JP7057973B2 - Microorganism identification device and microorganism identification method - Google Patents

Description

The present invention relates to a microorganism identification device and a microorganism identification method for identifying microorganisms.

MALDI-MS (matrix-assisted laser desorption / ionization mass spectrometry) is used to identify microorganisms. The MALDI-MS-based microbial identification system (hereinafter referred to as the MALDI-MS system) is excellent in speed and low cost, and has rapidly become widespread in clinical practice in recent years. At present, in clinical practice, microbial identification by the MALDI-MS system is limited to species-level identification. On the other hand, in academic studies, it has been reported that microorganisms were identified at the strain level (see, for example, Non-Patent Document 1).

In Non-Patent Document 1, 12 kinds of peaks corresponding to the mass-to-charge ratios of predetermined proteins are extracted from the mass spectra of various Salmonella as identification markers for identifying serotypes or strains. Here, each discriminant marker has a plurality of theoretical mass-to-charge ratios that differ from serotype or strain to strain. Therefore, the serotype or strain of Salmonella is identified based on the combination of 12 mass-to-charge ratios corresponding to each of the 12 extracted identification markers.

Teruyo Ojima-Kato, et al., "Application of proteotyping Strain SolutionTM ver. 2 software and theoretically calculated mass database in MALDI-TOF MS typing of Salmonella serotype," Appl Microbiol Biotechnol, 2017, 101 (23-24), 8557- 8569

When the peak corresponding to one mass-to-charge ratio of each identification marker appears in the mass spectrum and the peak corresponding to the other mass-to-charge ratio of the identification marker does not appear in the mass spectrum, one peak expressed appears. Can be identified with high accuracy by extracting as the identification marker. However, for some reason, a plurality of peaks corresponding to a plurality of mass-to-charge ratios of any of the identification markers may appear in the mass spectrum. In this case, one peak cannot be extracted as an identification marker. Therefore, the accuracy of serotype or strain identification is reduced.

An object of the present invention is to provide a microorganism identification device and a microorganism identification method capable of preventing a decrease in the accuracy of identification of a serotype or a strain.

The present inventors prepared a sample containing a microorganism belonging to more than 100 known serotypes or strains, and identified the serotype or strain of the microorganism using a plurality of identification markers. And, for a specific identification marker, although multiple peaks are expressed in the mass spectrum in many serotypes or strains, the peak that should be theoretically expressed as an identification marker has a higher detection intensity and higher reproduction than the other peaks. We obtained the finding that it is expressed by sex. Based on this finding, the following invention was conceived.

(1) The microorganism identification device according to the first invention is a microorganism identification device for identifying a serotype or strain of a microorganism whose species and genus is known and whose serotype or strain is unknown, and has a plurality of mass-charge ratios. Of the one or more marker proteins, the same as the theoretical information acquisition unit that acquires theoretical information indicating which theoretical value the one or more marker proteins having the theoretical values of are detected for each serotype or strain. The detection intensity of the peak at a specific theoretical value for a serotype or strain is greater than the detection intensity of the peak at other theoretical values. A peak list acquisition unit that acquires a peak list showing the correspondence between the mass charge ratio and the peak detection intensity, and a determination that only peaks with higher intensity are detected for the specified marker protein in the peak list. A unit and an identification unit that identifies the serotype or strain of the microorganism to be identified based on the theoretical information, the peak list, and the result of the determination by the determination unit.

In this microorganism identification device, theoretical information indicating which theoretical value one or more marker proteins having a plurality of theoretical values for mass-to-charge ratio is detected for each serotype or strain is acquired. Among one or more marker proteins, a marker protein in which the detection intensity of a peak at a specific theoretical value for the same serotype or strain is larger than the detection intensity of a peak at another theoretical value is specified. A peak list showing the correspondence between the mass-to-charge ratio of the peak and the detection intensity of the peak in the mass spectrum of the sample containing the microorganism to be identified is acquired.

Here, according to the findings of the present inventors, for the identified marker protein, when a plurality of peaks are detected in the mass spectrum, the peak having a higher intensity may be the peak that should be theoretically expressed. Is extremely high. Therefore, in the peak list, when a plurality of peaks are detected for the specified marker protein, it is determined that only the peak having a higher intensity is detected. The serotype or strain of the microorganism to be identified is identified based on theoretical information, a peak list, and the results of the determination.

