Preparation method of sample for microorganism analysis and analytical method of microorganism and identification method of microorganism and kit for microorganism analysis

Abstract

To detect a molecular included in a microorganism stably with good repeatability by mass spectroscopy.SOLUTION: A preparation method of a sample for microorganism analysis comprises preparing a sample solution with a concentration of a microorganism being higher than 1×106CFU / μL and less than 6×107CFU / μL, conducting centrifugal separation of the sample solution, and preparing a sample for mass spectroscopy using supernatant obtained by the centrifugal separation.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for preparing a sample for microbial analysis, a method for analyzing microorganisms, a method for identifying microorganisms, and a kit for microbial analysis. [Background technology]

[0002] Mass spectrometry has been used to analyze molecules contained in microorganisms. In particular, when identifying types of microorganisms using matrix assisted laser desorption / ionization (MALDI) mass spectrometry (MALDI mass spectrometry), it is extremely useful because it can be performed more quickly than a method in which DNA base sequences are determined and identification is performed based on the sequences. In this case, microorganisms are identified based on the positions of predetermined peaks in the obtained mass spectrum (see Patent Document 1). In order to improve the accuracy of identification of microorganisms, when a molecule such as a protein corresponding to a peak is present in the microorganism, it is necessary to detect the ion of the molecule as a peak with good reproducibility. Thus, in order to analyze molecules contained in microorganisms more precisely, it is desirable to improve the accuracy of mass spectrometry. In order to improve the accuracy of mass spectrometry, it is desirable to improve the sensitivity and reproducibility of peak detection.

[0003] Twelve types of marker proteins that are effective for identifying serotypes or strains of Salmonella and their analytical methods have been reported (Non-Patent Document 1). In order to perform more reproducible and stable analysis, it is considered to increase the sensitivity of the peaks corresponding to the marker proteins and reduce the variability in analytical conditions.

[0004] In order to increase the sensitivity in MALDI mass spectrometry, it has been proposed to use methylenediphosphonic acid (MDPNA) (Patent Document 2, Non-Patent Document 2) and the surfactant N-decyl-β-D-maltopyranoside (DMP) (Non-Patent Document 3, Non-Patent Document 4) as matrix additives.

[0005] The analytical conditions for MALDI mass spectrometry of microorganisms have been reported in several publications, including the reviews in Non-Patent Documents 5, 7, and 9, from the viewpoint of reproducibility or detection limit. Non-Patent Document 6 reports that MALDI mass spectrometry was performed on a sample solution containing Coxiella burnetti at different concentrations. Non-Patent Document 8 reports the detection limit of MALDI mass spectrometry combined with immunomagnetic separation for Salmonella enterica (serovar Choleraesuis; Salmonella Choleraesuis). Non-Patent Document 10 reports MALDI mass spectrometry at a predetermined concentration of Staphylococcus aureus, Bacillus cereus, and Enterococcus faecium. Non-Patent Documents 11 and 12 report MALDI mass spectrometry of a predetermined concentration of Escherichia coli combined with immunomagnetic separation and / or phage amplification. Non-Patent Documents 13 and 14 report the detection limits of MALDI mass spectrometry of Staphylococcus aureus, Staphylococcus saprophyticus, or Escherichia coli using magnetic nanoparticles.

[0006] Regarding the preparation of samples for MALDI mass spectrometry using ultrasonic treatment, its application to Escherichia coli is reported in Non-Patent Document 15. Furthermore, ultrasonic treatment is also used for the purpose of cell decomposition of Gram-positive and Gram-negative bacteria (Non-Patent Document 16). Non-Patent Document 17 reports an example of combining ultrasonic treatment with decomposition treatment using the hydrolase lysozyme.

[0007] Regarding the preparation of samples for mass spectrometry using centrifugation, a method has been proposed in which, when inactivating pathogenic bacteria, the bacterial cells are treated with a solution containing trifluoroacetic acid, and then the suspension is centrifuged and filtered (Non-Patent Document 18). [Prior art documents]

Patent Document

[0008]

Patent Document 1

Patent Document 2

Non - Patent Document

[0009]

Non - Patent Document 1

Non - Patent Document 2

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 17

Non-Patent Document 18

[0010] Peak detection in mass spectrometry of microorganisms was unstable, and the reproducibility of peak detection of molecules contained in microorganisms was sometimes low. [Means for solving the problem]

[0011] In the first aspect of the present invention, the concentration of microorganisms is 1×10 6 CFU / μL or more 6×10 7 The present invention relates to a method for preparing a sample for microbial analysis, comprising: preparing a sample solution having a CFU / μL or less; centrifuging the sample solution; and using the supernatant obtained by the centrifugation to prepare a sample for mass spectrometry. A second aspect of the present invention relates to a method for analyzing microorganisms, comprising preparing a sample for microbial analysis using the method for preparing a sample for microbial analysis of the first aspect, and performing a first mass spectrometry analysis of the prepared sample for microbial analysis. A third aspect of the present invention relates to a method for identifying a microorganism, which uses the method for analyzing a microorganism according to the second aspect to identify the microorganism. A fourth aspect of the present invention relates to a microbial analysis kit for carrying out the method for preparing a sample for microbial analysis of the first aspect, the method for analyzing a microorganism of the second aspect, or the method for identifying a microorganism of the third aspect. Effect of the Invention

[0012] According to the present invention, peak detection in mass spectrometry of microorganisms is stabilized, and the reproducibility of peak detection of molecules contained in microorganisms can be improved. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a conceptual diagram showing an analysis method according to one embodiment. [Diagram 2] FIG. 2 is a flow chart showing the flow of the analysis method according to one embodiment. [Diagram 3] Figure 3 (Table 1-A) is a table showing the detection rate of each marker for Salmonella enterica (serotype Orion, jfrlSe1402-15) when samples with multiple bacterial concentrations were subjected to MALDI mass spectrometry using the conventional method. [Figure 4] FIG. 4 (Table 1-B) is a table showing the detection rate of each marker when samples of Salmonella enterica (serotype Orion, jfrlSe1402-15) with multiple bacterial concentrations prepared by a method according to one embodiment were subjected to MALDI mass spectrometry. [Diagram 5] FIG. 5 (Table 1-C) is a table showing the detection rate of each marker in each tube when samples of Salmonella enterica (serotype Orion, jfrlSe1402-15) with a bacterial concentration of 1 x 107 cells / well prepared in multiple tubes (No. 1 to 8) using a method according to one embodiment were subjected to MALDI mass spectrometry. [Figure 6] FIG. 6 is a box plot showing the variation in bacterial concentration in each well for Salmonella enterica (serotype Orion, jfrlSe1402-15) when the bacterial concentration was not adjusted (Comparative Example 2) and when the bacterial concentration was adjusted (Example 2). [Figure 7]FIG. 7 shows crystal images on a MALDI plate of mass spectrometry samples containing two different strains of Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Enteritidis, NBRC3313), prepared by (a) a conventional method (Comparative Example 3) and (b) a method of one embodiment (Example 3). [Figure 8] FIG. 8 shows the peaks corresponding to SodA in mass spectra of six different strains of Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Enteritidis, NBRC3313; serovar Thompson, ATCC BAA-1738; serovar Choleraesuis, NBRC 105684; serovar Infantis, jfrlSe1402-4; serovar Orion, jfrlSe1402-15) after centrifugation (a) when an analytical sample was prepared using the obtained centrifugal supernatant and (b) when an analytical sample was prepared without centrifugation. [Figure 9] FIG. 9 shows the peak corresponding to YciF in the mass spectrum of Salmonella enterica (serotype Enteritidis, NBRC3313) when (a) an analytical sample was prepared by ultrasonic suspension and (b) an analytical sample was prepared by needle suspension. [Figure 10] FIG. 10 shows the peaks corresponding to YciF in the mass spectra of Salmonella enterica (serotype Enteritidis, NBRC3313) when (a) an analytical sample was prepared by ultrasonic suspension and (b) an analytical sample was prepared by suspension using a vortex mixer. [Figure 11] FIG. 11 shows mass spectra of (a) m / z 21500 to 23000 and (b) m / z 21500 to 24000 for Escherichia coli (E. coli DH5α Electro-Cells). [Figure 12]Figure 12 shows the peaks corresponding to SodA in the mass spectrum of Salmonella enterica (serotype Typhimurium, NBRC13245) when the detector voltage was set to (a) 90 V, (b) 70 V, (c) 50 V, (d) 30 V, and (e) 0 V higher than the reference voltage. [Figure 13] FIG. 13 shows the peaks corresponding to SodA in the mass spectrum of Salmonella enterica (serovar Choleraesuis, NBRC 105684) when the detector voltage was set to (a) 70 V, (b) 50 V, (c) 30 V, and (d) 0 V higher than the reference voltage. [Figure 14] Figure 14 shows the peak corresponding to SodA in the mass spectrum of Salmonella enterica (serotype Orion, jfrlSe1402-15) when the detector voltage was set to (a) 70 V, (b) 50 V, (c) 30 V, and (d) 0 V higher than the reference voltage. [Figure 15] FIG. 15 shows the peak corresponding to SodA in the mass spectrum of Salmonella enterica (serotype Orion, jfrlSe1402-15) when (a) processing was performed using a relatively low smoothing value (50) and (b) processing was performed using a relatively high smoothing value (200). [Figure 16] FIG. 16 shows a peak corresponding to SodA in mass spectra of Salmonella enterica (serotype Typhimurium, NBRC13245) when (a) an analytical sample was prepared by a conventional method and when (b) an analytical sample was prepared by a method according to one embodiment. [Figure 17] FIG. 17 shows a peak corresponding to SodA in mass spectra of Salmonella enterica (serotype Choleraesuis, NBRC 105684) when (a) an analytical sample was prepared by a conventional method and (b) an analytical sample was prepared by a method according to one embodiment. [Figure 18]FIG. 18 shows peaks corresponding to SodA in mass spectra of each well for Salmonella enterica (serotype Choleraesuis, NBRC 105684) when (a) an analytical sample was prepared by a conventional method and when (b) an analytical sample was prepared by a method according to one embodiment. [Figure 19] FIG. 19 shows the peaks corresponding to SodA in the mass spectra of each well for Salmonella enterica (serotype Infantis, jfrlSe1402-4) when (a) an analytical sample was prepared using a conventional method and when (b) an analytical sample was prepared using a method according to one embodiment of the present invention. [Figure 20] FIG. 20 shows peaks corresponding to SodA in the mass spectrum of each well when an analytical sample was prepared by the method of one embodiment for Salmonella enterica (serotype Typhimurium, NBRC13245). [Figure 21] FIG. 21 is a diagram showing peaks corresponding to SodA in the mass spectrum of each well when an analytical sample was prepared by the method of one embodiment for Salmonella enterica (serotype Enteritidis, 3313). [Figure 22] FIG. 22 shows peaks corresponding to SodA in the mass spectrum of each well when an analytical sample was prepared by the method of one embodiment for Salmonella enterica (serotype Infantis, jfrlSe1402-4). [Figure 23] FIG. 23 shows peaks corresponding to SodA in the mass spectrum of each well when an analytical sample was prepared by the method of one embodiment for Salmonella enterica (serotype Thompson, ATCC BAA-1738). [Figure 24] FIG. 24 (Table 12-A) is a table showing the detection rate of markers by date when analytical samples were prepared by the conventional method for Salmonella enterica (serotype Typhimurium, NBRC13245). [Diagram 25] FIG. 25 (Table 12-B) is a table showing the detection rate of markers by date when the analytical sample was prepared by the method of one embodiment for Salmonella enterica (serotype Typhimurium, NBRC13245). [Figure 26] FIG. 26 (Table 12-C) is a table showing the detection rate of markers by date when analytical samples were prepared by the conventional method for Salmonella enterica (serotype Choleraesuis, NBRC 105684). [Figure 27] FIG. 27 (Table 12-D) is a table showing the detection rate of markers by date when the analytical sample was prepared by the method of one embodiment for Salmonella enterica (serotype Choleraesuis, NBRC 105684). [Figure 28] FIG. 28 (Table 12-E) is a table showing the detection rate of markers by date when the analytical samples were prepared by the conventional method for Salmonella enterica (serotype Orion, jfrlSe1402-15). [Figure 29] FIG. 29 (Table 12-F) is a table showing the detection rate of markers by date when the analytical sample was prepared by the method of one embodiment for Salmonella enterica (serotype Orion, jfrlSe1402-15). [Diagram 30] Figure 30 (Table 13-A) is a table showing the detection rates of 12 markers when samples of six Salmonella enterica strains (serovar Typhimurium, NBRC13245; serovar Enteritidis, NBRC3313; serovar Thompson, ATCC BAA-1738; serovar Choleraesuis, NBRC 105684; serovar Infantis, jfrlSe1402-4; serovar Orion, jfrlSe1402-15) prepared by conventional methods were subjected to MALDI mass spectrometry. [Diagram 31]Figure 31 (Table 13-B) is a table showing the detection rates of 12 markers when samples of six Salmonella enterica strains (serovar Typhimurium, NBRC13245; serovar Enteritidis, NBRC3313; serovar Thompson, ATCC BAA-1738; serovar Choleraesuis, NBRC 105684; serovar Infantis, jfrlSe1402-4; serovar Orion, jfrlSe1402-15) prepared by the conventional method were subjected to MALDI mass spectrometry on a schedule different from that shown in Figure 30 (Table 13-A). [Diagram 32] FIG. 32 (Table 13-C) is a table showing the detection rates of 12 markers when samples of six Salmonella enterica strains (serotype Typhimurium, NBRC13245; serotype Enteritidis, NBRC3313; serotype Thompson, ATCC BAA-1738; serotype Choleraesuis, NBRC 105684; serotype Infantis, jfrlSe1402-4; serotype Orion, jfrlSe1402-15) prepared by a method according to one embodiment were subjected to MALDI mass spectrometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0015] -First embodiment- In the method for analyzing microorganisms according to the first embodiment, a sample solution containing a predetermined concentration of microorganisms is prepared, and a mass spectrometry sample is prepared using a supernatant obtained by centrifuging the sample solution. The prepared mass spectrometry sample is subjected to mass spectrometry.

