Microbiological analysis methods

Abstract

The invention relates to a microbial analysis method, which comprises an identification step, wherein the method comprises identifying which of the two serotypes the microorganisms contained in the sample are included according to whether one or more peaks with a specified mass-to-charge ratio are present in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of two serotypes of Salmonella bacteria, namely Abony and Pakistan; or identifying which of the three serotypes the microorganisms contained in the sample are included according to whether one or more peaks with a specified mass-to-charge ratio are present in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of three serotypes of Salmonella bacteria, namely Minnesota, Infantis and Brandenburg; or identifying which of the two serotypes the microorganisms contained in the sample are included according to whether one or more peaks with a specified mass-to-charge ratio are present in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of two serotypes of Salmonella bacteria, namely Schwarzengrund and Montevideo.

Description

Technical Field

[0001] The present invention relates to a method for analyzing microorganisms. Background Art

[0002] The matrix-assisted laser desorption / ionization (MALDI) method, which is one of the ionization methods in mass spectrometry, is the following method: in order to analyze substances that are difficult to absorb laser light, proteins and other substances that are easily damaged by laser light, a matrix substance that is easily absorbed and ionized under laser light is mixed with the substance to be analyzed, and the substance to be analyzed is ionized by irradiating it with laser light. Usually, the matrix substance is mixed with the substance to be analyzed in the form of a solution. Then, the solvent in the solution is vaporized to dry it, forming crystals containing the substance to be analyzed. When it is irradiated with laser light, the matrix substance absorbs the energy of the laser light and is rapidly heated, and vaporization occurs. At this time, the substance to be analyzed is also vaporized together with the matrix substance, and the substance to be analyzed is ionized in this process.

[0003] The mass spectrometer (MALDI-MS) using this MALDI method can perform analysis without substantially dissociating high molecular weight compounds such as proteins, and is also suitable for microanalysis, and is therefore widely used in the field of life sciences. One of the uses of MALDI-MS in the field of life sciences is the identification of microorganisms using MALDI-MS. It is a method for identifying microorganisms based on a mass spectrum obtained using the microorganism to be tested, and the analysis results can be obtained in a short time, so that simple and rapid identification of microorganisms is possible.

[0004] For example, one of the representative causative bacteria of food poisoning is Salmonella belonging to the family Enterobacteriaceae, which is a gram-negative facultative anaerobic bacillus. Three species of Salmonella (hereinafter abbreviated as "S.") enterica, S. bongori and S. subterranea belong to the genus Salmonella, and S. enterica is further classified into 6 subspecies. Most of the pathogenic Salmonella that cause food poisoning belong to S. enterica subsp. enterica, which is further classified into multiple serotypes. The identification of species, subspecies and serotypes of Salmonella is important for the elucidation of the infection route of food poisoning and the prevention of infection, so in recent years, the identification of Salmonella using MALDI-MS has been attempted.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Appl. Microbiol. Biotechnol., Applied Microbiology and Biotechnology, Vol. 101, No. 23-24, pp. 8557-8569, 2017 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] The identification of species, subspecies, and serotypes of Salmonella using MALDI-MS is performed by detecting peaks, i.e., biomarker peaks, that differ in position (mass-to-charge ratio, m / z value) and height (peak intensity, mV value) on the mass spectrum between bacteria of different species, subspecies, and serotypes. Protein peaks are often used as biomarker peaks for the identification of microorganisms represented by bacteria (Non-Patent Document 1).

[0010] Generally, in the case of closely related microorganisms, the mass-to-charge ratios of peaks derived from multiple proteins are the same or similar. Therefore, in order to accurately distinguish Salmonella bacteria at the serotype level, it is not enough to select only a peak derived from one protein as a biomarker peak, and it is necessary to select appropriate peaks derived from multiple proteins corresponding to the type of serotype.

[0011] For example, Non-Patent Document 1 describes that the mass-to-charge ratio values ​​of peaks derived from 12 proteins are read from a mass spectrum obtained by mass spectrometry analysis of a sample containing microorganisms, and the serotype of Salmonella bacteria that the microorganism contained in the sample is determined based on these values. However, even if the peaks derived from the 12 proteins are used as marker peaks, it is sometimes difficult to identify which serotype of Salmonella bacteria it is, even if any of a plurality of predetermined serotypes of Salmonella bacteria can be determined.

[0012] The problem to be solved by the present invention is to enable accurate identification of a specific serotype of Salmonella bacteria in a microbial analysis method using MALDI-MS.

[0013] Solutions for solving problems

[0014] The present invention made to solve the above problems is a microbial analysis method, which has the following steps:

[0015] The identification step includes identifying which of the two serotypes of Salmonella bacteria, namely, Abony and Pakistan, is included in the microorganism contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria,

[0016] The value of the aforementioned mass-to-charge ratio is selected from the group consisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803, 15990, and any combination thereof.

[0017] In addition, the present invention made to solve the above-mentioned problem is a microbial analysis method, which comprises the following steps:

[0018] an identification step of identifying which of the three serotypes of Salmonella bacteria, namely, Minnesota, Infantis, and Brandenburg, the microorganism contained in the sample is, based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the three serotypes of Salmonella bacteria, namely, Minnesota, Infantis, and Brandenburg;

[0019] The value of the mass-to-charge ratio specified above is selected from the group consisting of 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857, 12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766, 20838, and any combination thereof.