According to this configuration, even when an unnecessary peak corresponding to a specific marker protein is expressed, the probability that a strain is misidentified due to the unnecessary peak can be reduced. This can prevent the accuracy of serotype or strain identification from being reduced.

(2) The microorganism identification device includes a prior information acquisition unit that acquires prior information indicating the relationship between the type of serotype or strain and the multiple detection intensities of peaks at each theoretical value of the marker protein for the serotype or strain. Further, the specific unit may specify the marker protein based on the prior information. In this case, it is possible to easily identify a marker protein in which the detection intensity of the peak at a specific theoretical value for the same serotype or strain is larger than the detection intensity of the peak at another theoretical value.

(3) When the microorganism to be identified is Salmonella, the specific portion may specify at least gns marker protein among one or more marker proteins in the theoretical information. In this case, at least the gns marker protein can be easily identified as a marker protein in which the detection intensity of the peak at a specific theoretical value for the same serotype or strain is larger than the detection intensity of the peak at another theoretical value.

(4) The microorganism identification method according to the second invention is a microorganism identification method for identifying a serotype or strain of a microorganism whose species and genus is known and whose serotype or strain is unknown, and has a plurality of mass-charge ratios. Steps to obtain theoretical information indicating which theoretical value one or more marker proteins having the theoretical values of are detected for each serotype or strain, and one or more marker proteins of the same serotype or The steps to identify marker proteins whose peak detection intensity at a particular theoretical value for a strain is greater than the peak detection intensity at other theoretical values, and the mass-charge ratio of the peak in the mass spectrum of a sample containing the microorganism to be identified. A step to acquire a peak list showing the correspondence with the peak detection intensity, a step to determine that only a peak having a higher intensity is detected for the identified marker protein in the peak list, theoretical information, and a peak list. And the step of identifying the serotype or strain of the microorganism to be identified based on the result of the determination.

According to this method, even when an unnecessary peak corresponding to a specific marker protein is expressed, the probability that a strain is misidentified due to the unnecessary peak can be reduced. This can prevent the accuracy of serotype or strain identification from being reduced.

(5) The microbial identification method further comprises the step of acquiring prior information indicating the relationship between the type of serotype or strain and the multiple detection intensities of peaks at each theoretical value of the marker protein for the serotype or strain. The step of identifying the marker protein may include identifying the marker protein based on prior information. In this case, it is possible to easily identify a marker protein in which the detection intensity of the peak at a specific theoretical value for the same serotype or strain is larger than the detection intensity of the peak at another theoretical value.

(6) When the microorganism to be identified is Salmonella, the step of identifying the marker protein may include identifying at least the gns marker protein from one or more of the marker proteins in the theoretical information. In this case, at least the gns marker protein can be easily identified as a marker protein in which the detection intensity of the peak at a specific theoretical value for the same serotype or strain is larger than the detection intensity of the peak at another theoretical value.

According to the present invention, it is possible to prevent a decrease in the accuracy of serotype or strain identification.

It is a figure which shows the structure of the mass spectrometer including the microorganism identification apparatus which concerns on one Embodiment of this invention. It is a figure which shows an example of the identification marker list stored in the storage part. It is a figure which shows an example of the extracted peak. It is a figure which shows an example of the prior information when the protein gns is used as an identification marker. It is a figure which shows the structure of the microorganism identification apparatus. It is a flowchart which shows the algorithm of the microorganism identification processing performed by the microorganism identification program.

(1) Configuration of Mass Spectrometer The following, the microorganism identification apparatus and the microorganism identification method according to the embodiment of the present invention will be described in detail with reference to the drawings. FIG. 1 is a diagram showing a configuration of a mass spectrometer including a microorganism identification device according to an embodiment of the present invention. As shown in FIG. 1, the mass spectrometer 100 includes a processing device 10 and an analysis unit 20. The analysis unit 20 generates a mass spectrum by mass spectrometry of various samples containing microorganisms using MALDI (matrix-assisted laser desorption / ionization method).

