JP2021189089A - Method for identifying salmonella - Google Patents

Abstract

To provide a technique of identifying the type, the subspecies, the serotype, or the strain of salmonella easily and rapidly.SOLUTION: The present invention relates to a method for identifying salmonella that uses, as a marker, at least one protein selected from the group of the following proteins: S22; YcaR; L35; BcsR; SsaG; Nucletidyl transferase; YibJ; OadG; ChaB; ZapB; HU-1; YeiS; HU-2; IraP; S15; rpoZ; IHFb; IHFa; YgaM; RaiA; YifE; YeeX; Endolysin; RNaseP; CheY; and S5.SELECTED DRAWING: None

Description

The present invention relates to a method for identifying Salmonella. More specifically, the present invention relates to a method for identifying Salmonella species, subspecies, and serotypes using a specific protein as a marker.

Conventionally, homology analysis based on a DNA base sequence has been widely used as one of the methods for identifying the type of microorganism. In such a method using a DNA base sequence, it takes a relatively long time to extract DNA from a test microorganism and determine a DNA base sequence.

However, when suffering from a variety of disease-causing bacteria, rapid and accurate identification of the bacteria is crucial for the cure of the patient and the prevention of secondary infections. Therefore, there is a need for a rapid and accurate method for analyzing bacteria.

Therefore, in recent years, a method of identifying a microorganism based on a mass spectral pattern obtained by mass spectrometry of a test microorganism has been used. According to mass spectrometry, analysis results can be obtained in a short time using a very small amount of microbial samples, and continuous analysis of a large number of samples is easy, so that microbial identification can be performed easily and quickly. In particular, since the soft ionization method for ionizing biopolymers such as proteins without decomposing them as much as possible has been put into practical use, mass spectrometry has been widely applied to the analysis of microorganisms.

Among the soft ionization methods, mass spectrometry using an ionization method called matrix-assisted laser desorption / ionization mass spectrometry (hereinafter, may be referred to as MALDI-MS) has been attracting attention as a means for analyzing microorganisms in recent years. .. The test microorganism is identified by collating the mass spectrum pattern obtained by MALDI-MS with the mass spectrum pattern of a large number of known microorganisms recorded in advance in the database. Such a method is called a fingerprint method because it uses the mass spectrum pattern as information specific to each microorganism (that is, fingerprint).

In the identification of microorganisms using MALDI-MS, the fingerprint method is known for the analysis up to the species, and it has been put into practical use in some clinical fields. On the other hand, for analysis of subspecies and serotypes, for example, Patent Document 1 reports a method using a ribosome protein or the like as a marker. In the method of Patent Document 1, the detected peak information (m / z value, etc.) of the mass spectrum measured in advance is referred to the measured data of the analysis target, and the peak of the specific m / z value attributed to the marker. Microorganisms are identified by the presence or absence of.

Patent Document 2 describes a method of comparing the data of each group, performing a difference analysis, and searching for a marker for identifying each group in a plurality of groups composed of a plurality of data.
Further, the result of identifying the marker and identifying the microorganism by using the method of Patent Document 1 or 2 is also obtained.

For example, regarding Salmonella enterica subsp. Enterica of the genus Salmonella, a method has been reported in which up to species are identified by a fingerprint method and serotypes are identified using 12 markers (Patent Document 3, Non-Patent Document 3). Patent Document 1).
According to Non-Patent Document 1, by inquiring the detection peak information of the mass spectrum measured in advance such as the m / z value with the actual data by the method of Patent Document 1, it is first determined that the microorganism to be identified belongs to the genus Salmonella. I have specified. Next, the data of a plurality of strains grouped by serotype are compared to identify a marker for identifying each serotype. By such a method, 12 kinds of salmonella serotype identification markers have been identified, and it has been reported that 22 kinds of serotypes can be identified by these markers.
There are also reports of markers for other microorganisms (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, etc.).