[0020] Furthermore, the present invention made to solve the above-mentioned problem is a microbial analysis method, which comprises the following steps:

[0021] an identification step of identifying which of the two serotypes of Salmonella bacteria, Schwarzengrund and Montevideo, is included in the microorganism contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria, i.e., Schwarzengrund and Montevideo;

[0022] The aforementioned specified mass-to-charge ratio value is selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835, 19001, and any combination thereof.

[0023] Effects of the Invention

[0024] According to the present invention, when a sample contains any of two serotypes of Salmonella, namely Abony and Pakistan, it is possible to accurately identify which of the two serotypes the bacterium is. Furthermore, when a sample contains any of three serotypes of Salmonella, namely Minnesota, Infantis and Brandenburg, it is possible to accurately identify which of the three serotypes the bacterium is. Furthermore, when a sample contains any of two serotypes of Salmonella, namely Schwarzengrund and Montevideo, it is possible to accurately identify which of the two serotypes the bacterium is. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic overall configuration diagram of a microorganism analysis system used in the microorganism analysis method of the present invention.

[0026] Figure 2 This is a flowchart showing an example of the process of the microorganism analysis method.

[0027] Figure 3 This is a diagram showing the vicinity of the seven peaks in Table 2 that meet the second criterion among the peaks on the mass spectra obtained for samples containing each of the two serotypes of Salmonella bacteria, Abony and Pakistan (Group 1).

[0028] Figure 4 This is a graph showing the vicinity of seven peaks in Table 1 that do not meet the second criterion but meet the first criterion among the peaks on the mass spectrum obtained for samples containing each of the two serotypes of Salmonella bacteria, Abony and Pakistan (Group 1).

[0029] Figure 5 This is a diagram showing the vicinity of three peaks in Table 4 that meet the second criterion among peaks on the mass spectrum obtained for samples containing three serotypes of Salmonella bacteria, namely Minnesota, Infantis, and Brandenburg (Group 2).

[0030] Figure 6This is a graph showing the vicinity of three peaks in Table 3 that do not meet the second criterion but meet the first criterion among the peaks on the mass spectrum obtained for samples containing each of the three serotypes of Salmonella, Minnesota, Infantis, and Brandenburg (Group 2).

[0031] Figure 7 This is a graph showing the vicinity of the four peaks in Table 6 that meet the second criterion among the peaks on the mass spectrum obtained for samples containing two serotypes of Salmonella, Schwarzengrund and Montevideo (Group 3).

[0032] Figure 8 This is a graph showing the vicinity of three peaks in Table 5 that do not meet the second criterion but meet the first criterion among the peaks on the mass spectrum obtained for samples containing two serotypes of Salmonella, Schwarzengrund and Montevideo (Group 3). DETAILED DESCRIPTION

[0033] The microbial analysis method of the present invention is used as an object of the following sample: a sample in which it is known that the microorganisms contained therein include any of the three serotypes of Salmonella. Here, the three serotypes group refers to a group consisting of two serotypes, namely Abony and Pakistan (hereinafter referred to as Group 1); a group consisting of three serotypes, namely Minnesota, Infantis and Brandenburg (hereinafter referred to as Group 2); and a group consisting of two serotypes, namely Schwarzengrund and Montevideo (hereinafter referred to as Group 3). In other words, the following sample is used as an object: a sample in which it is known that Salmonella of Group 1 is included, but it is unknown which serotype of the bacterium is Abony and Pakistan; or a sample in which it is known that Salmonella of Group 2 is included, but it is unknown which serotype of the bacterium is Minnesota, Infantis and Brandenburg; or a sample in which it is known that Salmonella of Group 3 is included, but it is unknown which serotype of the bacterium is Schwarzengrund and Montevideo.

[0034] That is, the microbial analysis method of the present invention comprises the following steps:

[0035] The identification step includes identifying which of the two serotypes of Salmonella bacteria, namely, Abony and Pakistan, is included in the microorganism contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria,

[0036] The value of the aforementioned mass-to-charge ratio is selected from the group consisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803, 15990, and any combination thereof. Particularly preferably, it is selected from the group consisting of 3119, 4166, 5487, 6238, 6720, 7858, 8330, 8536, 8687, 9912, 12276, 15714, 15803, 15990, and any combination thereof.

[0037] The peak detection conditions of the two serotypes, Abony and Pakistan, for the above mass-to-charge ratios are shown in Tables 1 and 2. In the present invention, by using the peaks at the mass-to-charge ratios in Table 1 or Table 2 as marker peaks and confirming the detection conditions of these peaks, it is possible to discriminate which of the two serotypes, Abony and Pakistan, it is.

[0038] [Table 1]

[0039]

[0040] [Table 2]

[0041]

[0042] In addition, the microbial analysis method of the present invention comprises the following steps:

[0043] an identification step of identifying which of the three serotypes of Salmonella bacteria, namely, Minnesota, Infantis, and Brandenburg, the microorganism contained in the sample is, based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the three serotypes of Salmonella bacteria, namely, Minnesota, Infantis, and Brandenburg;

[0044] The value of the mass-to-charge ratio specified above is selected from the group consisting of 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857, 12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766, 20838, and any combination thereof. Particularly preferably, it is selected from the group consisting of 6483, 7940, 10990, 11808, 11821, 11848, 11857, 12209, 13406, 13445, 15803, 15878, 15895, 17713, 17735, and any combination thereof.

[0045] The peak detection conditions of the three serotypes, Minnesota, Infantis, and Brandenburg, for the above mass-to-charge ratios are shown in Tables 3 and 4. In the present invention, by using the peaks at the mass-to-charge ratios in Table 3 or Table 4 as marker peaks and confirming the detection conditions of these peaks, it is possible to determine which of the three serotypes, Minnesota, Infantis, and Brandenburg, it is.