[0016] FIG. 1 is a conceptual diagram showing a method for analyzing microorganisms according to the present embodiment. A sample solution MS containing a sample S containing microorganisms is prepared. The microorganisms are weighed or the turbidity of the sample solution MS is adjusted to a predetermined concentration, which will be described later. When weighing the microorganisms, mixing is performed after weighing (arrow A11), and an appropriate amount of matrix solution M and sample S based on the weighing result are mixed (arrow A12), and the sample solution MS is suspended to obtain a sample solution MS (arrow A21). The matrix solution is a solution containing a matrix, and preferably further contains a matrix additive. When adjusting the turbidity, the matrix solution M and sample S are mixed (arrow A13), and the turbidity of the obtained sample solution MS is measured and adjusted (arrow A14), and the sample solution MS is suspended to obtain a sample solution MS (arrow A22). In this case, the turbidity of the solution containing the sample S may be adjusted, and then the solution may be mixed with the matrix solution M to obtain the sample solution MS. Alternatively, the sample S may be weighed, and then a solution containing the sample S with an adjusted bacterial concentration may be prepared, and then the solution may be mixed with the matrix solution M to obtain the sample solution MS. Alternatively, the matrix solution M and the sample S may be mixed by dropping a solution containing the sample S that has been subjected to a series of processes including weighing or turbidity adjustment and at least one of ultrasonic treatment and centrifugation, which will be described later, onto a sample plate, dropping the matrix solution M onto the sample plate, and mixing the sample S and the matrix on the sample plate.

[0017] The sample solution MS is subjected to ultrasonic treatment (schematically shown by arrow A3), and then centrifuged (arrow A4). After centrifugation, the sample solution MS is separated into a supernatant Sn and a precipitate Pr. The supernatant Sn is dropped into a well 11 of a sample plate 10 (arrow A51) and then dried (arrow A5), completing the preparation of a sample for mass spectrometry. Then, the detector voltage is adjusted (arrow A6), and mass spectrometry using matrix-assisted laser desorption / ionization (hereinafter referred to as MALDI) (hereinafter referred to as MALDI mass spectrometry) is performed (arrow A7).

[0018] (About the sample) The sample S is not particularly limited as long as it contains microorganisms. When microorganisms contained in a collected object are subjected to analysis, a part containing the microorganisms is taken out of the object, and the part is applied to a culture medium to culture the microorganisms. The colonies obtained by this culture are collected, and the microorganisms contained in the colonies can be used as the sample S. For example, when specimens collected from the environment, food, or body fluids of living organisms are used as the objects, the type of microorganisms contained therein can be suitably identified by the method of this embodiment.

[0019] The sample S preferably contains bacteria belonging to the genus Salmonella, more preferably bacteria of the species Salmonella enterica, and even more preferably bacteria of the subspecies enterica of the species Salmonella enterica. Preferred serotypes of the subspecies enterica include S. Choleraesuis, S. Typhimurium, S. Enteritidis, S. Thompson, S. Infantis, and S. Orion.

[0020] (Bacteria concentration in sample solution) The bacterial concentration of the sample solution MS, adjusted by weighing or turbidity adjustment, is 1×10 6 CFU / μL or more is preferable, and 3×10 6 It is more preferable that the turbidity is 3 MFU or more, and more preferable that the turbidity is 1×10 MFU or more. Increasing the bacterial concentration makes it possible to more reliably detect peaks corresponding to larger m / z. From the same viewpoint, the turbidity of the sample solution MS is preferably 3 MFU or more, and more preferably 1×10 MFU or more. In the following, MFU refers to McFarland units.

[0021] The bacterial concentration of the sample solution MS, adjusted by weighing or turbidity adjustment, is 6 × 10 7 CFU / μL or less is preferable, and 3×10 7It is more preferable that the bacterial concentration is less than 2×10 CFU / μL. If the bacterial concentration is too high, the influence of ionization inhibitors may occur, and peaks may not be detected. From the same viewpoint, the turbidity of the sample solution MS is 2×10 2 It is preferable that the concentration is less than MFU, and 1×10 2 It is more preferable that the concentration is equal to or less than the MFU.

[0022] (Regarding adjustment of bacterial concentration) When weighing, the method is not particularly limited, but it is preferable to measure the amount of microorganisms using a balance such as a microbalance. A sample solution MS with an adjusted bacterial concentration is obtained by adding an appropriate amount of matrix solution to the amount of sample S obtained by weighing.

[0023] When adjusting the turbidity, the method is not particularly limited as long as the turbidity measurement can provide the bacterial concentration, but it is preferable to measure the turbidity by the McFarland turbidimetric method. By appropriately measuring the turbidity and adjusting the amount of the microorganism or the amount of the solution, the bacterial concentration can be adjusted to the above-mentioned preferred level.

[0024] (About ultrasonic treatment) The method of ultrasonic treatment is not particularly limited, and known methods can be used. The ultrasonic treatment can be performed by continuously or intermittently irradiating the sample solution MS with ultrasonic cleaner or ultrasonic crusher. The time for which ultrasonic treatment is performed is also not particularly limited, and can be appropriately set, for example, from 30 seconds to 5 minutes.

[0025] (About centrifugation) The method of centrifugation is not particularly limited, and can be performed by a known method using a centrifuge. The conditions of centrifugation are not particularly limited, but the rotation speed is preferably 6000 rpm or more, more preferably 10000 rpm or more. Furthermore, the rotation speed is preferably 15000 rpm or less, more preferably 13000 rpm or less. The time for centrifugation is also not particularly limited, and can be appropriately set to 1 minute to 5 minutes, etc.

