see Figure 2 ) and calculated value. For example, in the case of MALDI, for detecting univalent ions to which protons have been added to each subunit, the value obtained by adding the molecular weight of the proton to the molecular weight of each subunit can be used. It is also possible to add cations or anions other than protons. In this way, based on the mass-to-charge ratio of the detected Verotoxin, at least one of the types or subtypes of Verotoxin can be identified. In particular, when detecting univalent ions, it is possible to detect the B subunit with a small molecular weight with good accuracy, so it is preferred.

[0054] It should be noted that the control sample obtained by the operation of the same steps of the purification operation can also be supplied to mass spectral analysis without using the Verotoxin binding molecules that are combined with the Verotoxin of the detection object.For example, the molecule that is not combined with the Verotoxin can be added in the sample to replace the Verotoxin binding molecules, and carry out above-mentioned ultrafiltration, and the composition that is retained in the ultrafiltration membrane is supplied to mass spectral analysis.The mass spectral analysis of the above-mentioned step S1007 is used as the 1st mass spectral analysis, and the mass spectral analysis of the control sample is used as the 2nd mass spectral analysis.Based on the comparison of the mass spectral analysis data obtained in the 1st mass spectral analysis and the mass spectral analysis data obtained in the 2nd mass spectral analysis, it can be determined whether the sample comprises Verotoxin.For example, the mass spectrum obtained in the 1st mass spectral analysis and the mass spectrum obtained in the 2nd mass spectral analysis are compared, when the former has the peak corresponding to the Verotoxin and the latter does not have the peak corresponding to the Verotoxin, it can be considered that the sample comprises Verotoxin.Thus, by comparing with the control sample, the detection of more reliable Verotoxin can be carried out.

[0055] (Way)

[0056] Those skilled in the art will appreciate that the above-described various exemplary embodiments or modifications thereof are specific examples of the following aspects.

[0057] (Item 1) A method for detecting Verotoxin in one embodiment comprises: preparing a sample and a molecule that binds to Verotoxin; performing an operation of purifying the Verotoxin in the sample by utilizing the binding of the molecule to Verotoxin; and subjecting the sample obtained in the operation to a first mass spectrometry analysis. Thus, Verotoxin can be detected more accurately using mass spectrometry.

[0058] (Item 2) In another embodiment of the method for detecting Verotoxin, the method for detecting Verotoxin of the embodiment of Item 1 includes: identifying at least one of the type and subtype of Verotoxin based on the mass-to-charge ratio of Verotoxin detected in the first mass spectrometry analysis. Thus, the type or subtype of Verotoxin can be detected more accurately.

[0059] (Item 3) In another embodiment of the method for detecting Verotoxin, in the method for detecting Verotoxin of the embodiment of Item 1 or Item 2, the molecule that binds to the Verotoxin is at least one of an antibody and ceramide trihexoside. Thus, the specificity of the antigen-antibody reaction or the ligand-receptor reaction can be fully utilized to more reliably purify Verotoxin.

[0060] (Item 4) In another embodiment of the method for detecting Verotoxin, in the method for detecting Verotoxin according to Item 3, the antibody is a polyclonal antibody, which can be produced rapidly and easily compared to a monoclonal antibody.

[0061] (Item 5) In another embodiment of the method for detecting Verotoxin, in the method for detecting Verotoxin of the embodiment of Item 3 or Item 4, the molecule that binds to Verotoxin is an antibody that can bind to at least one of Verotoxin Type 1 and Verotoxin Type 2. Thus, it is possible to detect whether Verotoxin Type 1, Verotoxin Type 2, or both of them are present in the sample.

[0062] (Item 6) In another embodiment of the method for detecting Verotoxin, in the method for detecting Verotoxin of Item 4 or Item 5, during the purification, a plurality of antibodies are brought into contact with the sample. This allows for more reliable detection of Verotoxin.

[0063] (Item 7) In another embodiment of the method for detecting Verotoxin, in the method for detecting Verotoxin of any one of Items 1 to 6, in the aforementioned operation, the solution containing the binding molecule formed by the aforementioned molecule binding to Verotoxin is subjected to at least one of ultrafiltration and microfiltration. Thus, by utilizing the difference between the molecular weight of each subunit of Verotoxin and the molecular weight of the complex, molecules having a molecular weight near the molecular weight of each subunit of Verotoxin are removed from the sample, and mass spectrometry data can be analyzed with good accuracy.