[0046] [Table 3]

[0047]

[0048] [Table 4]

[0049]

[0050] In addition, the microbial analysis method of the present invention comprises the following steps:

[0051] an identification step of identifying which of the two serotypes of Salmonella bacteria, Schwarzengrund and Montevideo, is included in the microorganism contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria, i.e., Schwarzengrund and Montevideo;

[0052] The value of the mass-to-charge ratio is selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835, 19001, and any combination thereof. It is particularly preferably selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12276, 15820, 15835, and any combination thereof.

[0053] The peak detection conditions of the two serotypes, Schwarzengrund and Montevideo, for the above mass-to-charge ratios are shown in Tables 5 and 6. In the present invention, by using the peaks at the mass-to-charge ratios in Table 5 or 6 as marker peaks and confirming the detection conditions of these peaks, it is possible to discriminate which of the two serotypes, Schwarzengrund and Montevideo, it is.

[0054] [Table 5]

[0055]

[0056] [Table 6]

[0057]

[0058] As the standard for peak extraction in the above-mentioned microbial analysis method, for example, the following first standard or second standard can be used, but it is not limited to them. Compared with the first standard, the extraction standard of the peak of the second standard is more stringent. If the second standard is used, it is possible to reduce erroneous judgments. However, the intensity ratio of the peak sometimes changes with the strain and the culture conditions, so it is determined according to the purpose of determining the serotype to use the first standard and the second standard. In addition, the peak can be first extracted with the first standard, and then the peak that meets the second standard is further extracted from the extracted peak. Alternatively, the peak can be first extracted with the second standard, and then, when it is difficult to identify the target serotype, the peak that does not meet the second standard but meets the first standard is extracted and identified. The value of the mass-to-charge ratio of each peak is an approximate value, including a range that varies with the type of device used in mass spectrometry, analysis conditions, etc. Table 1, Table 3, and Table 5 show the peak detection status when the first standard is used, and Table 2, Table 4, and Table 6 show the peak detection status when the second standard is used. In each table, the circle mark indicates that the peak is detected, and the X mark indicates that the peak is not detected.

[0059] (1) Standard 1

[0060] In the case of a group consisting of two serotypes, the following peaks are extracted: in the mass spectrum obtained from a sample of Salmonella bacteria containing one serotype, the peak is detected as a peak with S / N ≥ 3, and in the mass spectrum obtained from a sample of Salmonella bacteria containing the other serotype, the peak is not detected or the difference in S / N of the peak is 5 times or more; or, in the mass spectrum of one, the peak is detected as a peak with S / N ≥ 3, and in the mass spectrum of the other, the peak is not detected or the difference in S / N is 10 times or more, and there is a neighboring peak with a mass-to-charge ratio m / z value close to that of the peak, or the peak partially overlaps with the neighboring peak.

[0061] In the case of a group consisting of three serotypes, the following peaks are extracted: in one of the three mass spectra obtained from a sample containing any one of the three serotypes, the peak is detected as a peak with S / N ≥ 3, and in the other two mass spectra, the peak is not detected or the difference in S / N of the peak is 5 times or more; or, in one of the three mass spectra, the peak is detected as a peak with S / N ≥ 3, and in the other two mass spectra, the peak is not detected or the difference in S / N of the peak is 10 times or more, and there is a neighboring peak with a mass-to-charge ratio m / z value close to that of the peak, or the peak partially overlaps with the neighboring peak.

[0062] (2) Second Standard

[0063] In the case of a group consisting of two serotypes, the following peaks are extracted: in the mass spectrum obtained from a sample of Salmonella bacteria containing one serotype, the peak is detected as a peak with S / N ≥ 10, and in the mass spectrum obtained from a sample of Salmonella bacteria containing the other serotype, the peak is not detected or the difference in S / N of the peak is 10 times or more.

[0064] In the case of a group consisting of three serotypes, the following peaks are extracted: in one of the three mass spectra obtained from a sample containing any one of the three serotypes, the peak is detected as a peak with S / N ≥ 10, and in the other two mass spectra, the peak is not detected or the difference in S / N of the peak is more than 10 times.

[0065] In either case, the following condition is satisfied: among the peaks extracted according to the second criterion, there are no adjacent peaks or partially overlapping peaks that may cause erroneous determination, and the peaks are independent peaks.

[0066] At this time, regarding the m / z value, the accuracy is, for example, 800 ppm, preferably 500 ppm. If a plurality of peaks exist within the accuracy, a marker peak with a closer m / z value is selected.

[0067] As to which group of Salmonella bacteria the microorganisms contained in the sample belong to among groups 1 to 3, the mass-to-charge ratio value of the peaks derived from 12 proteins (gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF, SodA) disclosed in non-patent document 1 can be used to identify the microorganisms contained in the sample.

[0068] In the microorganism identification method of the present invention, the serotype of bacteria in each group can be identified based on the presence or absence of a peak at one mass-to-charge ratio. However, by identifying the serotype of bacteria in each group based on the presence or absence of peaks at multiple mass-to-charge ratios, the accuracy of identification is improved.

[0069] As the mass spectrometer used in the microbial analysis method of the present invention, a mass spectrometer (MALDI-MS) using a matrix-assisted laser desorption / ionization (MALDI=Matrix Assisted Laser Desorption / Ionization) method is preferred. In addition, as MALDI-MS, a MALDI time-of-flight mass spectrometer (MALDI-TOFMS) is preferably used. Since MALDI-TOFMS can measure a very wide range of mass-to-charge ratios, it is possible to obtain a mass spectrum suitable for the analysis of high-mass molecules such as proteins that are constituent components of microorganisms.