[0026] (Mass spectrometry) Mass analysis is performed by a mass spectrometer. As long as the peak of the detection target can be detected, the method of mass analysis is not particularly limited. However, from the viewpoint of obtaining measurement data that is easy to analyze, it is preferable to perform ionization by the above-mentioned MALDI, which is easy to generate monovalent ions. Also, from the viewpoint of accurately analyzing high-mass ions corresponding to m / z of 10,000 or more, it is preferable to perform the above-mentioned time-of-flight mass analysis. The flight path of ions in time-of-flight mass analysis is not particularly limited, and a linear mode or a reflectron mode, etc. can be used as appropriate. It is more preferable to perform time-of-flight mass analysis of ions ionized by the MALDI method using a MALDI time-of-flight mass spectrometer (hereinafter referred to as MALDI-MS) capable of ionization by the MALDI method. The MALDI method is a type of soft ionization method, and is a method in which a matrix that easily absorbs laser light and is easily ionized is mixed with the analyte, and the analyte is irradiated with laser light to ionize the analyte, in order to analyze a substance that does not easily absorb laser light or a substance that is easily damaged by laser light such as a protein. In the ionization in mass analysis, either a positive ion mode or a negative ion mode may be used. Mass spectrometry may be performed using a single mass spectrometer, or fragment ions may be analyzed in multiple stages.

[0027] When performing MALDI, the matrix is ​​not particularly limited as long as it can detect the peak of the detection target, but it is preferable to use sinapinic acid from the viewpoint of efficiently generating high mass ions.

[0028] The matrix additive is not particularly limited as long as it is expected to have some effect in mass spectrometry, such as reducing noise or increasing the detection sensitivity of the ions to be analyzed. From the viewpoint of increasing the detection sensitivity, the additive preferably contains at least one of a compound containing a phosphonic acid group and a surfactant. The compound containing a phosphonic acid group is more preferably an alkylphosphonic acid, and further preferably methylenediphosphonic acid (MDPNA). The surfactant is preferably N-decyl-β-D-maltopyranoside (DMP).

[0029] The matrix solution is prepared by adding a matrix and an additive to a solvent. This solvent is preferably an organic solvent or a solvent obtained by mixing an organic solvent with an aqueous solvent, and the type of organic solvent or aqueous solvent is not particularly limited. As an example, this solvent is composed of a solution containing trifluoroacetic acid (TFA) prepared at a predetermined volume percent concentration, such as 0.1% to 3%, and acetonitrile (ACN) at an arbitrary concentration. The volume percent concentration of acetonitrile is preferably set to 30% to 70%. By preparing the matrix solution using a solvent prepared by containing an organic solvent in this way, the matrix and the like can be easily dissolved. Before dropping the mass spectrometry sample into the well of the sample plate, the well may be precoated. Precoating is performed, for example, by dropping a matrix solution containing an alcohol such as ethanol as a solvent into the well. Precoating makes it easier for the mass spectrometry sample to be stably held in an appropriate position after dropping, and also increases the ionization efficiency.

[0030] It is preferable to set the detector voltage when performing mass analysis on the target microorganisms based on data obtained by performing mass analysis on microorganisms different from the target microorganisms contained in the sample S. Hereinafter, the mass analysis of the target microorganisms is appropriately referred to as the first mass analysis, and the mass analysis of the microorganisms for adjusting the detector voltage is appropriately referred to as the second mass analysis, to distinguish between them.

[0031] In the second mass analysis, it is preferable to perform mass analysis on a mass analysis sample prepared containing E. coli. E. coli is a microorganism that is often used and easily available in the field of microbial mass analysis, and is also used for calibration in MALDI analysis, so there is an advantage that the calibration can be performed simultaneously. The method of preparing the mass analysis sample for the second mass analysis is not particularly limited, and for example, a colony obtained by culturing E. coli is added to the above-mentioned matrix solution, and after needle suspension or the like is performed, the suspension is dropped onto a sample plate and dried to obtain a mass analysis sample. This mass analysis sample corresponds to the microbial analysis sample prepared in this embodiment.

[0032] In adjusting the detector voltage, a detector voltage (hereinafter referred to as a reference voltage) at which a predetermined peak corresponding to a molecule contained in E. coli is detected at a predetermined magnitude by the second mass analysis is derived, and a voltage different from the reference voltage by a predetermined voltage is set as the detector voltage in the first mass analysis. For example, the predetermined peak is at least one of the peak with the highest intensity in the range of m / z 22000 to 23000 in E. coli and the peak with the highest intensity in the range of m / z 23000 to 24000 in E. coli. A detector voltage at which this peak is detected, for example, at 0.1 mV to 0.5 mV, preferably the former at 0.1 to 0.3 mV and the latter at 0.1 to 0.4 mV, is derived as the reference voltage, and a voltage 50 V to 70 V higher than the reference voltage is set as the detector voltage in the first mass analysis. When the detector voltage is set to a voltage 50 V to 70 V higher than the reference voltage, the S / N ratio in the mass analysis is higher than the S / N ratio when a voltage outside this range is set. This adjustment of the detector voltage is performed by a processing device equipped with a CPU or the like in a control device installed in the mass spectrometer or communicably connected to the mass spectrometer. The adjustment of the detector voltage may be omitted as appropriate.

[0033] The data obtained by the first mass analysis is input to a processor equipped with a CPU, which is disposed in the analyzer, and this processor becomes the subject of various data analyses performed by the analyzer, such as generating data corresponding to the mass spectrum (hereinafter referred to as mass spectrum data) based on the data. The method of this data analysis is not particularly limited. The analyzer can be constituted by any information processor such as a computer. The analyzer may constitute the above-mentioned control device.

[0034] (Smoothing process) It is preferable that the above-mentioned processing device performs a smoothing process on the mass spectrum data obtained by the first mass analysis. In this smoothing process, different smoothing processes are performed depending on the value of m / z. For example, the threshold value is set to any value between 15000 and 20000. Here, in the smoothing process of data points corresponding to m / z equal to or greater than the threshold value, smoothing can be performed using more or a wider range of data points for one data point than in the smoothing process of data points corresponding to m / z less than the threshold value. In this way, the processing device modifies the intensity corresponding to each data point using more data points in the second range having a higher m / z than the first range of m / z than in the smoothing in the first range of m / z. The first range is set, for example, from a range equal to or less than the threshold value, and the second range is set from a range greater than the threshold value.

[0035] In general, in mass spectrometry, one smoothing value is applied to one mass spectrum. However, when a relatively wide mass range such as m / z 6000 to 23000 is targeted, as in the case of the Salmonella marker protein (Table 1) described later, one smoothing value may not be able to perform accurate analysis over the entire mass spectrum. By applying appropriate smoothing values ​​to the first range on the low mass side and the second range on the high mass side, more reliable and stable m / z values ​​corresponding to peaks can be obtained over the entire mass spectrum. In particular, when analyzing markers using analysis software, a list of m / z values ​​corresponding to markers is used for analysis as described later, so that more reliable analysis can be performed by appropriately correcting the smoothing value before analysis. In this way, by performing appropriate smoothing processing according to the m / z value, it is possible to reduce the shift in the position of the peak due to noise, etc. The smoothing process may be omitted as appropriate.

[0036] (Microorganism Identification) When identifying a microorganism, the peaks in the mass spectrum obtained by the first mass spectrometry are compared with the peaks in the mass spectra of proteins contained in a plurality of microorganisms stored in a database. The database stores data linking the type of microorganism (genus name, species epithet, subspecies name, serotype, etc.) with the m / z of the peak observed in the mass spectrum.

[0037] The processing device determines whether a peak corresponding to the m / z indicated in the database is present in the mass spectrum obtained by the first mass analysis based on an error range determined based on the accuracy of the mass analysis, and calculates the similarity based on the determination. The similarity is a parameter indicating the degree of similarity between the mass spectrum of the microorganism in the database and the microorganism to be analyzed, and it is defined that the higher the similarity, the more similar the mass spectra are. The processing device identifies the type of microorganism in the database whose calculated similarity is equal to or higher than a certain level and is the highest as the type of microorganism contained in the sample S. The method of identifying microorganisms based on the data obtained in the first mass analysis is not particularly limited. For example, the database may contain weighting information based on the ratio or probability that a peak corresponding to each m / z is observed in the mass spectrum of each type of microorganism, and the similarity may be calculated based on the weighting information.

[0038] In the case of Salmonella, 12 proteins, gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF and SodA, are known as markers. In the first mass spectrometry, the serotype of Salmonella can be classified based on whether or not these proteins are detected. In other words, the serotype of Salmonella can be classified based on whether or not peaks are detected within the error range based on the accuracy of mass spectrometry (e.g., 800 ppm on the high mass side and the low mass side, respectively) from the m / z corresponding to these proteins. Table 1 shows an example of theoretical values ​​of m / z of peaks corresponding to Salmonella marker proteins. The values ​​shown in Table 1 are examples of values ​​found in at least one strain of Salmonella. For the m / z of the marker in each strain, please refer to Non-Patent Document 1.

[0039] [Table 1]

[0040] FIG. 2 is a flow chart showing the flow of the microorganism analysis method of this embodiment. In step S1001, a sample containing microorganisms is prepared. In the following, the mass spectrometer will be described as including a control unit that controls each part of the mass spectrometer and an analysis device. When step S1001 is completed, step S1003 is started. In step S1003, a sample solution MS containing microorganisms and a matrix is ​​prepared so that the concentration of the microorganisms is a predetermined concentration. When step S1003 is completed, step S1005 is started.

[0041] In step S1005, the sample solution MS is subjected to ultrasonic treatment. After step S1005 is completed, step S1007 is started. In step S1007, the sample solution MS is subjected to centrifugation. After step S1007 is completed, step S1009 is started.

[0042] In step S1009, the supernatant Sn obtained by centrifugation is dropped onto the sample plate 10 and dried to prepare a mass spectrometry sample. When step S1009 is completed, step S1011 is started. In step S1011, the mass spectrometer performs mass analysis (second mass analysis) on the mass spectrometry sample containing E. coli, and sets the detector voltage based on the obtained data. When step S1011 is completed, step S1013 is started.

[0043] In step S1013, the mass spectrometer performs mass analysis (first mass analysis) on the mass analysis sample obtained in step S1009 to obtain mass spectrum data. After step S1013 is completed, step S1015 is started. In step S1015, smoothing processing is appropriately performed on the mass spectrum data. After step S1015 is completed, step S1017 is started.