[0064] (Item 8) In another embodiment of the method for detecting Verotoxin, the method for detecting Verotoxin in any one of the embodiments of Items 1 to 7 comprises: subjecting a solution obtained by performing the same operation as the above operation without using the above molecule to a second mass spectrometry analysis; and determining whether Verotoxin is contained in the above sample based on a comparison of the data obtained in the above first mass spectrometry analysis with the data obtained in the above second mass spectrometry analysis. Thus, it is possible to confirm whether the peak corresponding to the m / z of the ion derived from Verotoxin is a peak corresponding to Verotoxin, and Verotoxin detection can be performed more reliably.

[0065] The present invention is not limited to the contents of the above-mentioned embodiment, and other aspects that can be considered within the scope of the technical concept of the present invention are also included in the scope of the present invention.

[0066] [Example]

[0067] Examples of the present embodiment are shown below, but the present invention is not intended to be limited to the following examples.

[0068] (Example 1)

[0069] In Example 1, an unknown sample was purified using anti-Verotoxin 1 antibody and anti-Verotoxin 2 antibody, and Verotoxin was detected by mass spectrometry. Each operation was performed in the order of 1 to 5 below.

[0070] 1. An Escherichia coli strain derived from a patient with enterohemorrhagic Escherichia coli infection was cultured in brain heart infusion (BHI) agar medium. The colonies formed by the culture were suspended in a polymyxin B solution for 30 minutes and then centrifuged to obtain a supernatant as a sample solution.

[0071] 2. The sample solution obtained in 1. was dispensed into two tubes at 150 μL per tube, and 150 μL of a PBS buffer solution containing 5 mM n-octyl-β-D-glucoside was added to each tube. The obtained solution was used in an ultrafiltration device (NMWL: 100 KDa, Merck Millipore, UFC510096), and the two tubes were simultaneously subjected to centrifugal separation (14000G, 5 minutes).

[0072] 3. To one of the filtrates obtained in the centrifugation in 2., 0.5 μg of anti-Verotoxin 1 antibody (Nacalai Tesque, 01770-74) was added, and to the other, 0.5 μg of anti-Verotoxin 2 antibody (Nacalai Tesque, 01771-64) was added, and each was incubated for 30 minutes.

[0073] 4. The incubated solution in 3. was used in a new ultrafiltration device (NMWL: 100K Da), and two tubes were simultaneously subjected to centrifugation. After centrifugation, 100 μL of PBS buffer solution containing 2.5 mM n-octyl-β-D-glucoside was added to the residual liquid in the ultrafiltration filter and the entire amount was recovered.

[0074] 5. The solution recovered in 4. was desalted using a solid phase extraction chip (Agilent Technologies, Bond Elut OMIX, A57009100). The desalted eluate was dropped onto a stainless steel plate and subjected to mass spectrometry analysis using a mass spectrometer (Shimadzu Corporation, MALDI-8020) to obtain a mass spectrum.

[0075] Figure 3 This is the mass spectrum obtained by mass spectrometry analysis during purification using anti-Verotoxin 2 antibody. Figure 4 This is a mass spectrum obtained by mass spectrometry analysis when using anti-Verotoxin 1 antibody for purification. A mass spectrum is a graph in which the horizontal axis represents the m / z of the detected ions and the vertical axis represents the intensity of the detection signal of the ions. Figure 3 to Figure 6 The same is true in each figure of . Figure 3 and Figure 4 In the comparison, only Figure 4 The peak P1 of m / z 7692.1 was observed with high intensity, and the other peaks had almost the same intensity. This peak P1 was used as a candidate for the peak derived from Verotoxin and was compared with the peak P1 of Figure 2By comparing the peak P1 with the table of , it was determined that the peak P1 was derived from the B subunit of the Stx1a type of verotoxin. That is, it was concluded that verotoxin was detected from the sample used in Example 1, and its type and subtype were Stx1a.

[0076] (Example 2)

[0077] In Example 2, known samples were purified using anti-Verotoxin 1 antibody and anti-Verotoxin 2 antibody, and Verotoxin was detected by mass spectrometry. Each operation was performed in the order of 1 to 5 below.