JP-A-2015-184020 Japanese Unexamined Patent Publication No. 2018-505063 WO2017 / 168740 Gazette

Applied Microbiology and Biotechnology, 2017, Vol. 101, No. 23-24 PLOS ONE. 2014; 9: e113458. J. Microbiol Methods 2015; 119: 233 PLOS ONE. 2016; 11: e0159730

It is known that even if microorganisms belong to the same genus, they are finely classified into their species, subspecies, serotypes, strains, etc., and their properties are different. For example, in the case of the above-mentioned Salmonella genus, there are two species, enterica and bongori, or three species in which subterranea is added, and there are six subspecies of enterica. Furthermore, there are different serotypes and strains for each subspecies. For example, it is said that there are more than 2000 serotypes in the subspecies enterica.

Furthermore, the serotypes of the genus Salmonella have different biological properties and may or may not be pathogenic to humans, for example. Therefore, it is necessary to identify Salmonella species, variants, serotypes and strains as easily and quickly as possible.

When there are many serotypes and strains, such as Salmonella, it is not easy to analyze all of them. In the analysis of microorganisms that are pathogenic to humans, it is important to reduce the number of actual measurements as much as possible from the viewpoint of the safety of the inspector. In addition, the types of samples that can be purchased are limited. Therefore, there is a limit to the marker search based only on the measured data as in the above method. Therefore, the number of markers reported so far has been limited, and the species, subspecies, and serotypes that can be identified by the markers have also been limited.

In addition, in the case of microorganisms with a huge number of serotypes and strains, there are few actual measurement data that can be used for identification, and the database of actual measurement data has not yet been sufficiently prepared.

Therefore, a method for identifying Salmonella by a quick and simple method based on the specified marker based on as little actual measurement data as possible has been desired.

The present inventors have found that a specific protein can be used as a marker capable of distinguishing a species, subspecies, and serotype of Salmonella, and have completed the present invention.

That is, the present invention
The present invention relates to a method for identifying Salmonella using at least one protein selected from the group consisting of the following proteins as a marker.
S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5

According to the present invention, by using a specific protein as a marker for distinguishing Salmonella, Salmonella can be identified quickly and in a simpler manner.

It is a flowchart which shows the method of specifying the identification marker of Salmonella of this invention. It is a schematic diagram of the database of this invention.

In the present invention, a marker is a characteristic protein used to identify various species, variants, serotypes or strains of Salmonella. The marker does not necessarily have to identify a serotype or a strain, and may be a marker that identifies a species depending on the purpose, or a marker that identifies a subspecies or a serotype. The markers used to identify Salmonella of the present invention are the following 26 proteins.
S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5

The above-mentioned protein is a protein contained in Salmonella, and is a protein commonly contained regardless of species, subspecies, and serotype. Furthermore, even if these proteins are the same protein, if they have different species, subspecies or serum types, when mass spectrometric analysis is performed, a molecule in which a proton is added to the neutral protein M (hereinafter, [M + H] ⁺ ) The theoretical m / z value of the molecular weight-related ion peak based on the amino acid sequence of the protein such as) is different. Therefore, the theoretical m / z value of the molecular weight-related ion peak attributed to the above protein is obtained from the peak of the molecular weight-related ion obtained by mass spectrometry of the salmonella to be identified (hereinafter, may be referred to as the molecular weight-related ion peak). If it is specified in advance which species, subspecies or serotype the protein having the obtained m / z value belongs to, which species, subspecies or serotype these salmonellas to be identified belong to. Prove. At this time, it is desirable to use the calculated mass obtained by translating the base sequence of each protein into an amino acid sequence as the value of the mass-to-charge ratio m / z of the protein. Furthermore, when determining the calculated mass from the amino acid sequence, it is desirable to consider 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 theoretical value is calculated assuming that the N-terminal methionine is cleaved.

The above 26 kinds of marker proteins in the present invention were identified by the following procedure as shown in the flowchart of FIG. The specific method is not limited to this method. For example, real data may be compared to identify these marker proteins.
Step 1: Select a Salmonella strain whose entire genome has been deciphered.
Step 2: Mass spectrometry of the Salmonella strain selected in step 1 above is performed to obtain peaks of molecular weight-related ions such as ^{[M + H] + based on the amino acid sequence of the protein.}
Step 3: The m / z value of each peak is obtained as an actually measured value from the molecular weight-related ion peak obtained in step 2 above (hereinafter, may be referred to as an actually measured m / z value).
Step 4: For the Salmonella strain selected in Step 1, obtain the protein and amino acid sequence of the Salmonella from the gene database, and calculate the theoretical m / z value of the protein from the amino acid sequence information (hereinafter). , Sometimes referred to as the theoretical m / z value).