The processing device 10 includes a CPU (central processing unit) 11, a RAM (random access memory) 12, a ROM (read-only memory) 13, a storage unit 14, an operation unit 15, a display unit 16, and an input / output I / F (interface). It is composed of 17. The CPU 11, RAM 12, ROM 13, storage unit 14, operation unit 15, display unit 16, and input / output I / F 17 are connected to the bus 18. The CPU 11, RAM 12 and ROM 13 constitute the microorganism identification device 1.

The RAM 12 is used as a working area of the CPU 11. The system program is stored in the ROM 13. The storage unit 14 includes a storage medium such as a hard disk or a semiconductor memory, and stores a microorganism identification program. By executing the microorganism identification program stored in the storage unit 14 on the RAM 12, the CPU 11 performs the microorganism identification process described later. In addition, the storage unit 14 stores an identification marker list for identifying the serotype or strain of the microorganism contained in the sample. The details of the identification marker list will be described later.

The operation unit 15 is an input device such as a keyboard, a mouse, or a touch panel. The display unit 16 is a display device such as a liquid crystal display device. The user can give various instructions to the microorganism identification device 1 by using the operation unit 15. The display unit 16 can display the identification result by the microorganism identification device 1. The input / output I / F 17 is connected to the analysis unit 20.

FIG. 2 is a diagram showing an example of an identification marker list stored in the storage unit 14. As shown in FIG. 2, the identification marker list is a list showing the correspondence between the type of serotype or strain and one or more identification markers for identifying the serotype or strain. The identification marker is a marker protein, and the identification marker list is created by genetic analysis of the marker protein or the like. In the present embodiment, the method for identifying the strain will be described, but the same applies to the method for identifying the serotype.

In the example of FIG. 2, the identification markers Ma and Mb are included as the two types of identification markers. Further, the identification marker Ma has a theoretical mass-to-charge ratio "A1" or "A2" that differs depending on the type of strain. The identification marker Mb has a theoretical mass-to-charge ratio "B1", "B2" or "B3" which varies depending on the type of strain. A theoretical value of "1" or "0" is given to each mass-to-charge ratio for each strain. At the mass-to-charge ratio to which the theoretical value "1" is given, a peak appears in the mass spectrum of the corresponding strain. On the other hand, in the mass-to-charge ratio to which the theoretical value "0" is given, no peak appears in the mass spectrum of the corresponding strain.

Specifically, in "Strain X", peaks appear in the mass-to-charge ratios "A1" and "B1" as identification markers Ma and Mb, respectively, and the mass-to-charge ratios "A2", "B2" and "B3" respectively. Does not show a peak. In "Strain Y", peaks appear in the mass-to-charge ratios "A1" and "B2" as identification markers Ma and Mb, respectively, and no peaks appear in the mass-to-charge ratios "A2", "B1" and "B3". .. In "Strain Z", peaks appear in the mass-to-charge ratios "A2" and "B3" as identification markers Ma and Mb, respectively, and no peaks appear in the mass-to-charge ratios "A1", "B1" and "B2". ..

A method for identifying strains using the identification marker list of FIG. 2 will be described. First, the analysis unit 20 generates a mass spectrum of a sample containing a microorganism derived from the strain to be identified. Next, a peak list is created based on the generated mass spectrum. The peak list is a list showing the correspondence between the mass-to-charge ratio of the peak and the detection intensity of the peak. Subsequently, the peaks included in the tolerance range of the mass-to-charge ratio of the identification markers Ma and Mb are extracted from the created peak list.

FIG. 3 is a diagram showing an example of the extracted peak. In the example of FIG. 3, for "Sample 1", peaks are extracted at the mass-to-charge ratios "A1" and "B2", respectively, and no peaks are extracted at the mass-to-charge ratios "A2", "B1" and "B3". .. For "Sample 2", peaks are extracted at the mass-to-charge ratios "A2" and "B3", respectively, and no peaks are extracted at the mass-to-charge ratios "A1", "B1" and "B2". For "Sample 3", peaks are extracted at the mass-to-charge ratios "A1", "B1" and "B2", respectively, and no peaks are extracted at the mass-to-charge ratios "A2" and "B3".