[0070] Next, one embodiment of a microorganism analysis system used in the microorganism analysis method of the present invention will be described.

[0071] Figure 1 The overall structure of the microorganism analysis system is shown. The system generally includes a mass spectrometer 10 and a microorganism identification unit 20. The mass spectrometer 10 includes: an ionization unit 11, which ionizes molecules and atoms in a sample by matrix-assisted laser desorption / ionization (MALDI); and a time-of-flight mass separator (TOF) 12, which separates various ions emitted from the ionization unit 11 according to the mass-to-charge ratio.

[0072] The TOF 12 includes an extraction electrode 13 for extracting ions from the ionization unit 11 and introducing the ions into an ion flight space in the TOF 12 , and a detector 14 for detecting the ions separated by mass in the ion flight space.

[0073] The entity of the microorganism identification unit 20 is a computer such as a workstation or a personal computer, and a memory 22, a display unit 23 composed of an LCD (Liquid Crystal Display), an input unit 24 composed of a keyboard, a mouse, and a storage unit 30 composed of a large-capacity storage device such as a hard disk and an SSD (Solid State Drive) are connected to a central processing unit, i.e., a CPU (Central Processing Unit) 21. The storage unit 30 stores an OS (Operating System) 31, a spectrum creation program 32, a species determination program 33, and a serotype determination program 35 (programs of the present invention), and stores a first database 34 and a second database 36. The microorganism identification unit 20 further includes an interface (I / F) 25 for managing direct connection with an external device and connection with an external device via a network such as a LAN (Local Area Network), and is connected to the mass spectrometer analysis unit 10 via a network cable NW (or a wireless LAN) from the interface 25.

[0074] Figure 1 In the embodiment, as the serotype determination program 35, a spectrum acquisition unit 37, an m / z reading unit 38, and a serotype determination unit 39 are shown. These are basically functional means implemented in a software manner by the CPU 21 executing the serotype determination program 35. It should be noted that the serotype determination program 35 does not have to be a separate program, and can also be a function built into a part of a program for controlling the mass spectrometer 10, such as the species determination program 33, and its form is not particularly limited. It should be noted that as the species determination program 33, for example, a conventional program for performing microbial identification by fingerprinting can be used.

[0075] in addition, Figure 1 In the configuration, the spectrum creation program 32, the species determination program 33, the serotype determination program 35, the first database 34, and the second database 36 are installed on a terminal operated by a user, but it can also be set as follows: at least a part or all of them are set in another device connected to the aforementioned terminal through a computer network, and processing based on the program set in the aforementioned other device and / or access to the database is performed according to instructions from the aforementioned terminal.

[0076] A plurality of mass lists of known microorganisms are registered in the first database 34 of the storage unit 30. The mass list lists the mass-to-charge ratios of ions detected when a certain microbial cell is subjected to mass spectrometry analysis, and includes at least information (classification information) of the classification unit (family, genus, species, etc.) to which the aforementioned microbial cell belongs in addition to the information of the mass-to-charge ratio. Such a mass list is preferably prepared based on data (actually measured data) obtained by actually performing mass spectrometry analysis on various microbial cells in advance using the same ionization method and mass separation method as when using the aforementioned mass spectrometry unit 10.

[0077] When making a mass list based on the aforementioned measured data, first, extract the peaks that appear in a specified mass-to-charge ratio range from the mass spectrum obtained as the aforementioned measured data. At this time, by setting the aforementioned mass-to-charge ratio range to about 4000 to 30000, it is possible to mainly extract peaks derived from proteins. In addition, by only extracting peaks whose peak heights (relative intensities) are above a specified threshold, unnecessary peaks (noise) can be excluded. In addition, the mass-to-charge ratios (m / z) of the extracted peaks are tabulated for each cell and the aforementioned classification information is added, and then registered in the first database 34. It should be noted that in order to suppress the deviation of gene expression caused by culture conditions, it is ideal that the culture environment of each microbial cell used for the collection of measured data is standardized in advance.

[0078] The second database 36 of the storage unit 30 stores information related to marker proteins for identifying known microorganisms by serotypes, which are lower-level classifications than species. The information related to the marker proteins includes at least information on the mass-to-charge ratio (m / z) of the marker proteins in the known microorganisms. It should be noted that the second database 36 may also store information related to marker proteins for identification by lower-level classifications other than serotypes (e.g., subspecies, pathogens, strains, etc.).

[0079] The second database 36 in the present embodiment stores the following information: mass-to-charge ratio values ​​of peaks derived from 12 marker proteins (gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF, and SodA) for determining the serotype of the detected microorganism when the detected microorganism is a Salmonella bacterium (see Non-Patent Document 1), and information related to the marker proteins for identifying the serotype of the detected microorganism when the detected microorganism is determined to be a serotype belonging to any of the first to third groups among Salmonella bacterium. Information, specifically, the mass-to-charge ratio value of a prescribed marker peak for identifying which of the two serotypes it is when it is determined to belong to the first group, the mass-to-charge ratio value of a prescribed marker peak for identifying which of the three serotypes it is when it is determined to belong to the second group, or the mass-to-charge ratio value of a prescribed marker peak for identifying which of the two serotypes it is when it is identified to belong to the third group (specifically, a combination of the mass-to-charge ratio values ​​recorded in Tables 1 to 6).