Step 5: A protein having a theoretical m / z value that matches the measured m / z value by comparing the theoretical m / z value calculated in step 4 with the measured m / z value obtained in step 3 and the protein thereof. The measured m / z value is assigned to the amino acid sequence.
Step 6: Obtain an amino acid sequence similar to the protein assigned in step 5 above from the database.
Step 7: Salmonella is selected from the microorganisms having a similar amino acid sequence obtained in step 6, and the theoretical m / z value of the amino acid sequence of the protein possessed by Salmonella is calculated.
Step 8: The theoretical m / z value of the amino acid sequence calculated in step 7 is compared for each identification classification of Salmonella species, subspecies, serotypes, strains, etc., and the identification classification of species, subspecies, serotypes, strains, etc. Proteins with different theoretical m / z values are identified as markers for the identification of Salmonella.

The strain of salmonella whose entire genome has been decoded selected in step 1 may be selected based on a known database such as a gene database, for example, UniProt (registered trademark aka The Universal Protein Resource), NCBI, or supplier information. .. Once the strain of salmonella whose entire genome has been deciphered is selected, the salmonella is obtained, and mass spectrometry is performed in step 2 to obtain a molecular weight-related ion peak based on the amino acid sequence of the protein. The above-mentioned MALDI-MS is preferable as the mass spectrometry method. In MALDI-MS, a molecular weight-related ion peak based on the amino acid sequence is obtained for each protein, and the measured m / z value is obtained for each peak in step 3.

On the other hand, since the whole genome of the Salmonella strain selected in step 1 has been decoded, the protein present in the Salmonella strain and its amino acid sequence are known and are usually recorded in the gene database. Therefore, in step 4, the amino acid sequences of all the proteins of Salmonella selected in step 1 can be obtained from the database, and the theoretical m / z values of each can be calculated. Examples of the database to be used include UniProt (registered trademark aka The Universal Protein Resource), NCBI and the like.

In step 5, all the molecular weight-related ion peaks obtained in the mass spectrometry in step 2 are known by comparing the measured m / z value obtained in step 3 with the theoretical m / z value calculated in step 4. It can be attributed to a protein and its amino acid sequence.

In the case of microorganisms, the mass of protein may vary depending on the genus, species, subspecies, serotype, or strain. This difference in mass is thought to be due to the mutation of the amino acids that make up the protein. In step 6, in order to search for a variant of the amino acid sequence, an amino acid sequence similar to the assigned protein is searched.

The protein having a similar amino acid sequence in step 6 above can be identified by searching, for example, from a database of existing microorganisms. As a search method, for example, a Simulity search (homology search) using UniProt, NCBI, or the like as a database can be mentioned. When performing a Simulity search, for example, the search is performed under the condition that the sequence similarity is 50% or more. The sequence similarity may be appropriately set according to the purpose of identification and the like.

Since the protein having a similar amino acid sequence selected in step 6 above includes amino acid sequences other than salmonella, only the amino acid sequence of salmonella was selected from among them in step 7 based on the existing database, and the theory m. Find the / z value.

In this way, by mass spectrometry of the Salmonella strain whose entire genome has been deciphered in steps 4 to 7, the protein to which the peak detected is assigned and the theoretical m / z value of its amino acid sequence can be obtained. At the same time, the theoretical m / z value of the salmonella protein having an amino acid sequence similar to that of the salmonella whose entire genome has been deciphered is obtained. In step 8, for example, the theoretical m / z values based on the amino acid sequences of the proteins possessed by different species of the same genus are compared, and if a protein having an m / z value having a difference between species can be identified, the protein is used as the species of Salmonella. It can be specified as a marker for discrimination. Similarly, in the subspecies or serotype, if a protein having a theoretical m / z value different between the subspecies or serotype can be identified, the protein can be specified as a marker for discriminating the subspecies or serotype.

By the above-mentioned method, the following 26 kinds of proteins were identified as markers for Salmonella (hereinafter, may be referred to as marker proteins).
S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5

The method for identifying Salmonella of the present invention is a method for identifying a species, subspecies, and serotype of Salmonella using at least one of the above marker proteins as a marker. By using these markers, it is possible to easily distinguish the species, subspecies, and serotype of Salmonella, and to easily and quickly identify the species, subspecies, and serotype of Salmonella.