[0044] In step S1017, the microorganism is identified based on whether a peak is detected corresponding to the m / z corresponding to a known marker protein. After step S1017, the entire process is completed.

[0045] (About Microbiological Analysis Kits) A microorganism analysis kit suitable for use in the preparation of a sample for microorganism analysis, mass spectrometry of microorganisms, and identification of microorganisms according to the present embodiment is provided. The analysis kit includes any consumables and the like used in the preparation of the sample for microorganism analysis, mass spectrometry, and identification of microorganisms described above. Furthermore, the microorganism analysis kit includes documents, electronic data, and the like showing a method for preparing the sample for microorganism analysis or a method for analyzing microorganisms. By using the microorganism analysis kit, the preparation of the sample for microorganism analysis, analysis, and identification of microorganisms can be efficiently performed.

[0046] According to the above-described embodiment, the following advantageous effects can be obtained. (1) In one embodiment, a method for preparing a sample for microbial analysis is provided, the concentration of microorganisms being 1×10 6 CFU / μL or more 6×10 7 The method includes preparing a sample solution (MS) of CFU / μL or less, centrifuging the sample solution (MS), and preparing a mass spectrometry sample using the supernatant (Sn) obtained by the centrifugation. This stabilizes peak detection in mass spectrometry and improves the reproducibility of peak detection of molecules contained in microorganisms. Conventionally, there have been cases where the detection status of a peak corresponding to a marker protein was not reproduced between wells or dates. In addition, marker proteins for distinguishing not only the genus and species of microorganisms but also subspecies, serotypes, strains, etc. often have low peak detection sensitivity and low reproducibility (compared to marker proteins for distinguishing genus and species). In the analysis method of the present embodiment, for example, it is possible to improve the detection sensitivity and reproducibility of peaks corresponding to such marker proteins.

[0047] (2) In another embodiment, the method for preparing a sample for microbial analysis according to the first aspect of the present invention further comprises subjecting the sample solution (MS) to ultrasonic treatment prior to the centrifugation, which can further stabilize peak detection in mass spectrometry and increase the likelihood of detecting, for example, a YciF peak in Salmonella.

[0048] (Item 3) In another embodiment of the method for preparing a sample for microbial analysis according to the method for preparing a sample for microbial analysis described in items 1 or 2, the microorganism is added to a solution containing a matrix, and then the turbidity of the solution is adjusted, or the turbidity of the solution containing the microorganism is adjusted, and then the solution containing the microorganism is mixed with the solution containing a matrix, or the microorganism is weighed, and the weighed microorganism is added to a solution containing a matrix, or the microorganism is weighed, and a solution containing the weighed microorganism is prepared, and then the solution containing the weighed microorganism is mixed with the solution containing a matrix to prepare the sample solution. This reduces the variation in the bacterial concentration, and allows peaks to be detected with good reproducibility in mass spectrometry.

[0049] (Item 4) In another embodiment of the method for preparing a sample for microbial analysis according to the method for preparing a sample for microbial analysis described in item 3, the sample for mass spectrometry is prepared using at least one of a compound containing a phosphonic acid group and a surfactant as an additive to the matrix, thereby improving detection sensitivity and further increasing the possibility of detecting a peak.

[0050] (Item 5) In another embodiment of the method for preparing a sample for microbial analysis, the additive is methylenediphosphonic acid (MDPNA) and N-decyl-β-D-maltopyranoside (DMP), which further enhances the detection sensitivity.

[0051] (Item 6) In another embodiment of the method for preparing a sample for microbial analysis according to any one of items 3 to 5, the matrix is ​​sinapic acid, which allows efficient generation of high mass ions.

[0052] (Item 7) In another embodiment of the method for preparing a sample for microbial analysis according to any one of items 1 to 6, the microorganism is a bacterium of the genus Salmonella. This makes it easier to detect a marker protein for distinguishing the serotype of Salmonella, and can stabilize peak detection in mass spectrometry of Salmonella.

[0053] (Item 8) A method for analyzing microorganisms according to one embodiment includes preparing a sample for microbial analysis using the method for preparing a sample for microbial analysis according to any one of the first to seventh embodiments, and performing a first mass spectrometry analysis of the prepared sample for microbial analysis. This allows mass spectrometry with stable peak detection to be performed, and improves the reproducibility of peak detection of molecules contained in the microorganisms.

[0054] (Item 9) In another embodiment of the method for analyzing microorganisms, the first mass analysis ionizes the sample for microorganism analysis by matrix-assisted laser desorption / ionization (MADI), which makes it easier to detect monovalent ions and obtain a mass spectrum that is easy to analyze.

[0055] (Item 10) In another embodiment of the method for analyzing microorganisms, the first mass spectrometry detects a peak corresponding to an m / z of 10,000 or more, in the method for analyzing microorganisms described in item 8 or 9. This can increase the possibility of detecting high-mass ions that are difficult to detect.

[0056] (Item 11) In another aspect, the method for analyzing microorganisms according to item 10 further comprises performing a second mass analysis of a mass analysis sample prepared from a microorganism different from the microorganism, and setting a detector voltage in the first mass analysis based on data obtained by the second mass analysis. This makes it possible to further stabilize peak detection in mass analysis and improve the reproducibility of peak detection of SodA in Salmonella, for example.

[0057] (Item 12) In another embodiment of the method for analyzing microorganisms according to item 11, the second mass analysis ionizes the sample for microbial analysis by matrix-assisted laser desorption / ionization, which makes it easy to set the detector voltage using a mass spectrum that is easy to analyze.

[0058] (Item 13) In another embodiment of the method for analyzing microorganisms, in the method for analyzing microorganisms described in item 11 or 12, in the second mass analysis, the voltage of the detector is set based on the intensity of at least one of the most intense peaks in the range of m / z 22,000 to 23,000 in the mass spectrum of E. coli and the most intense peak in the range of m / z 23,000 to 24,000 in the mass spectrum. This improves the reproducibility of peak detection for high-mass ions that are difficult to detect, and makes it possible to provide mass spectra with high stability (reproducibility) over a wide m / z range.

[0059] (Item 14) In another embodiment of the method for analyzing microorganisms, the method for analyzing microorganisms according to any one of items 8 to 13 includes obtaining mass spectrum data corresponding to a mass spectrum by the first mass analysis, and performing different smoothing on the mass spectrum data based on m / z. This allows appropriate smoothing processing to be performed on data points of various m / z, and provides a highly accurate mass spectrum for a wide range of m / z.

[0060] (Item 15) In another embodiment of the method for analyzing microorganisms, the method for analyzing microorganisms described in item 14 further includes, in the mass spectrum data, smoothing the intensities corresponding to each data point in a second range having a higher m / z than the first range using more or a wider range of data points. This makes it possible to perform an appropriate smoothing process on data points corresponding to high-mass ions that are difficult to detect, and to provide a highly accurate mass spectrum for a wide m / z range.

[0061] (Item 16) In another embodiment of the method for analyzing microorganisms according to item 15, the first range and the second range are set based on a threshold value, and the threshold value is m / z 15000 or more and 20000 or less. This makes it possible to appropriately perform smoothing processing on data points corresponding to ions having m / z equal to or more than the threshold value, which are particularly difficult to detect, and to provide a highly accurate mass spectrum for a wide m / z range.

[0062] (Item 17) In one embodiment of the method for identifying microorganisms, the method for analyzing microorganisms according to any one of items 8 to 16 is used to identify the microorganisms. This makes it possible to reproducibly detect ions corresponding to a marker when the microorganism has the marker, including cases where the ions have a high mass, and to identify the microorganisms with high accuracy.

[0063] (Item 18) In another embodiment of the method for identifying a microorganism, the microorganism is a bacterium of the genus Salmonella, and the first mass spectrometry detects at least one protein derived from Salmonella selected from the group consisting of gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF, and SodA. This allows accurate discrimination of the serotype of Salmonella. In this case, peak detection (or non-detection) can be performed for a plurality of proteins derived from Salmonella at m / z values ​​corresponding to at least one of a plurality of mass value candidates for each protein. The combination of m / z values ​​(or non-detection) of peaks detected as each marker protein of the group of these marker proteins allows accurate discrimination of the serotype of Salmonella.

[0064] (Item 19) A kit for microbial analysis according to one embodiment is for carrying out the method for preparing a sample for microbial analysis described in any one of items 1 to 7, the method for analyzing a microorganism described in any one of items 8 to 16, and the method for identifying a microorganism described in item 17 or 18. This allows mass spectrometry with improved stability (reproducibility) of peak detection to be carried out efficiently.

[0065] The present invention is not limited to the above-described embodiment, and other aspects that are conceivable within the scope of the technical concept of the present invention are also included within the scope of the present invention. EXAMPLES

[0066] Examples of the present embodiment will be described below, but the present invention is not limited to the numerical values ​​or device conditions of the following examples. EXAMPLES

[0067] In Example 1, the optimal bacterial concentration was evaluated by performing MALDI mass spectrometry on a microbial analysis sample (hereinafter simply referred to as the analytical sample) prepared without centrifugation using a matrix solution containing no additives, and an analytical sample prepared by centrifugation using a matrix solution containing additives, and the relationship between the bacterial concentration and the detection rate of the marker peak was evaluated.

[0068] <Method> The following operations 1 to 9 were carried out in chronological order. Similarly, in each of the following Examples, operations were carried out in the order of the numbers. 1. Salmonella enterica (serotype Orion, jfrlSe1402-15) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 solutions were prepared. The composition of each matrix solution is as follows. SA-1: 25 mg / mL; sinapinic acid (SA) in ethanol (EtOH) (hereinafter, the liquid after "in" indicates the solvent), SA-2: 25 mg / mL; SA in 0.6% trifluoroacetic acid (TFA), 50% acetonitrile (ACN), SA-3: 25 mg / mL; SA in 1% MDPNA, 1 mM DMP, 0.6% TFA 50% ACN. As in the following examples, SA was manufactured by Wako, and MDPNA and DMP were manufactured by Sigma-Aldrich.