[0080] As the value of the mass-to-charge ratio of the marker protein stored in the second database 36, it is ideal to select by comparing the calculated mass obtained by translating the base sequence of each marker protein into an amino acid sequence with the mass-to-charge ratio detected by actual measurement. It should be noted that, in addition to determining the base sequence of the marker protein by sequence, the base sequence obtained from a public database, such as the database of NCBI (National Center for Biotechnology Information, USA) can also be used. When calculating the mass from the aforementioned amino acid sequence, it is ideal to consider the cleavage of the N-terminal methionine residue as a post-translational modification. Specifically, when the penultimate amino acid residue is Gly, Ala, Ser, Pro, Val, Thr, or Cys, the aforementioned theoretical value is calculated in the form of the N-terminal methionine being cut off. In addition, what is actually observed by MALDI-TOF MS is a molecule with a proton added, so it is ideal to also consider the amount of the proton to obtain the aforementioned calculated mass (i.e., the theoretical value of the mass-to-charge ratio of the ion obtained when each protein is analyzed by MALDI-TOF MS).

[0081] Next, the analysis process of the serotype of Salmonella bacteria using the above-mentioned microorganism analysis system will be described with reference to the flowchart.

[0082] First, the user prepares a sample containing constituent components of the microorganism to be tested, installs it in the mass spectrometry unit 10, and performs mass spectrometry analysis. In this case, as the sample, in addition to cell extracts or cell constituent components such as ribosomal proteins purified from cell extracts, bacterial cells or cell suspensions can also be used directly.

[0083] The spectrum creation program 32 acquires the detection signal obtained by the detector 14 of the mass spectrometer 10 via the interface 25, and creates a mass spectrum of the test microorganism based on the detection signal (step 101).

[0084] Next, the species determination program 33 compares the mass spectrum of the aforementioned microorganism to be tested with the mass list of known microorganisms included in the first database 34, and extracts the mass list of known microorganisms having a mass-to-charge ratio pattern similar to the mass spectrum of the microorganism to be tested, for example, a mass list containing multiple peaks that are consistent with each peak in the mass spectrum of the microorganism to be tested within a specified error range (step 102). The species determination program 33 then identifies the biological species to which the known microorganism corresponding to the mass list belongs by referring to the classification information stored in the first database 34 corresponding to the mass list extracted in step 102 (step 103). Then, if the biological species is not Salmonella, the biological species is output to the display unit 23 as the biological species of the microorganism to be tested (step 114), and the analysis process is terminated. On the other hand, if the aforementioned biological species is Salmonella, then the analysis process using the serotype determination program 35 is then advanced. It should be noted that if it is determined in advance that the sample contains Salmonella by other methods, the mass spectrum species determination program is not used, but the serotype determination program 35 can be advanced.

[0085] In the serotype determination program 35, the mass spectrum of the microorganism to be tested is compared with the mass-to-charge ratio value of the marker protein included in the second database to determine the serotype of the microorganism to be tested (step 104). Specifically, first, the serotype determination unit 39 reads the mass-to-charge ratio value of the peak derived from the above-mentioned 12 marker proteins (gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF, SodA)) from the second database 36. Next, the spectrum acquisition unit 37 acquires the mass spectrum of the microorganism to be tested prepared in step 101. Then, the m / z reading unit 38 selects the peak that appears in the mass-to-charge ratio range stored in the second database 36 in association with the above-mentioned marker proteins as the peak corresponding to each marker protein on the mass spectrum, and determines the serotype according to the mass-to-charge ratio value of the peak.

[0086] As a result, when the serotype of the test microorganism is determined to be a serotype belonging to the first group (Abony, Pakistan) (step 105), the mass-to-charge ratio value of the predetermined marker peak corresponding to the serotype belonging to the first group is acquired from the second database (step 109).

[0087] On the other hand, when the serotype of the microorganism under test is determined to be a serotype belonging to the second group (Minnesota, Infantis, Brandenburg) (step 106), the mass-to-charge ratio value of the specified marker peak corresponding to the serotype belonging to the second group is obtained from the second database (step 110).

[0088] When the serotype of the test microorganism is determined to be a serotype belonging to Group 3 (Schwarzengrund, Montevideo) (Step 107), the mass-to-charge ratio value of a predetermined marker peak corresponding to the serotype belonging to Group 3 is acquired from the second database (Step 111).

[0089] Here, when it is determined that the serotype of the microorganism to be tested does not belong to any of the serotypes in Groups 1 to 3 (Step 108), the serotype of the microorganism to be tested is output to the display unit 23 as a serotype that does not belong to any of the serotypes in Groups 1 to 3 (Step 114).

[0090] Then, when it is determined to be a serotype belonging to any one of the 1st to 3rd groups and the mass-to-charge ratio values ​​of the specified marker peaks corresponding to the serotypes belonging to each group are obtained, the presence or absence of peaks appearing within the mass-to-charge ratio range stored in the second database 36 in association with these mass-to-charge ratio values ​​is confirmed (step 112), and the serotype of the tested microorganism is determined based on the situation (step 113), and the result is output to the display unit 23 as an identification result of the tested microorganism (step 114).

[0091] Example

[0092] Hereinafter, experiments for actually proving the effect of the microorganism analysis method according to the present embodiment will be described, but these are merely illustrative and the present invention is not limited to them.

[0093] 1. Cultivation of Salmonella

[0094] A total of seven species of Salmonella enterica, including two species of Salmonella enterica belonging to Group 1, three species of Salmonella enterica belonging to Group 2, and two species of Salmonella enterica belonging to Group 3, were cultured on an LB agar medium at 37°C for 20 hours.