Strains are identified by comparing the peak in FIG. 3 with the identification marker list in FIG. Specifically, as a result of comparison, the strain of "Sample 1" is identified as "Strain Y", and the strain of "Sample 2" is identified as "Strain Z". However, for "Sample 3", the peak corresponding to the identification marker Mb is extracted in both the mass-to-charge ratios "B1" and "B2". Therefore, it is not possible to distinguish whether the strain of "Sample 3" is "Strain X" or "Strain Y". In this case, the accuracy of stock identification is reduced.

On the other hand, the present inventors say that for a specific identification marker, even when a plurality of peaks are expressed, the peak that should be theoretically expressed is expressed with a higher detection intensity and higher reproducibility than the other peaks. I got the knowledge. Therefore, when a plurality of peaks are detected corresponding to a specific identification marker, the microorganism identification device 1 selects the peaks based on the magnitude of the detection intensity of each peak, thereby expressing unnecessary peaks. Prevents a decrease in the accuracy of stock identification due to.

(2) Prior information The microorganism identification device 1 targets a microorganism whose species and genus are known and whose strain is unknown, but before identifying the strain of the microorganism to be measured, a plurality of known strains are used. Get the generated advance information. The prior information is information including the relationship between the type of strain and the plurality of detection intensities of peaks at each mass-to-charge ratio of the discrimination marker with respect to the strain for one or more identification markers.

FIG. 4 is a diagram showing an example of prior information when the protein gns (N-acetylglucosamine-6-sulfatase) is used as a discrimination marker. The prior information in FIG. 4 is generated by mass spectrometry of a plurality of samples containing Salmonella belonging to "Strain 1" to "Strain 33".

As shown in FIG. 4 (a), the gns identification marker theoretically has a mass-to-charge ratio of "6483.51" and "6511.56". Hereinafter, the mass-to-charge ratios “6483.51” and “6511.56” are referred to as mass-to-charge ratios “M1” and “M2”, respectively. Further, in FIG. 4A, the detection probabilities of the peaks at the mass-to-charge ratios “M1” and “M2” corresponding to “Strain 1” to “Strain 33” are shown.

Specifically, four samples are prepared for each strain, and the detection probability is evaluated based on the number of samples in which the peak at each mass-to-charge ratio is detected among the four samples. When the number of samples in which peaks are detected at each mass-to-charge ratio is 4, 3, 2, 1, and 0, the detection probabilities of peaks at the mass-to-charge ratio are 100%, 75%, and 50, respectively. %, 25% and 0%.

FIGS. 4 (b) and 4 (c) show the intensity of the peaks detected in "Strain 1" and "Strain 18", respectively. In the example of FIG. 4B, peaks were detected at the mass-to-charge ratios “M1” and “M2” in the four samples for “Strain 1”, so that the mass-to-charge ratios “M1” and “M2” were determined. The detection probability of the peak in each is 100%.

In the example of FIG. 4C, since the peak was detected in the mass-to-charge ratio “M1” in the four samples of “Strain 18”, the detection probability of the peak in the mass-to-charge ratio “M1” is 100%. .. On the other hand, a peak was detected in the mass-to-charge ratio "M2" in one sample of "Strain 18", but no peak was detected in the mass-to-charge ratio "M2" in the other three samples. The detection probability of the peak at the mass-to-charge ratio "M2" is 25%.

Further, in FIG. 4A, the frame corresponding to the mass-to-charge ratio of the peak theoretically to be expressed as an identification marker is highlighted by the hatch pattern. For example, for "Strain 1", the peak of the mass-to-charge ratio "M1" is the peak that should be theoretically expressed. Therefore, the frame corresponding to the mass-to-charge ratio “M1” is highlighted by the hatch pattern.

Here, according to the prior information, regarding the identification marker in FIG. 4, even when a peak is detected in both the mass-to-charge ratios “M1” and “M2” with a 100% probability as in “Strain 1”, FIG. As shown in 4 (b), it is shown that the detection intensity of the peak that should theoretically appear is larger than the detection intensity of the other peaks. The same applies to "stock 18" in FIG. 4 (c) and other "stocks 2" to "17" and "stock 19" to "stock 33".

Therefore, the microorganism identification device 1 has a larger detection intensity when peaks are detected in both the mass-to-charge ratios “M1” and “M2” for the identification marker (specifically, the gns identification marker) in FIG. Select a peak and eliminate the other peaks. This makes it possible to reduce the probability of misidentification of strains due to unnecessary peaks.