[0078] 1. The E. coli strain identified by gene detection as having the Stx1c gene was cultured in TSA (Trypticase soy agar) plate medium. The colonies formed by the culture were suspended in physiological saline and lysed using an ultrasonic disruptor to obtain a sample solution.

[0079] 2. The sample solution obtained in 1. was dispensed into two tubes at 150 μL per tube, and 150 μL of a PBS buffer solution containing 5 mM n-octyl-β-D-glucoside was added to each tube. The obtained solution was used in an ultrafiltration device (NMWL: 100 KDa, Merck Millipore, UFC510096), and the two tubes were simultaneously subjected to centrifugal separation (14000G, 5 minutes).

[0080] 3. To one of the filtrates obtained in the centrifugation in 2., 0.5 μg of anti-Verotoxin 1 antibody (Nacalai Tesque, 01770-74) was added, and to the other, 0.5 μg of anti-Verotoxin 2 antibody (Nacalai Tesque, 01771-64) was added, and each was incubated for 30 minutes.

[0081] 4. The incubated solution in 3. was used in a new ultrafiltration device (NMWL: 100K Da), and two tubes were simultaneously subjected to centrifugation. After centrifugation, 100 μL of PBS buffer solution containing 2.5 mM n-octyl-β-D-glucoside was added to the residual liquid in the ultrafiltration filter and the entire amount was recovered.

[0082] 5. The solution recovered in 4. was desalted using a solid phase extraction chip (Agilent Technologies, Bond Elut OMIX, A57009100). The desalted eluate was dropped onto a stainless steel plate and subjected to mass spectrometry analysis using a mass spectrometer (Shimadzu Corporation, MALDI-8020) to obtain a mass spectrum.

[0083] Figure 5This is the mass spectrum obtained by mass spectrometry analysis during purification using anti-Verotoxin 2 antibody. Figure 6 This is a mass spectrum obtained by mass spectrometry analysis during purification using an anti-Verotoxin 1 antibody. Figure 5 and Figure 6 In the comparison, only Figure 6 Peak P2 of m / z 7662.4 was observed with high intensity, and the other peaks had almost the same intensity. Peak P2 was considered as a candidate for the peak derived from Verotoxin and was compared with the peak of Figure 2 By comparing the peak P2 with the table of , it was determined that the peak P2 originated from the B subunit of the Stx1c type viroxin. That is, it was concluded that the Stx1c viroxin was detected from the sample used in Example 2, and the same type and subtype of viroxin as the result of the gene detection was expressed. Sequence Listing <110> SHIMADZU CORPORATION OSAKA INSTITUTE OF PUBLICHEALTH <120> Verotoxin Detection Method <130> SP20190684 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 69 <212> PRT <213> Escherichia coli <400> 1 Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr Lys Tyr Asn Asp 1 5 10 15 Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu Leu Phe Thr Asn 20 25 30 Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln Ile Thr Gly Met 35 40 45 Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly Gly Gly Phe Ser 50 55 60 Glu Val Ile Phe Arg 65 <210> 2 <211> 69 <212> PRT <213> Escherichia coli <400> 2 Ala Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr Lys Tyr Asn Asp 1 5 10 15 Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu Leu Phe Thr Asn 20 25 30 Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln Ile Thr Gly Met 35 40 45 Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly Gly Gly Phe Ser 50 55 60 Glu Val Ile Phe Arg 65 <210> 3 <211> 69 <212> PRT <213> Escherichia coli <400> 3 Ala Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr Lys Tyr Asn Asp 1 5 10 15 Asp Asp Thr Phe Thr Val Lys Val Ala Asp Lys Glu Leu Phe Thr Asn 20 25 30 Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln Ile Thr Gly Met 35 40 45 Thr Val Thr Ile Lys Thr Thr Ala Cys His Asn Gly Gly Gly Phe Ser 50 55 60 Glu Val Ile Phe Arg 65 <210> 4 <211> 70 <212> PRT <213> Escherichia coli <400> 4 Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asp 1 5 10 15 Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp Thr Ser Arg 20 25 30 Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr 35 40 45 Val Thr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly Phe Ala Glu 50 55 60 Val Gln Phe Asn Asn Asp 65 70 <210> 5 <211> 68 <212> PRT <213> Escherichia coli <400> 5 Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asn 1 5 10 15 Asp Thr Phe Thr Val Lys Val Ala Gly Lys Glu Tyr Trp Thr Asn Arg 20 25 30 Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr 35 40 45 Val Thr Ile Lys Ser Asn Thr Cys Ala Ser Gly Ser Gly Phe Ala Glu 50 55 60 Val Gln Phe Asn 65 <210> 6 <211> 70 <212> PRT <213> Escherichia coli <400> 6 Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asn 1 5 10 15 Asp Thr Phe Thr Val Lys Val Ala Gly Lys Glu Tyr Trp Thr Ser Arg 20 25 30 Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr 35 40 45 Val Thr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly Phe Ala Glu 50 55 60 Val Gln Phe Asn Asn Asp 65 70 <210> 7 <211> 70 <212> PRT <213> Escherichia coli <400> 7 Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asn 1 5 10 15 Asp Thr Phe Thr Val Lys Val Ala Gly Lys Glu Tyr Trp Thr Ser Arg 20 25 30 Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr 35 40 45 Val Thr Ile Lys Ser Ser Thr Cys Ala Ser Gly Ser Gly Phe Ala Glu 50 55 60 Val Gln Phe Asn Asn Asp 65 70 <210> 8 <211> 68 <212> PRT <213> Escherichia coli <400> 8 Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asp 1 5 10 15 Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn Arg 20 25 30 Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr 35 40 45 Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala Gln 50 55 60 Val Lys Phe Asn 65 <210> 9 <211> 68 <212> PRT <213> Escherichia coli <400> 9 Ala Asp Cys Ala Val Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asp 1 5 10 15 Asp Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn Arg 20 25 30 Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr 35 40 45 Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala Gln 50 55 60 Val Lys Phe Asn 65 <210> 10 <211> 70 <212> PRT <213> Escherichia coli <400> 10 Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Gly Asp 1 5 10 15 Asn Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp Thr Asn Arg 20 25 30 Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr 35 40 45 Val Thr Ile Lys Ser Asn Thr Cys Glu Ser Gly Ser Gly Phe Ala Glu 50 55 60 Val Gln Phe Asn Asn Asp 65 70