[0095] The following Salmonella bacteria belong to Groups 1 to 3, respectively.

[0096] (1) Group 1

[0097] S.Abony,NBRC100797

[0098] S.Pakistan,GTC09493

[0099] (2) Group 2

[0100] S.Minnesota,NBRC15182

[0101] S.Infantis,ATCC BAA-1675

[0102] S. Brandenburg, jfrlSe1402-3

[0103] (3) Group 3

[0104] S. Schwarzengrund, Hyogo SO12004

[0105] S.Montevideo,jfrlSe1409-6

[0106] 2. Preparation of Matrix Solution

[0107] The following two base solutions were prepared.

[0108] (2-1) Sinapic acid (SA) as a matrix substance was dissolved in ethanol so as to contain 25 mg / mL to prepare a matrix solution (saturated solution). This matrix solution was designated as "SA-1".

[0109] (2-2) SA 25 mg / mL, methylene diphosphonic acid (MDPNA) 1%, and decyl-β-D-maltopyranoside (DMP) 1 mM as a surfactant were dissolved in an aqueous solution containing 50% acetonitrile (ACN) and 0.6% trifluoroacetic acid (TFA) to prepare a matrix solution. This matrix solution is referred to as "SA-2".

[0110] SA used in the substrate solutions SA-1 and SA-2 was manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., and MDPNA and DMP were manufactured by Sigma-Aldrich Japan.

[0111] 3. Preparation of Matrix / Microbial Suspension

[0112] (3-1) Using a microbalance, approximately 1 mg of each of the seven types of Salmonella cultured on LB agar medium was weighed and placed in a tube, and the matrix SA-2 solution was added thereto to a bacterial concentration of 1 mg / 0.075 mL (1×10 7 CFU / μL) and suspended with a needle.

[0113] (3-2) Ultrasonic vibration was applied to the tube for 1 minute, and the obtained suspension was centrifuged (12000 rpm, 5 min) to obtain a centrifugal supernatant.

[0114] 4. Analysis using MALDI-MS

[0115] (4-1) 0.5 μL of the matrix solution SA-1 (pre-coated) was added dropwise to each well of the MALDI sample plate.

[0116] (4-2) Next, 1 μL of the centrifugation supernatant was added dropwise to each well pre-coated with the substrate solution SA-1, and the wells were allowed to dry naturally.

[0117] (4-3) The MALDI sample plate obtained in (4-2) is inserted into a MALDI-MS (AXIMA Performance, manufactured by Shimadzu Corporation) and measured in linear mode (positive ion mode). All measurement data are obtained by grid analysis. Grid analysis is an automatic measurement function of the mass spectrometer, which is a method of obtaining mass spectrum data by laser irradiating the sample in each hole of the sample plate with a preset number of points and shots.

[0118] 5. Peak Extraction

[0119] Self-calibration of Salmonella bacteria (specifically, calibration is performed using several peaks of Salmonella itself as internal standards) is applied to the measurement data, and peaks useful for serotype identification are extracted from the obtained mass spectrum. For the extracted peaks, the mass-to-charge ratio values ​​of the peaks derived from 12 marker proteins shown in Non-Patent Document 1 are read, and the serotype of Salmonella bacteria that the microorganism contained in the sample is determined from these values.

[0120] When the serotype is determined by using the above 12 marker proteins, when any of the two serotypes of Salmonella, Abony and Pakistan, is determined, the mass spectrum obtained is confirmed to be selected from 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 74 53, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803, and 15990, for peak detection conditions of one or more mass-to-charge ratio values.

[0121] When the serotype is determined by using the above 12 marker proteins, if any of the three serotypes of Salmonella, namely Minnesota, Infantis and Brandenburg, is determined, the mass spectrum obtained is confirmed to be selected from 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990 , 11808, 11821, 11848, 11857, 12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766, and 20838, respectively.

[0122] When the serotype is identified using the above 12 marker proteins, if it has been identified as any of the two serotypes of Salmonella, namely Schwarzengrund and Montevideo, for the obtained mass spectrum, the peak detection status of one or more mass-to-charge ratio values ​​selected from 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835, and 19001 is confirmed.

[0123] The m / z value is evaluated with an accuracy of 800 ppm, preferably 500 ppm, and when a plurality of peaks exist within this accuracy, a marker peak with a closer m / z value is selected for evaluation.

[0124] 5. Results

[0125] (1) Group 1

[0126] When the mass spectrum obtained from the sample containing any of the two species of Salmonella bacteria (S. Abony and S. Pakistan) belonging to Group 1 was confirmed for the peak detection status of the mass-to-charge ratio (m / z value), the detection status of Tables 1 and 2 was confirmed. In Tables 1 and 2, "○" indicates that the peak marked with the mass-to-charge ratio was detected, and "×" indicates that it was not detected.

[0127] Figure 3 Among the m / z values ​​of the peaks meeting the second criterion (i.e., the m / z values ​​shown in Table 2), the mass spectra near 4165.7, 5487.4, 6238.3, 7858.2, 8329.5, 9911.9, and 15713.9 are shown. Figure 4 Among the m / z values ​​of the peaks that meet the first criterion but do not meet the second criterion, mass spectra near 5506.7, 6261.2, 7588.5, 9669.0, 11847.4, 13444.3, and 15990.2 are shown. Figure 3 and Figure 4 In the figure, the upper part shows the mass spectrum of S. Abony, and the lower part shows the mass spectrum of S. Pakistan. Figure 3 and Figure 4 In the figure, arrows indicate extracted peaks.