[0069] 3. In Comparative Example 1, approximately 1 mg of the Salmonella from 1. was weighed out on a microbalance, and matrix solution SA-2 from 2. was added to prepare sample solutions with multiple bacterial concentrations. The samples were then suspended with a needle to prepare suspensions. 4. In Example 1, approximately 1 mg of the Salmonella from 1. was weighed out using a microbalance, and matrix solution SA-3 from 2. was added to prepare sample solutions with multiple bacterial concentrations. The bacteria were suspended using a needle, and the resulting suspensions were centrifuged (12,000 rpm, 5 min).

[0070] 5. 0.5 μL of the matrix solution SA-1 from step 1 was dropped onto a MALDI sample plate (hereinafter referred to as MALDI plate) (precoat). 6. As Comparative Example 1, 1 μL of the suspension prepared in 3 was dropped onto each of the wells precoated in 5. 7. In Example 1, 1 μL of the supernatant obtained by the centrifugation in 4. was dropped onto each of the wells precoated in 5.

[0071] 8. The droplets dropped in steps 6 and 7 were allowed to dry naturally to obtain samples for mass spectrometry, after which the MALDI plate was inserted into a MALDI-MS (AXIMA Performance, Shimadzu Corporation). The crystals in the wells were then irradiated with a laser by a raster analysis method that automatically irradiates the same number of points at the same intervals, and measurements were performed in linear and positive ion modes. 9. After the measurement, self-calibration was applied to the obtained measurement data, and the obtained mass spectra were compared and evaluated. For peaks corresponding to high mass ions with split peak tops (upper parts of the peaks), smoothing correction was performed (the smoothing setting of Axima Performance's Peak Processing was changed from 50 to 200). Whether or not the marker was detected was determined based on whether or not the peak was picked within 800 ppm of the theoretical m / z value corresponding to the peak of the 12 Salmonella markers (simply referred to as markers in the following examples). The detection rate of the marker was calculated as the percentage (%) of wells in which the peak was detected among multiple wells for each sample.

[0072] <Result> Table 1-A in FIG. 3 and Table 1-B in FIG. 4 show the detection rates (n=4) of the peaks corresponding to the 12 markers at multiple bacterial concentrations (turbidity) in Comparative Example 1 and Example 1, respectively. In Comparative Example 1 and Example 1, it was confirmed that the detection rate decreases when the bacterial concentration (turbidity) is too high or too low, while there is an optimal bacterial concentration (optimum turbidity) at which the detection rate is relatively high. In Tables 1-A and 1-B, the optimal bacterial concentration with a particularly high detection rate is enclosed in a thick frame F. In Comparative Example 1, the optimal bacterial concentration was 6×10 6 ~1×10 7 / well (2 × 10 to 3 × 10 MFU), 3 × 10 in Example 1 6 ~3×10 7 Pieces / well (1×10 to 1×10 2 MFU) and Example 1 had a wider optimum range. Overall, a tendency for the detection rate to be higher was confirmed in Example 1 compared to Comparative Example 1. This was thought to be due to at least one of the effects of the additive improving sensitivity including the high mass range and the effect of improving sensitivity and crystal uniformity mainly by using the centrifugal supernatant described later in Examples 3 and 4. In Example 1, the detection rate of 1×10 7 The sensitivity of the peak corresponding to the marker was particularly high at 3 × 10 cells / well (3 × 10 MFU).

[0073] Table 1-C in FIG. 5 shows the results of the analysis samples prepared in Example 1 with a bacterial concentration of 1×10 7 The detection rate of peaks corresponding to the 12 markers in 8 tubes (n=8) of analytical samples containing bacteria / well (turbidity 3 x 10 MFU) is shown. Detection of all 12 markers was confirmed in all 8 tubes (n=8). It was confirmed that analysis at the optimal bacterial concentration stabilizes marker peak detection and improves reproducibility.

[0074] As described later in Example 2, the converted optimal concentration was confirmed to be the same for four strains of Salmonella enterica (serotype Typhimurium, NBRC13245; serotype Enteritidis, NBRC3313; serotype Thompson, ATCC BAA-1738; serotype Orion, jfrlSe1402-15). EXAMPLES

[0075] In Example 2, the effect of adjusting the bacterial concentration by weighing the bacterial cells was evaluated.

[0076] <Method> 1. Salmonella enterica (serotype Orion, jfrlSe1402-15) was cultured on LB agar medium at 37°C for 20 hours. 2. In Comparative Example 2, each strain colony was randomly taken with a needle to about 1 cm in order to eliminate the intuition of an expert, and suspended in physiological saline to prepare a suspension (n=4). The above-mentioned about 1 cm of bacterial cells was prepared to be contained in 10 μL of suspension. 3. In Example 2, approximately 1 mg of a colony of each strain was weighed out, suspended in physiological saline, and serially diluted to prepare suspensions of different concentrations (n=4). 4. The turbidity of the suspensions prepared in Comparative Example 2 and Example 2 was measured using a turbidity meter (DensiCHEK (registered trademark) plus; BIOMERIEUX). 5. From the turbidity measured in step 4, the number of bacteria per well (x 10 7 The optimal bacterial concentration (1 μL of a concentration of 1 mg / 0.075 mL) and the variation per well in Comparative Example 2 and Example 2 were evaluated.

[0077] <Result> The bacterial concentration and turbidity shown in each Example, including the multiple bacterial concentrations shown in Table 1-A and Table 1-B, were converted by the McFarland turbidimetric method. At this time, it was confirmed that the conversion value of the optimal concentration was the same for four strains of Salmonella enterica (serotype Typhimurium, NBRC13245; serotype Enteritidis, NBRC3313; serotype Thompson, ATCC BAA-1738; serotype Orion, jfrlSe1402-15).

[0078] FIG. 6 is a graph (box plot) showing the variation in the amount of bacteria per well. The CV (Coefficient of Variation) values ​​of the amount of bacteria per well in Comparative Example 2 and Example 2 were 0.90 and 0.07, respectively. It is clear that the variation in the amount of bacteria was reduced in Example 2 compared to Comparative Example 2. This confirmed that by adjusting the bacterial concentration (or turbidity), the variation in the number of bacteria per well was reduced, improving the reproducibility of the amount of bacteria of the microorganism to be measured. EXAMPLES

[0079] In Example 3, homogenization of crystals of a mass spectrometry sample was evaluated.

[0080] <Method> 1. Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Enteritidis, NBRC3313) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 described above were prepared. 3. In Comparative Example 3, for the Salmonella of 1., a suspension was prepared by suspending approximately 1 cm of a colony in 10 μL of the matrix SA-2 solution of 2. with a needle. 4. In Example 3, approximately 1 mg of the Salmonella from 1 was weighed on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1×10 7 The matrix SA-3 solution from step 2 was added so that the concentration of the particles became 100% (particles / μL), and the mixture was suspended with a needle to prepare a suspension. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12000 rpm, 5 min).

[0081] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. As Comparative Example 3, the suspension prepared in 3 was dropped in 1.2 μL portions onto the wells precoated in 5. 7. In Example 3, 1 μL of the supernatant from the centrifugation in 4 was dropped onto the wells precoated in 5. 8. The droplets dispensed in steps 6 and 7 were allowed to dry naturally to obtain samples for mass spectrometry, and then photographs of the crystals on the MALDI plate were taken.

[0082] <Result> FIG. 7 shows the crystal photographs obtained in (a) Comparative Example 3 and (b) Example 3. In Comparative Example 3, relatively thick crystals with uneven surfaces were formed, whereas in Example 3, relatively uniform thin-film crystals were formed. This was thought to be due to the fact that in Comparative Example 3, a suspension of bacteria that had not been subjected to centrifugation was dropped, whereas in Example 3, the centrifugal supernatant was dropped. Also, for the same reason, in Comparative Example 3, crystals were prone to vary between wells, whereas in Example 3, relatively identical crystals tended to be obtained. This improved the non-uniformity of the crystals, and improved reproducibility on the well and between different wells. EXAMPLES

[0083] In Example 4, the effect of using the centrifugal supernatant was evaluated, particularly with respect to the detection status of high mass marker peaks.

[0084] <Method> 1. Six strains of Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Enteritidis, NBRC3313; serovar Thompson, ATCC BAA-1738; serovar Choleraesuis, NBRC 105684; serovar Infantis, jfrlSe1402-4; serovar Orion, jfrlSe1402-15) were cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1 and SA-3 described above were prepared.

[0085] 3. As an example of not performing centrifugation (Example 4-1), approximately 1 mg of the Salmonella from 1. was weighed on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 107 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became 100% (particles / μL), and the mixture was suspended with a needle to prepare a suspension. The resulting suspension was subjected to ultrasonic treatment (1 min). 4. As an example of centrifugation (Example 4-2), approximately 1 mg of the Salmonella from 1 was weighed on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 10 7 The matrix SA-3 solution from step 2 was added so that the concentration of the particles became 100% (particles / μL), and the mixture was suspended with a needle to prepare a suspension. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12000 rpm, 5 min).

[0086] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. In Example 4-1, the suspension prepared in 3 was dropped in an amount of 1 μL onto the wells precoated in 5. 7. In Example 4-2, 1 μL of the centrifugal supernatant obtained in 4 was dropped onto the wells precoated in 5. 8. The droplets dropped in steps 6 and 7 were allowed to dry naturally to obtain samples for mass spectrometry, and then mass spectrometry was carried out under the same conditions as in Example 1. After mass spectrometry, self-calibration was applied to the obtained data, and the obtained mass spectra were compared and evaluated.