Two or more of the marker proteins identified by the above method may be combined and used to identify Salmonella. From the viewpoint of identification accuracy, it is preferable to combine two or more types.

In the method for identifying Salmonella of the present invention, the above-mentioned marker protein may be used in combination with other markers to identify Salmonella. Examples of other markers include markers described in known documents and the like.

Examples of known documents include Non-Patent Document 1.
For example, Non-Patent Document 1 proposes the following 12 proteins as markers for Salmonella. At least one of the following 12 proteins and at least one of the above proteins may be combined and used for identification of Salmonella.
Gns, YaiA, YibT, PPIase, L25, L21, S8,
L17, L15, S7, YciF, SodA
Further, it may be used for identification in combination with other markers.

The above marker protein and, if necessary, other markers are used to identify the Salmonella to be identified. As the identification method, for example, a method of mass spectrometric analysis of Salmonella to be identified can be adopted. In particular, it is preferable to use MALDI-MS which employs a soft ionization method for ionizing a polymer without decomposing it as much as possible.

The salmonella to be identified is mass-analyzed to obtain the molecular weight-related ion peak of the protein. From the obtained molecular weight-related ion peaks, the presence or absence of a peak at the theoretical m / z value attributed to the protein identified as the marker protein is confirmed. Alternatively, it is confirmed at which theoretical m / z value the peak of the marker protein is detected. If the presence of a peak at the theoretical m / z value attributed to the protein identified as the marker protein is confirmed in the Salmonella to be identified, the species, subspecies, serotype, etc. having the protein can be identified. Alternatively, Salmonella species, subspecies or serotypes are identified based on the m / z value of the peak of the marker protein.

As described above, by selecting a marker that can be used to identify Salmonella in advance, mass spectrometry of the Salmonella to be identified can identify the species, subspecies, serotype, etc. to which the Salmonella belongs. can. Further, it is not necessary to analyze all the assumed species, subspecies, and serotypes separately and compare them with the Salmonella to be identified, and it is sufficient to analyze only the Salmonella to be identified.

The selected marker facilitates the discrimination of Salmonella species, subspecies and serotypes, and can easily and quickly identify Salmonella species, subspecies and serotypes. In addition, a marker is specified for each species, subspecies, and serotype, and a database consisting of at least one of the identified markers and its theoretical m / z value, and at least one of various, each subspecies, and each serotype. Can also be built. For example, it is possible to identify a marker that can identify a subspecies and construct a database of each subspecies and the marker. The database may have markers other than marker proteins.

An example of the database format is shown in FIG. FIG. 2 is a schematic diagram of a database showing the theoretical m / z value of the marker protein when the species is known.

By constructing the database as shown in Fig. 2 above, the measured m / z value is obtained from the molecular weight-related ion peak obtained as a result of mass spectrometry of the salmonella to be identified, and the obtained measured m / z value and the database are obtained. The theoretical m / z values can be compared and the corresponding species, subspecies, or serotype can be identified from the matching theoretical m / z values.
From the viewpoint of improving the identification accuracy, other markers may be recorded in the above database in the same format.

By constructing a database as described above, the species, subspecies, and serotype can be immediately identified from the results of mass spectrometry of the Salmonella to be identified.

Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited thereto.

The marker protein of Salmonella was identified by the following procedure, and the results were summarized.

Mass spectrometry was performed on some Salmonellas with MALDI-MS. The device used for MALDI-MS is AXIMA (registered trademark) Performance manufactured by Shimadzu Corporation, and the measurement conditions are as follows.

[Mass spectrometry conditions]
Equipment: AXIMA (registered trademark) Performance manufactured by Shimadzu Corporation
Condition: positive mode. Lin mode. Raster analysis.

[procedure]
1. A strain of Salmonella enterica subsp. Enterica serovar Abaetetuba (hereinafter referred to as S. Abaetetuba) was selected as a whole-genome decoding strain of Salmonella, and cultured on LB agar medium at a temperature of 37 ° C. for 20 hours. Similarly, each of the two serotypes of the genus Salmonella: S. Enteritidis (strains: GTC00131, GTC09491, Hyogose11002, Hyogose12001) and S. Cyphimurium (strains: NBRC14210, NBRC15181, NBRC12529) The cells were cultured at a temperature of 37 ° C. for 20 hours.