(3) Microorganism identification process FIG. 5 is a diagram showing a configuration of a microorganism identification device 1. FIG. 6 is a flowchart showing an algorithm of the microorganism identification process performed by the microorganism identification program. As shown in FIG. 5, the microorganism identification device 1 includes a prior information acquisition unit A, a theoretical information acquisition unit B, a specific unit C, a peak list acquisition unit D, a determination unit E, and an identification unit F as functional units.

The functional unit of the microorganism identification device 1 is realized by the CPU 11 of FIG. 1 executing the microorganism identification program stored in the storage device 14. A part or all of the functional part of the microorganism identification device 1 may be realized by hardware such as an electronic circuit. Hereinafter, the microorganism identification process will be described with reference to the microorganism identification device 1 of FIG. 5 and the flowchart of FIG.

The advance information acquisition unit A acquires the advance information of FIG. 4 from the analysis unit 20 (step S1). When the prior information is stored in the storage unit 14 or another storage device, the prior information acquisition unit A may acquire the prior information from the storage unit 14 or another storage device. The theoretical information acquisition unit B acquires an identification marker list from the storage unit 14 (step S2). When the identification marker list is stored in another storage device, the theoretical information acquisition unit B may acquire the identification marker list from the other storage device.

Either of steps S1 and S2 may be executed first, or may be executed at the same time. After steps S1 and S2, the identification unit C identifies the identification marker included in the prior information acquired in step S1 among the one or more identification markers in the identification marker list acquired in step S2 (step S3). ..

The peak list acquisition unit D acquires the mass spectrum of the sample containing the microorganism to be identified generated by the analysis unit 20 (step S4). Further, the peak list acquisition unit D acquires the peak list by detecting the peak from the mass spectrum acquired in step S4 (step S5). Steps S4 and S5 may be executed before any one of steps S1 to S3, or may be executed at the same time.

The determination unit E determines whether or not a peak at both the mass-to-charge ratios “M1” and “M2” is detected for the identification marker identified in step S3 in the peak list acquired in step S5 (step). S6). If the peaks in both the mass-to-charge ratios "M1" and "M2" are not detected and only the peaks in one of the mass-to-charge ratios "M1" and "M2" are detected, the determination unit E proceeds to step S10. When both peaks are detected, the determination unit E determines whether or not the detection intensity of the peak at the mass-to-charge ratio “M1” is larger than the detection intensity of the peak at the mass-to-charge ratio “M2” (step S7). ..

When the detection intensity of the peak at the mass-to-charge ratio “M1” is larger than the detection intensity of the peak at the mass-to-charge ratio “M2”, the determination unit E erases the peak at the mass-to-charge ratio “M2” (step S8). Proceed to S10. When the detection intensity of the peak at the mass-to-charge ratio “M1” is smaller than the detection intensity of the peak at the mass-to-charge ratio “M2”, the determination unit E erases the peak at the mass-to-charge ratio “M1” (step S9). Proceed to S10.

In step S10, the identification unit F identifies the strain of the microorganism to be identified based on the identification marker list acquired in step S2, the peak list acquired in step S5, and the determination results in steps S8 and S9 (step S10). ). Further, the identification unit F causes the display unit 16 to display the identification result (step S11), and ends the microorganism identification process.

(4) Effect In the microorganism identification device 1 according to the present embodiment, identification indicating which mass-to-charge ratio is detected for each strain of one or more identification markers having a theoretical plurality of mass-to-charge ratios. The marker list is acquired by the theoretical information acquisition unit B. Among one or more identification markers, the identification marker in which the detection intensity of the peak at a specific mass-to-charge ratio for the same strain is larger than the detection intensity of the peak at another mass-to-charge ratio is specified by the specific unit C. The peak list in the mass spectrum of the sample containing the microorganism to be identified is acquired by the peak list acquisition unit D.