[0087] <Result> FIG. 8 shows mass spectra including peaks corresponding to SodA of the above six strains of Salmonella enterica in (a) Example 4-2 (supernatant: after centrifugation, the supernatant was used) and (b) Example 4-1 (no centrifugation: suspension was used without centrifugation). For all six strains, when the supernatant was used, the peak of SodA was clearly detected with higher sensitivity than when centrifugation was not performed. This is thought to be because ionization inhibitors in the suspension were precipitated by centrifugation and separated from the marker protein, improving the sensitivity of the peaks corresponding to the marker in the supernatant. In particular, high mass marker peaks such as SodA, which were unstable to detect, became stably and reproducibly detected. Similar experiments were performed under different centrifugation conditions. Specifically, centrifugation was performed at 6000 rpm, 12000 rpm, or 15000 rpm for 1 or 5 minutes. Although a certain degree of sensitivity and stability was obtained at 6000 rpm and 15000 rpm, the marker peak was detected more sensitively and more stably when centrifugation was performed at 12000 rpm for 5 minutes. EXAMPLES

[0088] In Example 5, the effect of ultrasonic treatment was evaluated by comparing the case where ultrasonic suspension was performed with the case where needle suspension was performed without ultrasonic suspension.

[0089] <Method> 1. Salmonella enterica (serovar Enteritidis, NBRC3313) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1 and SA-3 described above were prepared. 3. As an example of a case where ultrasonic treatment was not performed and only suspension was performed using a needle (Example 5-1), approximately 1 mg of the Salmonella from 1. was weighed out using a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 10 7 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was then centrifuged (12,000 rpm, 5 min). 4. As an example of ultrasonic suspension (Example 5-2), approximately 1 mg of the Salmonella from 1 was weighed out on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 10 7 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min).

[0090] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. 1 μL each of the suspensions from 3. and 4. was dropped onto the wells precoated in 5. 7. The droplets dropped in step 6 were allowed to dry naturally to obtain a sample for mass spectrometry. Mass spectrometry was then carried out in the same manner as in Example 4, and the obtained mass spectra were compared and evaluated.

[0091] <Result> FIG. 9 shows mass spectra including a peak corresponding to the marker YciF for Salmonella enterica (serovar Enteritidis, NBRC3313) in the case of (a) ultrasonic suspension (Example 5-2) and (b) needle suspension without ultrasonic treatment (Example 5-1). Conventionally, the marker YciF for Salmonella enterica (serovar Enteritidis, NBRC3313) had low sensitivity, particularly for the marker peak, and detection was unstable. In this example, the YciF peak was more stably detected by preparing the analytical sample through ultrasonic suspension. It was considered possible that more YciF was eluted into the solution when ultrasonic suspension was performed compared to when ultrasonic suspension was not performed. EXAMPLES

[0092] In Example 6, the effect of ultrasonic treatment was evaluated by comparing the case where ultrasonic suspension was performed with the case where suspension was performed using a vortex mixer without ultrasonic suspension.

[0093] 1. Salmonella enterica (serovar Enteritidis, NBRC3313) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1 and SA-3 described above were prepared. 3. As an example of suspending using a vortex mixer without ultrasonic treatment (Example 6-1), approximately 1 mg of the Salmonella from 1. was weighed out on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 10 7 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became 100% (particles / μL), and the mixture was suspended using a needle. The mixture was then suspended in a vortex mixer (5 min) to prepare a suspension. The resulting suspension was centrifuged (12,000 rpm, 5 min). 4. As an example of ultrasonic suspension (Example 6-2), approximately 1 mg of the Salmonella from 1 was weighed out on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 10 7 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became 100% (particles / μL), and the mixture was suspended using a needle. The mixture was then subjected to ultrasonic treatment (1 min) to prepare a suspension. The resulting suspension was subjected to centrifugation (12000 rpm, 5 min).

[0094] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. 1 μL of each of the suspensions obtained in 3 and 4 was dropped onto the wells precoated in 5. 7. The droplets dropped in step 6 were allowed to dry naturally to obtain a sample for mass spectrometry. Mass spectrometry was then carried out in the same manner as in Example 4, and the obtained mass spectra were compared and evaluated.

[0095] <Result> FIG. 10 shows mass spectra including a peak corresponding to the marker YciF for Salmonella enterica (serotype Enteritidis, NBRC3313) in (a) the case of ultrasonic suspension (Example 6-2) and (b) the case of suspension using a vortex mixer (Example 6-1). Compared to suspension using a vortex mixer, the peak corresponding to YciF was detected with higher sensitivity in the case of ultrasonic suspension. Improved peak sensitivity was also confirmed in the entire mass spectrum including peaks other than YciF. It was considered that more proteins including YciF were eluted in the suspension in the case of ultrasonic suspension compared to suspension using a vortex mixer. EXAMPLES

[0096] In Example 7, the effect of adjusting the detector voltage of the mass spectrometer was evaluated.

[0097] 1. Three strains of Salmonella enterica (serotype Typhimurium, NBRC13245; serotype Choleraesuis, NBRC 105684; serotype Orion, jfrlSe1402-15) were cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 described above were prepared. 3. As a standard suspension of E. coli for adjusting the detector voltage, a suspension of E. coli DH5α Electro-Cells (TKR 9027, Takara Bio) was prepared by diluting it 1 / 50-fold with the SA-2 solution from 2.

[0098] 4. To prepare the Salmonella suspension according to Example 7, approximately 1 mg of the Salmonella from step 1 was weighed out using a microbalance, and the optimal bacterial concentration (1 mg / 0.075 mL; 1×10 7 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min). 5. 0.5 μL of the SA-1 solution was dropped onto a MALDI plate to perform precoating, and then 1 μL of the supernatant from the centrifugation in step 4 was dropped onto the precoated wells. 6. 1 μL of the E. coli suspension obtained in 3 was dropped onto a MALDI plate.

[0099] 7. After the droplets from steps 5 and 6 were allowed to dry naturally to obtain samples for mass spectrometry, the plate was inserted into a MALDI-MS (AXIMA Performance, Shimadzu Corporation) and measurements were performed in linear and positive ion modes using raster analysis. The mass spectra of the E. coli and Salmonella samples were evaluated after applying self-calibration.

[0100] In this mass analysis, mass analysis of E. coli was performed, and then mass analysis of the three Salmonella strains was performed. In the mass analysis of E. coli, multiple detector voltages were used, focusing on the peaks around m / z 23000, and as shown in FIG. 11 described later, the detector voltage values ​​at which the maximum peak in m / z 22000-23000 could be stably detected at a peak intensity of 0.1-0.3 mV, or the maximum peak in m / z 23000-24000 at a peak intensity of 0.1-0.4 mV were confirmed. The above three Salmonella strains were measured using detector voltage values ​​0-90 V higher than the confirmed detector voltage values.

[0101] <Result> Figure 11 shows mass spectra obtained by mass spectrometry of E. coli, (a) m / z 21500 to 23000, and (b) m / z 21500 to 24000. At the detector voltage when the mass spectrum in Figure 11 was obtained, the peak intensity near m / z 22000 was 0.1 to 0.3 mV, and the peak intensity near m / z 23000 was 0.1 to 0.4 mV, and they were detected relatively stably.

[0102] Figure 12 shows mass spectra including a peak corresponding to the marker SodA of Salmonella enterica (serovar Typhimurium, NBRC13245) obtained by using a detector voltage (a) 90, (b) 70, (c) 50, (d) 30, or (e) 0 V higher than the detector voltage (EC (V)) used to obtain the mass spectrum shown in Figure 11. As a result, the peaks with the highest sensitivity and S / N ratio were confirmed at detector voltages 50 V and 70 V higher than EC V (EC+50, EC+70). At a detector voltage 90 V higher than EC V (EC +90), the S / N ratio was slightly lower than that at EC+50 V or EC+70 V.

[0103] Figures 13 and 14 show mass spectra including a peak corresponding to the marker SodA for Salmonella enterica (serovar Choleraesuis, NBRC 105684; serovar Orion, jfrlSe1402-15) obtained by using detector voltages (EC (V)) that were (a) 70, (b) 50, (c) 30, or (d) 0 V higher than the detector voltage used to obtain the mass spectra shown in Figure 11. For both strains, mass spectra with the highest sensitivity and S / N ratio were obtained at detector voltages that were 50 and 70 V higher than the EC V (EC+50 and EC+70).

[0104] This embodiment confirmed that the detector voltage contributes particularly to the detection of high mass marker peaks, and that by establishing adjustment standards such as those in this embodiment, more stable and reproducible detection of marker peaks is possible. EXAMPLES

[0105] In Example 8, the effect of smoothing treatment on mass spectra was evaluated.

[0106] 1. Salmonella enterica (serotype Orion, jfrlSe1402-15) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1 and SA-3 described above were prepared. 3. Weigh out approximately 1 mg of the Salmonella from step 1 on a microbalance and determine the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 10 7 The matrix SA-3 solution from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min).

[0107] 4. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 5. 1 μL of the supernatant obtained in 3. was dropped onto the pre-coated wells in 4. 6. The droplets dropped in 5. were naturally dried to obtain a mass spectrometry sample, and then mass spectrometry was performed under the same conditions as in Example 1. After mass spectrometry, self-calibration was applied to the obtained data, and the obtained mass spectrum was evaluated. It was determined whether or not a marker peak was detected using the same criteria as in Example 1. For peaks corresponding to high mass ions with split peak tops, smoothing correction was performed (the smoothing setting of Peak Processing in Axima Performance was changed from 50 to 200), and the effect on peak detection before and after correction was evaluated.

[0108] <Result> Figure 15 shows mass spectra containing a peak corresponding to the marker SodA for Salmonella enterica (serotype Orion, jfrlSe1402-15) using (a) a smoothing value of 50 and (b) a smoothing value of 200 in the Peak Processing settings of Axima Performance.

[0109] The theoretical m / z value for the peak of SodA from Salmonella enterica (serotype Orion, jfrlSe1402-15) is 22948.82. In (a), the SodA peak was judged to be undetected because it was 800 ppm outside the theoretical value. After smoothing correction in (b), the SodA peak was judged to be detected because it was within 800 ppm. This is thought to be because the peak corresponding to SodA in (a) with a smoothing value of 50 splits at the top and the m / z value corresponding to the peak leans toward the high mass side, whereas in (b) with a smoothing value of 200, the shape of the peak is rounded and the m / z value corresponding to the peak is corrected toward the center (centroid) of the peak, making the m / z value corresponding to the peak more accurate. That is, when comparing (a) and (b) in Figure 15, it was considered that (b) applied a smoothing value suitable for ions with higher mass, resulting in a more accurate m / z value of the peak. EXAMPLES

[0110] In Example 9, the effectiveness of the method of the present embodiment was evaluated in terms of sensitivity to high mass marker peaks.