2. As a matrix solution, the following sinapinic acid (manufactured by Wako, hereinafter, SA) solution was prepared and used in the following procedure.
SA-1: SA 25 mg / mL ethanol (hereinafter, EtOH) solution SA-2: SA 25 mg / mL methylene diphosphonic acid (methylenediphosphoric acid, manufactured by Sigma-Aldrich, hereinafter MDPNA) 1% by weight, n-decyl- β-D-maltopyranoside (Sigma-Aldrich, DMP) 1 mM, trifluoroacetic acid (Wako, trifluoroacetic acid, TFA) 0.6% by weight, and acetonitrile (Wako, acenotrile, hereinafter, ACN) An aqueous solution consisting of 50% by weight.

3. Weigh about 1 mg of salmonella in step 1 above with a microbalance, add the SA-2 solution prepared in step 2 above, and the concentration of salmonella becomes 1 mg / 0.075 mL (1 x 10 ⁷ pieces / μL). After suspending with a needle, the suspension was subjected to ultrasonic waves for 1 min, and the obtained suspension was centrifuged at 12000 rpm and 5 min.

4. The SA-1 solution prepared in step 2 above was added dropwise to a MALDI plate in an amount of 0.5 μL each and precoated. Then, 1 μL of the supernatant after centrifugation in 3. was added dropwise onto the precoated wells. After air drying, a plate was inserted into MALDI-MS and measured by raster analysis in positive and Lin modes. The n number was set to 4. After the measurement, self-calibration of Salmonella was applied, the obtained mass spectrum was evaluated, and the m / z value of the peak of the detected protein was confirmed.

5. All amino acid sequences and protein names were obtained from the public gene information of S. Abaetuba, which is a whole genome decoding strain. From this amino acid sequence information, the theoretical m / z value based on the amino acid sequence of each protein was calculated.

6. Of the peaks of the mass spectrum obtained in 4. above, the m / z value is in the range of 3000 to 20000 and the peak signal / noise ratio (S / N) is 3 or more with respect to the protein obtained in step 5 above. , The mass accuracy is within 500 ppm, it is detected 3 times or more out of n number 4, and the approximate value of the theoretical m / z value of the protein obtained from step 5 is attributed to a peak in which two or more do not exist for one peak. The protein to be used was selected.

7. For each protein in step 6 above, search for similar amino acid sequence information by a similarity search (sequence similarity of 50% or more) of public gene information, and use the theoretical m / z value in each strain of Salmonella spp. , Subspecies, and serotype information.

8. Compare the theoretical m / z values obtained in step 7 above for each species, subspecies, and serotype of the genus Salmonella, and use markers that show different m / z values for each species, subspecies, and serotype. The following 26 species were identified as.

S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5

9. Theoretical information on species, subspecies, serotypes, and strains when the above 26 marker proteins and the 12 marker proteins reported in Non-Patent Document 1 are used is summarized. In addition, each strain of two serotypes of the genus Salmonella: S. Enteritidis (strains: GTC00131, GTC09491, Hyogose11002, Hyogose12001) and S. Typhurium (strains: NBRC14210, NBRC15181, NBRC15181, NBRC125181, NBRC125181, In order to confirm whether the identified marker candidates are correct, the peak detection status was confirmed from the measured mass spectrum.

[result]
In the mass spectra of S. Enterica (strains: GTC00131, GTC09491, Hyogose11002, Hyogose12001) and S. Typhimurium (strains: NBRC14210, NBRC15181, NBRC12529, NBRC13245), the same as in step 6 above. Table 1 summarizes the peak detection status. The measured data almost reflected the m / z value information of the protein calculated from the genetic information.

The detection rate was calculated as follows.
Each of the above strains was measured 4 times for each serotype, and when the protein was detected 3 times or more within S / N> 3 and mass accuracy of 500 ppm, it was determined to be the strain in which the protein was detected. The number of detected strains was divided by the total number of measured strains to obtain the detection rate. For example, if the number of strains in which the protein is detected is 4 out of 4, the detection rate is 100%, and if 3 out of 4 strains, the detection rate is 75%.