Here, for the identification marker specified by the specific unit C, when a plurality of peaks are detected in the mass spectrum, it is highly possible that the peak having a higher intensity is the peak that should be theoretically expressed. Therefore, for the identification marker specified by the specific unit C in the peak list, when a plurality of peaks are detected, the determination unit E determines that only the peak having a higher intensity is detected. The strain of the microorganism to be identified is identified by the identification unit F based on the identification marker list, the peak list, and the result of the determination by the determination unit E.

According to this configuration, even when an unnecessary peak corresponding to a specific identification marker appears, the probability of misidentification of the strain due to the unnecessary peak can be reduced. As a result, it is possible to prevent the accuracy of stock identification from being lowered.

(5) Other Embodiments In the above embodiment, the microorganism identification device 1 includes a prior information acquisition unit A, but the present invention is not limited thereto. In the identification of the serotype or strain of Salmonella, when gns is used as an identification marker, prior information may not be acquired, and the microorganism identification device 1 may not include the prior information acquisition unit A.

1 ... Microbial strain identification device, 10 ... Processing device, 11 ... CPU, 12 ... RAM, 13 ... ROM, 14 ... Storage unit, 15 ... Operation unit, 16 ... Display unit, 17 ... Input / output I / F, 18 ... Bus , 20 ... Analytical unit, 100 ... Mass spectrometer, A ... Prior information acquisition unit, B ... Theoretical information acquisition unit, C ... Specific unit, D ... Peak list acquisition unit, E ... Judgment unit, F ... Identification unit, Ma, Mb ... Identification marker

Claims (6)

A microbial identification device that identifies serotypes or strains of microorganisms whose species and genus are known and whose serotype or strain is unknown.
A theoretical information acquisition unit that acquires theoretical information indicating which theoretical value one or more marker proteins having a plurality of theoretical values for mass-to-charge ratio are detected for each serotype or strain, and
Among the above-mentioned one or more marker proteins, a specific portion that identifies a marker protein whose peak detection intensity at a specific theoretical value for the same serotype or strain is larger than that at another theoretical value.
A peak list acquisition unit that acquires a peak list showing the correspondence between the mass-to-charge ratio of the peak and the detection intensity of the peak in the mass spectrum of the sample containing the microorganism to be identified, and the peak list acquisition unit.
In the peak list, for the specified marker protein, a determination unit for determining that only a peak having a higher intensity is detected, and a determination unit.
A microorganism identification device comprising the identification unit for identifying the serotype or strain of the microorganism to be identified based on the theoretical information, the peak list, and the result of determination by the determination unit. Further provided with a prior information acquisition unit for acquiring prior information indicating the relationship between the type of serotype or strain and the multiple detection intensities of peaks at each theoretical value of the marker protein for the serotype or strain.
The microorganism identification device according to claim 1, wherein the specific unit identifies a marker protein based on the prior information. The microorganism identification device according to claim 1 or 2, wherein when the microorganism to be identified is Salmonella, the specific portion identifies at least the gns marker protein among the one or more marker proteins in the theoretical information. A method for identifying a microorganism that identifies a serotype or strain of a microorganism whose species and genus are known and whose serotype or strain is unknown.
Steps to obtain theoretical information indicating at which theoretical value one or more marker proteins having multiple theoretical values for mass-to-charge ratio are detected for each serotype or strain, and
Among the above-mentioned one or more marker proteins, a step of identifying a marker protein whose peak detection intensity at a specific theoretical value for the same serotype or strain is larger than that at another theoretical value.
The step of acquiring a peak list showing the correspondence between the mass-to-charge ratio of the peak and the detection intensity of the peak in the mass spectrum of the sample containing the microorganism to be identified, and
In the peak list, for the identified marker protein, a step of determining that only a peak having a higher intensity is detected, and
A method for identifying a microorganism, comprising the step of identifying the serotype or strain of the microorganism to be identified based on the theoretical information, the peak list, and the result of the determination. It further comprises the step of obtaining prior information indicating the relationship between the type of serotype or strain and the multiple detection intensities of peaks at each theoretical value of marker protein for that serotype or strain.
The microorganism identification method according to claim 4, wherein the step of identifying the marker protein comprises identifying the marker protein based on the prior information. 4. The step of specifying the marker protein, wherein when the microorganism to be identified is Salmonella, at least gns marker protein is specified among the one or more marker proteins in the theoretical information, according to claim 4 or 5. Microbial identification method.

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