[0128] like Figure 3 and Figure 4 As shown in Table 1, the peak extracted according to the first standard and the second standard only appears in either the mass spectrum of S.Abony or the mass spectrum of S.Pakistan, so it is possible to identify which of S.Abony and S.Pakistan the Salmonella contained in the sample is based on the presence or absence of the peak, in other words, the serotype of the Salmonella can be identified. In particular, the peak that meets the second standard has a larger S / N than the peak that only meets the first standard, and there is no adjacent peak, so the serotype can be identified more accurately. It should be noted that any of the peaks of the 42 mass-to-charge ratios (m / z values) shown in Table 1 can be used as a marker peak for identifying the serotype of the Salmonella contained in the sample, but if the peaks of multiple mass-to-charge ratios (m / z values) are used as marker peaks, the serotype of the Salmonella contained in the sample can be identified with better accuracy.

[0129] Thus, by confirming the peak detection status of the mass-to-charge ratio (m / z value) in Table 1 or Table 2, it is possible to identify which serotype of the two Salmonella bacteria (S. Abony and S. Pakistan) belonging to Group 1.

[0130] (2) Group 2

[0131] When the peak detection conditions of the mass-to-charge ratios (m / z values) were confirmed for the three mass spectra obtained from the samples containing any of the three Salmonella species belonging to the second group (S. Minnesota, S. Infantis, and S. Brandenburg), the detection conditions shown in Tables 3 and 4 were confirmed.

[0132] Figure 5 Among the mass-to-charge ratios (m / z values) of the peaks meeting the second criterion (ie, the m / z values ​​shown in Table 4), mass spectra in the vicinity of 7939.5, 13406.3, and 15878.4 / 15894.7 are shown.

[0133] in addition, Figure 6 Among the mass-to-charge ratios (m / z values) of the peaks that meet the first criterion but do not meet the second criterion, mass spectra in the vicinity of 7947.5, 9322.0, and 13366.5 / 13376.0 are shown. Figure 5 and Figure 6 In the figure, the upper part shows the mass spectrum obtained for the sample containing S. Minnesota, the middle part shows the mass spectrum obtained for the sample containing S. Infantis, and the lower part shows the mass spectrum obtained for the sample containing S. Brandenburg. Figure 5 and Figure 6 In the figure, arrows indicate extracted peaks.

[0134] like Figure 5 and Figure 6 As shown, the peak extracted according to the first standard and the second standard exists or does not exist in the mass spectrum obtained by the sample containing any one of S.Minnesota, S.Infantis, and S.Brandenburg, so it is possible to identify which of S.Minnesota, S.Infantis, and S.Brandenburg the Salmonella contained in the sample is according to the presence or absence of the peak, in other words, it is possible to identify the serotype of the Salmonella. In particular, the peak that meets the second standard has a larger S / N than the peak that only meets the first standard, and there is no adjacent peak, so the serotype can be identified more accurately. It should be noted that any of the peaks of the 35 mass-to-charge ratios (m / z values) shown in Table 1 can be used as a marker peak for identifying the serotype of the Salmonella contained in the sample, but if the peaks of multiple mass-to-charge ratios (m / z values) are used as marker peaks, the serotype of the Salmonella contained in the sample can be identified with better accuracy.

[0135] Thus, by confirming the peak detection status of the mass-to-charge ratio (m / z value) in Table 3 or Table 4, it is possible to identify which serotype of the three Salmonella bacteria (S. Minnesota, S. Infantis, S. Brandenburg) belonging to the second group.

[0136] (3) Group 3

[0137] When the mass spectrum obtained from a sample containing any of the two species of Salmonella bacteria belonging to Group 3 (S. Schwarzengrund and S. Montevideo) is checked for the peak detection status of the mass-to-charge ratio (m / z value), it is confirmed that Figure 7 and Figure 8 Detection status.

[0138] Figure 7 Among the m / z values ​​of the peaks meeting the second criterion (ie, the m / z values ​​shown in Table 6), mass spectra near 5096.4, 6830.8, 12276.1, and 15834.5 are shown.

[0139] in addition, Figure 8 Among the m / z values ​​of the peaks that meet the first criterion but do not meet the second criterion, mass spectra near 12261.9, 13074.0, and 19001.0 are shown. Figure 7 and Figure 8 The upper part is the mass spectrum obtained for the sample containing S. Schwarzengrund, and the lower part is the mass spectrum obtained for the sample containing S. Montevideo. Figure 7 and Figure 8 In the figure, arrows indicate extracted peaks.

[0140] like Figure 7 and Figure 8 As shown in the figure, the peak extracted according to the first standard and the second standard only appears in the mass spectrum of the sample containing S. Schwarzengrund and the mass spectrum of the sample containing S. Montevideo, so it is possible to identify which of S. Schwarzengrund and S. Montevideo the Salmonella contained in the sample is based on the presence or absence of the peak, that is, the serotype of the Salmonella can be identified. In particular, the peak that meets the second standard has a larger S / N than the peak that only meets the first standard, and there is no adjacent peak, so the serotype can be identified more accurately. It should be noted that any of the peaks of the 11 mass-to-charge ratio m / z values ​​shown in Table 5 can be used as a marker peak for identifying the serotype of the Salmonella contained in the sample, but if the peaks of multiple mass-to-charge ratio m / z values ​​are used as marker peaks, the serotype of the Salmonella contained in the sample can be identified with better accuracy.