Claims

1. A method for detecting Verotoxin, comprising: preparing a test sample and a molecule that binds to the A subunit or B subunit of verotoxin; performing an operation of purifying the Verotoxin in the sample by utilizing the binding of the molecule to Verotoxin to obtain a purified sample; and subjecting the purified sample obtained by the operation to a first mass spectrometry analysis; In the operation, a solution containing the binding molecule formed by binding the molecule to the A subunit or B subunit of virulence toxin is subjected to at least one of ultrafiltration and microfiltration. In at least one of the ultrafiltration and microfiltration, a filter membrane is used which is impermeable to the binding molecule but permeable to the A subunit or B subunit of the verotoxin. The purified sample subjected to the first mass spectrometry analysis includes the binding molecules that have not passed through the filter membrane.

2. The method for detecting Verotoxin according to claim 1, comprising: At least one of the type and subtype of Verotoxin is identified based on the mass-to-charge ratio of Verotoxin detected in the first mass spectrometry analysis.

3. The method for detecting Verotoxin according to claim 1, wherein: The molecule that binds to Verotoxin is at least one of an antibody and ceramide trihexoside.

4. The method for detecting Verotoxin according to claim 3, wherein: The antibody is a polyclonal antibody.

5. The method for detecting Verotoxin according to claim 3, wherein: The molecule that binds to Verotoxin is an antibody that can bind to at least one of Verotoxin type 1 and Verotoxin type 2.

6. The method for detecting Verotoxin according to claim 4, wherein: In the purification, a plurality of antibodies are brought into contact with the sample.

7. The method for detecting Verotoxin according to any one of claims 1 to 6, comprising: subjecting a solution obtained by performing the same operation as the above operation without using the above molecule to a second mass spectrometry analysis; Based on the comparison between the data obtained in the first mass spectrometry analysis and the data obtained in the second mass spectrometry analysis, it is determined whether or not the sample contains verotoxin.

Images