[0111] <Method> 1. Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Choleraesuis, NBRC 105684) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 described above were prepared. 3. In Comparative Example 9, for the Salmonella of 1., a suspension was prepared by suspending approximately 1 cm of a colony in 10 μL of matrix solution SA-2 of 2. with a needle. 4. In Example 9, approximately 1 mg of the Salmonella from 1 was weighed on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1×10 7The matrix SA-3 solution from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min).

[0112] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. In Comparative Example 9, the suspension obtained in 3. was dropped in 1.2 μL portions onto the wells precoated in 5. 7. In Example 9, 1 μL of the centrifugation supernatant obtained in 4 was dropped onto the wells precoated in 5. 8. The droplets of 6. and 7. were naturally dried to obtain samples for mass spectrometry, and then mass spectrometry was performed in the same manner as in Example 4, and the mass spectrum obtained was evaluated. The same detector voltage was used in the mass spectrometry in Comparative Example 9 and Example 9.

[0113] <Result> FIG. 16 shows mass spectra including a peak corresponding to the marker SodA by (a) Comparative Example 9 and (b) Example 9 for Salmonella enterica (serotype Typhimurium, NBRC13245) and FIG. 17 shows mass spectra including a peak corresponding to the marker SodA by (a) Comparative Example 9 and (b) Example 9 for Salmonella enterica (serotype Choleraesuis, NBRC 105684). In the case of both strains, it was confirmed that the peak of SodA, which was not easy to detect in Comparative Example 9, was stably and highly sensitively detected in Example 9. Conventionally, the peak of SodA is known to be the highest mass protein among the Salmonella markers shown in Table 1, and has a particularly low sensitivity and is unstable to detect. It was considered that the improvement in sensitivity in Example 9 was due to multiple factors, such as the sensitivity improvement effect of the additive, the sensitivity improvement effect of separation from ionization inhibitors by using the centrifugal supernatant, and the sensitivity improvement effect of eluting the marker protein by ultrasound. EXAMPLES

[0114] In Example 10, the effect of the method of this embodiment was evaluated in terms of the reproducibility between wells of the detection status of high mass marker peaks.

[0115] <Method> 1. Salmonella enterica (serovar Choleraesuis, NBRC 105684; serovar Infantis, jfrlSe1402-4) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 described above were prepared. 3. As Comparative Example 10, for the Salmonella of 1., a suspension was prepared by suspending approximately 1 cm of a colony with a needle in 10 μL of the matrix solution SA-2 of 2. 4. In Example 10, approximately 1 mg of the Salmonella from step 1 was weighed out on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1×10 7 The matrix SA-3 solution from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min).

[0116] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. In Comparative Example 10, the suspension obtained in 3. was dropped in 1.2 μL portions onto the wells precoated in 5. 7. In Example 10, 1 μL of the centrifugation supernatant obtained in 4 was dropped onto the wells precoated in 5. 8. The droplets dropped in 6. and 7. were naturally dried to obtain samples for mass spectrometry, and then mass spectrometry was performed in the same manner as in Example 4, and the obtained mass spectrum was evaluated. The same detector voltage was used in the mass spectrometry in Comparative Example 10 and Example 10.

[0117] <Result> FIG. 18 shows the mass spectrum including the peak corresponding to SodA obtained for Salmonella enterica (serotype Choleraesuis, NBRC 105684), and FIG. 19 shows the mass spectrum obtained for Salmonella enterica (serotype Infantis, jfrlSe1402-4). In Comparative Example 10 and Example 10, mass spectra obtained in each of 8 wells (n=8) are shown. In both FIG. 18 and FIG. 19, the sensitivity or S / N ratio, etc., varies between wells in Comparative Example 10, whereas the peak detection conditions were almost reproduced in all 8 wells in Example 10. As described above, it has been known that the SodA peak is the highest mass protein among the Salmonella markers shown in Table 1, and is particularly low in sensitivity and unstable in detection. The improvement in reproducibility in Example 10 is influenced by the effect of improving sensitivity by additives, centrifugal supernatant, or ultrasonic treatment, or the effect of homogenizing crystals or improving reproducibility between wells by centrifugal supernatant, etc. It was thought that. EXAMPLES

[0118] In Example 11, the effectiveness of the method of this embodiment was evaluated in terms of the detection status of high mass marker peaks and reproducibility between wells. In the following examples, similarly to Example 7, the detector voltage was set based on the data obtained by mass spectrometry of the E. coli sample.

[0119] <Method> 1. Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Enteritidis, GTC00131; serovar Infantis, ATCC BAA-1675; serovar Thompson, ATCC BAA-1738) were cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 described above were prepared. 3. Weigh out approximately 1 mg of the Salmonella from step 1 on a microbalance and determine the optimal bacterial concentration (1 mg / 0.075 mL; 1 × 10 7The matrix solution SA-3 from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min).

[0120] 4. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 5. 1 μL of the supernatant obtained in step 3 was dropped onto the wells precoated in step 4. 6. The droplets dropped in step 5 were allowed to dry naturally to obtain a sample for mass spectrometry. Mass spectrometry was then performed in the same manner as in Example 4, except that the detector voltage was set in the same manner as in Example 7, and the obtained mass spectrum was evaluated.

[0121] <Result> 20, 21, 22 and 23 show mass spectra including a peak corresponding to the marker SodA of Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Enteritidis, GTC00131; serovar Infantis, ATCC BAA-1675; serovar Thompson, ATCC BAA-1738), respectively. Each mass spectrum in the figure corresponds to each of 4 wells (n=4). In all strains, the sensitivity and reproducibility were high, and the peak of SodA was stably detected. The improvement in sensitivity and reproducibility in Example 11 was considered to be influenced by the sensitivity improvement effect by the additive, the centrifugal supernatant, ultrasound, or the adjustment of the detector voltage, and the homogenization of crystals or the effect of improving reproducibility between wells by the centrifugal supernatant. EXAMPLES

[0122] In Example 12, the effectiveness of the method of this embodiment was evaluated from the viewpoint of whether peaks corresponding to the 12 Salmonella markers shown in Table 1 could be detected reproducibly on different days.

[0123] <Method> 1. Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Choleraesuis, NBRC 105684; serovar Orion, jfrlSe1402-15) was cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 described above were prepared. 3. As Comparative Example 12, for the Salmonella of 1., about 1 cm of colonies were added to 10 μL of the matrix solution SA-2 of 2. and suspended with a needle to prepare a suspension. 4. In Example 12, approximately 1 mg of the Salmonella from step 1 was weighed out on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1×10 7 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min).

[0124] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. In Comparative Example 12, 1 or 1.2 μL of the suspension obtained in 3 was dropped onto the wells precoated in 5. 7. In Example 12, 1 μL of the centrifugation supernatant obtained in 4. was dropped onto the wells precoated in 5. 8. The droplets dropped in 6. and 7. were naturally dried to obtain samples for mass spectrometry, and mass spectrometry was performed in the same manner as in Example 4, except that the detector voltage was set in the same manner as in Example 7, and the obtained mass spectrum was evaluated. Whether or not the marker was detected was determined based on whether or not the peak was picked within 800 ppm of the theoretical value of m / z corresponding to the marker peak. The detection rate of the marker was calculated as the percentage (%) of wells in which the marker was detected out of 4 or 8 wells for each sample.

[0125] <Result> Table 12-A in FIG. 24 and Table 12-B in FIG. 25 show the daily detection rates of the 12 markers for Salmonella enterica (serotype Typhimurium, NBRC13245) in Comparative Example 12 and Example 12, respectively. Table 12-C in FIG. 26 and Table 12-D in FIG. 27 show the daily detection rates of the 12 markers corresponding to Salmonella enterica (serotype Choleraesuis, NBRC 105684) in Comparative Example 12 and Example 12, respectively. Table 12-E in FIG. 28 and Table 12-F in FIG. 29 show the daily detection rates of the 12 markers corresponding to Salmonella enterica (serotype Orion, jfrlSe1402-15) in Comparative Example 12 and Example 12, respectively. The six-digit number for the measurement date has the first two digits indicating the last two digits of the year, the next two digits indicating the month, and the last two digits indicating the day. When the date is connected with a hyphen, such as "180117-0309" in Table 12-B (Figure 25), it indicates that the measurement was performed on any day between the previous date and the later date connected with the hyphen.

[0126] For both strains, the detection rate of the marker varied for each date in Comparative Example 12, whereas the detection rate showed less variation and was detected with good reproducibility in Example 12. The improvement in reproducibility in Example 12 was thought to be influenced by the effects of adjusting the bacterial concentration, additives, centrifugal supernatant, ultrasound, or detector voltage to improve sensitivity, and the effect of homogenizing the crystals or improving reproducibility between wells by the centrifugal supernatant. EXAMPLES

[0127] In Example 13, the effectiveness of the method of this embodiment was evaluated in terms of the detection rate of marker peaks of six Salmonella strains.

[0128] <Method> 1. Six strains of Salmonella enterica (serovar Typhimurium, NBRC13245; serovar Enteritidis, NBRC3313; serovar Thompson, ATCC BAA-1738; serovar Choleraesuis, NBRC 105684; serovar Infantis, jfrlSe1402-4; serovar Orion, jfrlSe1402-15) were cultured on LB agar medium at 37°C for 20 hours. 2. As matrix solutions, SA-1, SA-2 and SA-3 described above were prepared. 3. As Comparative Example 13, for Salmonella in 1., about 1 cm of a colony was added to 10 μL of matrix solution SA-2 in 2., and suspended with a needle to prepare a suspension. 4. In Example 13, approximately 1 mg of the Salmonella from step 1 was weighed out on a microbalance and the optimal bacterial concentration (1 mg / 0.075 mL; 1×10 7 The matrix solution SA-3 from step 2 was added so that the concentration of the particles became equal to 100 particles / μL, and the particles were suspended using a needle. The resulting suspension was subjected to ultrasonic treatment (1 min) and then centrifuged (12,000 rpm, 5 min).