[0141] Thus, by confirming the peak detection status of the mass-to-charge ratio (m / z value) in Table 5 or Table 6, it is possible to identify which serotype of the two Salmonella bacteria (S. Schwarzengrund and S. Montevideo) belonging to Group 3 it is.

[0142] [Way]

[0143] Those skilled in the art will appreciate that the above-described multiple exemplary embodiments are specific examples of the following aspects.

[0144] (Item 1) A method for analyzing microorganisms according to one embodiment, comprising the following steps:

[0145] The identification step includes identifying which of the two serotypes of Salmonella bacteria, namely, Abony and Pakistan, is included in the microorganism contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria,

[0146] The value of the mass-to-charge ratio specified above is selected from the group consisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803, 15990 and any combination thereof.

[0147] (Item 2) The microbial analysis method according to Item 1, wherein:

[0148] The aforementioned specified mass-to-charge ratio value is particularly selected from the group consisting of 3119, 4166, 5487, 6238, 6720, 7858, 8330, 8536, 8687, 9912, 12276, 15714, 15803, 15990 and any combination thereof.

[0149] According to the microbial analysis method described in item 1, when it is known that the serotype of Salmonella bacteria contained in the sample is any one of two serotypes (Abony and Pakistan), it is possible to identify which serotype is Abony or Pakistan. According to the microbial analysis method described in item 2, it is possible to more accurately identify which serotype is Abony or Pakistan.

[0150] (Item 3) Another method of microbial analysis comprises the following steps:

[0151] an identification step of identifying the microorganism contained in the sample as any of the three serotypes of Salmonella, namely, Minnesota, Infantis and Brandenburg, based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the three serotypes of Salmonella, namely, Minnesota, Infantis and Brandenburg;

[0152] The value of the mass-to-charge ratio specified above is selected from the group consisting of 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857, 12209, 13366, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766, 20838 and any combination thereof.

[0153] (Item 4) The microbial analysis method according to Item 3, wherein:

[0154] The aforementioned specified mass-to-charge ratio value is particularly selected from 6483, 7940, 10990, 11808, 11821, 11848, 11857, 12209, 13406, 13445, 15803, 15878, 15895, 17713, and 17735.

[0155] According to the microbial analysis method described in Item 3, when it is known that the serotype of Salmonella bacteria contained in the sample is any one of the three serotypes (Minnesota, Infantis, Brandenburg), it is possible to identify which serotype is Minnesota, Infantis, or Brandenburg. According to the microbial analysis method described in Item 4, it is possible to more accurately identify which serotype is Minnesota, Infantis, or Brandenburg.

[0156] (Item 5) A microbial analysis method according to another embodiment comprises the following steps:

[0157] an identification step of identifying which of the two serotypes of Salmonella bacteria, Schwarzengrund and Montevideo, is included in the microorganism contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria, i.e., Schwarzengrund and Montevideo;

[0158] The aforementioned specified mass-to-charge ratio value is selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835, 19001 and any combination thereof.

[0159] (Item 6) The microbial analysis method according to Item 5, wherein:

[0160] The aforementioned specified mass-to-charge ratio value is particularly selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12276, 15820, 15835 and any combination thereof.

[0161] According to the microbial analysis method described in Item 5, when it is known that the serotype of Salmonella bacteria contained in the sample is any one of the two serotypes (Schwarzengrund and Montevideo), it is possible to identify which serotype is Schwarzengrund or Montevideo. According to the microbial analysis method described in Item 6, it is possible to more accurately identify which serotype is Schwarzengrund or Montevideo.

[0162] (Item 7) The second aspect is a program for causing a computer to execute each step according to any one of Items 1 to 6 above.

Claims

1. A microbial analysis method comprising the following steps: an identification step of identifying which of the two serotypes of Salmonella bacteria, namely, Abony and Pakistan, is included in the microorganisms contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria, The value of the specified mass-to-charge ratio is selected from the group consisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803, 15990 and any combination thereof.

2. The microbial analysis method according to claim 1, wherein: The specified mass-to-charge ratio value is selected from the group consisting of 3119, 4166, 5487, 6238, 6720, 7858, 8330, 8536, 8687, 9912, 12276, 15714, 15803, 15990 and any combination thereof.

3. A microbial analysis method comprising the following steps: an identification step, based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the three serotypes of Salmonella bacteria, namely, Minnesota, Infantis, and Brandenburg, identifying which of the three serotypes the microorganisms contained in the sample include; The value of the specified mass-to-charge ratio is selected from the group consisting of 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857, 12209, 13366, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766, 20838 and any combination thereof.

4. The microbial analysis method according to claim 3, wherein: The specified mass-to-charge ratio value is selected from the group consisting of 6483, 7940, 10990, 11808, 11821, 11848, 11857, 12209, 13406, 13445, 15803, 15878, 15895, 17713, 17735 and any combination thereof.

5. A microbial analysis method comprising the following steps: an identification step of identifying which of the two serotypes of Salmonella bacteria, Schwarzengrund and Montevideo, is included in the microorganism contained in the sample based on the presence or absence of a peak at a predetermined mass-to-charge ratio in a mass spectrum obtained by mass spectrometry analysis of a sample containing any of the two serotypes of Salmonella bacteria, The specified mass-to-charge ratio value is selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835, 19001 and any combination thereof.

6. The microbial analysis method according to claim 5, wherein: The specified mass-to-charge ratio value is selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12276, 15820, 15835 and any combination thereof. 7 . A non-transitory computer-readable medium storing a program for causing a computer to execute the microorganism analysis method according to claim 1 .

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