[0129] 5. 0.5 μL of the SA-1 solution was dropped onto the MALDI plate for precoating. 6. As Comparative Example 13, 1 or 1.2 μL of the suspension obtained in 3 was dropped onto the wells precoated in 5. 7. In Example 13, 1 μL of the centrifugation supernatant obtained in 4 was dropped onto the wells precoated in 5. 8. The droplets dropped in 6. and 7. were naturally dried to obtain samples for mass spectrometry, and mass spectrometry was performed in the same manner as in Example 4, except that the detector voltage was set in the same manner as in Example 7, and the obtained mass spectrum was evaluated. Whether or not the marker peak was detected was determined based on whether or not the peak was picked within 800 ppm of the theoretical m / z value for the marker peak. The detection rate of the marker peak was calculated as the percentage (%) of the number of wells in which the marker peak was detected among the four wells corresponding to each sample.

[0130] <Result> Table 13-A in FIG. 30, Table 13-B in FIG. 31, and Table 13-C in FIG. 32 show the detection rates of 12 markers for 6 representative strains of Salmonella enterica (serotype Typhimurium, NBRC13245; serotype Enteritidis, NBRC3313; serotype Thompson, ATCC BAA-1738; serotype Choleraesuis, NBRC 105684; serotype Infantis, jfrlSe1402-4; serotype Orion, jfrlSe1402-15). Table 13-A and Table 13-B show the results of Comparative Example 13 on different experimental days. Table 13-C shows the results of Example 13.

[0131] In Comparative Example 13, the detection rate varied for each strain and each marker, whereas in Example 13, 100% detection rate was observed for all 12 markers for 6 strains, confirming stable detection of 12 marker peaks for 6 strains in Example 13. [Explanation of symbols]

[0132] 10: sample plate, 11: well, M: matrix solution, MS: sample solution, Pr: precipitate, S: sample, Sn: supernatant.

Claims

1. The concentration of microorganisms is 1 x 10 ６ CFU / μL or more 6×10 ７ Prepare a sample solution with a CFU / μL or less; Centrifugation of the sample solution is performed; The supernatant obtained by the centrifugation is used to prepare a sample for microbial analysis; A method for identifying a microorganism, comprising ionizing the sample for microorganism analysis by matrix-assisted laser desorption / ionization and performing a first mass spectrometry analysis, preparing the sample solution by any one of the following: adding the microorganism to a solution containing a matrix, and then adjusting the turbidity of the solution; adjusting the turbidity of the solution containing the microorganism, and then mixing the turbidity-adjusted solution containing the microorganism with a solution containing a matrix; weighing the microorganism, and mixing the weighed solution containing the microorganism with a solution containing a matrix; and weighing the microorganism, preparing a solution containing the weighed microorganism, and then mixing the prepared solution containing the microorganism with a solution containing a matrix; The method for identifying microorganisms, wherein the sample for microbial analysis is prepared using methylenediphosphonic acid and N-decyl-β-D-maltopyranoside as additives to the matrix.

2. The concentration of microorganisms is 1 x 10 ６ CFU / μL or more 6×10 ７ Prepare a sample solution with a CFU / μL or less; Centrifugation of the sample solution is performed; The supernatant obtained by the centrifugation is used to prepare a sample for microbial analysis; A method for identifying a microorganism, comprising ionizing the sample for microorganism analysis by matrix-assisted laser desorption / ionization and performing a first mass spectrometry analysis, In the first mass analysis, a peak corresponding to an m / z of 10,000 or more is detected; performing a second mass spectrometry analysis of a microbial analysis sample prepared from a microorganism different from the microorganism; and setting a detector voltage for the first mass spectrometry analysis based on data obtained by the second mass spectrometry analysis; the microorganism different from said microorganism is Escherichia coli; A method for identifying a microorganism, wherein in the second mass spectrometry, the voltage of the detector is set based on the intensity of at least one of the most intense peaks in the m / z range of 22,000 to 23,000 in the mass spectrum of E. coli and the most intense peak in the m / z range of 23,000 to 24,000 in the mass spectrum.

3. The method for identifying a microorganism according to claim 1 or 2, A method for identifying microorganisms, comprising subjecting the sample solution to ultrasonic treatment prior to the centrifugation.

4. The method for identifying a microorganism according to claim 2, A method for identifying microorganisms, which prepares a sample solution by any one of the following: adding the microorganisms to a solution containing a matrix and then adjusting the turbidity of the solution; adjusting the turbidity of the solution containing the microorganisms and then mixing the turbidity-adjusted solution containing the microorganisms with a solution containing a matrix; weighing the microorganisms and mixing the weighed solution containing the microorganisms with a solution containing a matrix; and weighing the microorganisms and preparing a solution containing the weighed microorganisms and then mixing the prepared solution containing the microorganisms with a solution containing a matrix.

5. The method for identifying a microorganism according to claim 4, The method for identifying microorganisms, wherein the sample for microbial analysis is prepared using at least one of a compound containing a phosphonic acid group and a surfactant as an additive to the matrix.

6. The method for identifying a microorganism according to any one of claims 1, 3, 4 and 5, A method for identifying a microorganism, wherein the matrix is ​​sinapic acid.

7. The method for identifying a microorganism according to any one of claims 1 to 6, The method for identifying a microorganism, wherein the microorganism is a bacterium of the genus Salmonella.

8. The method for identifying a microorganism according to any one of claims 1, 3, 6 and 7, The method for identifying a microorganism, wherein the first mass spectrometry detects a peak corresponding to an m / z of 10,000 or more.

9. The method for identifying a microorganism according to claim 8, A method for identifying a microorganism, comprising: performing a second mass analysis of a sample for microbial analysis prepared from a microorganism different from the microorganism; and setting a detector voltage in the first mass analysis based on data obtained by the second mass analysis.

10. The method for identifying a microorganism according to claim 9, The second mass analysis is a method for identifying microorganisms, in which the sample for microorganism analysis is ionized by matrix-assisted laser desorption / ionization.

11. The method for identifying a microorganism according to claim 9 or 10, the microorganism different from said microorganism is Escherichia coli; A method for identifying a microorganism, wherein in the second mass spectrometry, the voltage of the detector is set based on the intensity of at least one of the most intense peaks in the m / z range of 22,000 to 23,000 in the mass spectrum of E. coli and the most intense peak in the m / z range of 23,000 to 24,000 in the mass spectrum.

12. The concentration of microorganisms is 1 x 10 ６ CFU / μL or more 6×10 ７ Prepare a sample solution with a CFU / μL or less; Centrifugation of the sample solution is performed; The supernatant obtained by the centrifugation is used to prepare a sample for microbial analysis; A method for identifying a microorganism, comprising ionizing the sample for microorganism analysis by matrix-assisted laser desorption / ionization and performing a first mass spectrometry analysis, Identifying at least one of the subspecies, serotypes, and strains of the microorganisms contained in the sample solution based on the presence or absence of a peak derived from a marker protein for discriminating at least one of the subspecies, serotypes, and strains of the microorganisms; In the first mass spectrometry, a peak corresponding to an m / z of 10,000 or more is detected as a peak derived from the marker protein, A method for identifying a microorganism, comprising: performing a second mass analysis of a sample for microbial analysis prepared from Escherichia coli, which is a microorganism different from the microorganism; and setting a detector voltage in the first mass analysis based on the intensity of at least one of the most intense peaks in the m / z range of 22,000 or more and 23,000 or less in the mass spectrum of Escherichia coli and the most intense peak in the m / z range of 23,000 or more and 24,000 or less in the mass spectrum in the second mass analysis.

13. The method for identifying a microorganism according to claim 12, The second mass analysis is a method for identifying microorganisms, in which the sample for microorganism analysis is ionized by matrix-assisted laser desorption / ionization.

14. The method for identifying a microorganism according to any one of claims 1 to 13, further comprising: obtaining mass spectrum data corresponding to a mass spectrum by the first mass analysis; and performing different smoothing on the mass spectrum data based on the m / z value.

15. The method for identifying a microorganism according to claim 14, A method for identifying microorganisms, in which smoothing is performed on the mass spectral data in a second range having an m / z higher than a first range of m / z, and the intensities corresponding to each data point are corrected using more or a wider range of data points than when smoothing is performed on the mass spectral data in a first range of m / z.

16. The method for identifying a microorganism according to claim 15, A method for identifying a microorganism, wherein the first range and the second range are set based on a threshold, and the threshold is m / z 15,000 or more and 20,000 or less.

17. A method for identifying a microorganism according to any one of claims 1 to 16, The microorganism is a bacterium of the genus Salmonella, A method for identifying a microorganism, wherein the first mass spectrometry detects at least one protein derived from a bacterium of the genus Salmonella selected from the group consisting of gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF and SodA.

18. The method for identifying a microorganism according to claim 1 or 2, A method for identifying microorganisms, which identifies at least one of the subspecies, serotypes and strains of microorganisms contained in a sample solution based on the presence or absence of detection of a peak derived from a marker protein for discriminating at least one of the subspecies, serotypes and strains of the microorganisms.

19. The method for identifying a microorganism according to claim 1 or 2, The concentration of the microorganism is 3×10 ６ CFU / μL or more 3×10 ７ A method for identifying microorganisms, which involves preparing a sample solution with a CFU / μL or less.

20. The method for identifying a microorganism according to claim 2 or 3, A method for identifying a microorganism, wherein the centrifugation speed is 6,000 rpm or more and 15,000 rpm or less.

21. The method for identifying a microorganism according to claim 1 or 2, The method for identifying a microorganism, wherein the centrifugation speed is 10,000 rpm or more and 13,000 rpm or less.

22. The method for identifying a microorganism according to claim 20 or 21, A method for identifying microorganisms, wherein the centrifugation time is from 1 to 5 minutes.

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