As can be seen from Table 1, the peaks are detected almost according to the theoretical values except for a part. The reason why the detection rate may be low depending on the protein is that the peak of the protein is insensitive. From this result, it was confirmed that it is possible to predict the m / z value of the detection peak from the genetic information. Therefore, it is considered that the marker protein can be predicted based on the genetic information. In addition, the marker protein identified by the above method was considered to be effective in identifying Salmonella.

Examples of the theoretical m / z values of the 26 species, subspecies, and serotypes of the 26 proteins identified as markers above are shown in Tables 2, 4, and 6. In addition, Tables 3, 5 and 7 show the results of summarizing the theoretical m / z values of the 12 marker proteins for salmonella serotyping reported in Non-Patent Document 1.

Table 2 shows 17 representative species (S15, S22, YeiS, YcaR, YgaM, RaiA, IraP, HU2) among the 26 proteins identified as markers above for 2 species of the genus Salmonella (S. bongori, S. enterica). , BcsR, IHFa, CheY, rpoZ, YifE, IHFb, YeeX, HU1, Endolysin) theoretical m / z values are shown. The underlined numbers in the table indicate the theoretical m / z values confirmed only for that species. However, if there is an m / z value within 500 ppm between species, it is not underlined. When there are multiple m / z values, the values other than the representative values are shown as "etc.". In that case, as the representative value, the representative m / z value having a difference between the species and the m / z value that is close to the species are given priority and exemplified.
If a peak is confirmed at the underlined value of m / z, it indicates that the species may be identified only by the peak. Further, it can be seen that even when it is difficult to distinguish with one protein, it may be possible to distinguish by checking the m / z values of a plurality of proteins. At the same time, by combining with the detection status of the m / z value of the known marker protein as shown in Table 3 described later, there is a possibility that identification that is difficult with the known marker protein alone may be possible.

Table 3 shows the theoretical m / z values of 6 representative species out of 12 known marker proteins for serotyping reported in Non-Patent Document 1 for 2 species of Salmonella (S. bongori and S. enterica). Is shown. The underlined numbers indicate the theoretical m / z values confirmed only for that species. However, if there is an m / z value within 500 ppm between species, it is not underlined. When there are multiple m / z values, the values other than the representative values are shown as "etc.". In that case, as the representative value, the representative m / z value having a difference between the species and the m / z value that is close to the species are given priority and exemplified.
The 6 kinds of proteins exemplified are 6 of the 12 markers for serotype discrimination reported in Non-Patent Document 1, and are not known as the markers for discriminating the species exemplified in Table 3.
If a peak is confirmed at the m / z value of the underlined value in Table 3, it indicates that the species may be identified only by the peak. Further, even when it is difficult to distinguish one protein, it can be understood that it may be possible to distinguish by checking the m / z values of a plurality of proteins. In addition, by combining with the detection status of the m / z value of the new marker protein as shown in Table 2 above, there is a possibility that identification that was difficult with the known marker protein alone can be performed.

Table 4 shows the theory m of 6 representative species (ChaB, YeiS, SsaG, IraP, BcsR, Endolysin) out of 26 novel marker proteins for 6 subspecies of Salmonella enterica (houtenae, salamae, indica, dializonae, arizonae, enterica). Shows the / z value. Hyphens in the table indicate that they are not listed in the database. The underlined numbers indicate the theoretical m / z values confirmed only for that variant. However, if there is an m / z value within 500 ppm between species, it is not underlined. When there are multiple m / z values, the values other than the representative values are shown as "etc.". In that case, as the representative value, the representative m / z value having a difference between the species and the m / z value that is close to the species are given priority and exemplified.
If a peak is confirmed at the underlined value of m / z, it indicates that the species may be identified only by the peak. Further, even when it is difficult to distinguish one protein, it can be understood that it may be possible to distinguish by checking the m / z values of a plurality of proteins. At the same time, by combining with the detection status of the m / z value of the known marker protein as shown in Table 5 described later, there is a possibility that identification that was difficult with the known marker protein alone can be performed.

Table 5 shows 4 representative species (4 species) out of 12 known marker proteins for serotyping reported in Non-Patent Document 1 for 6 subspecies of Salmonella enterica (houtenae, salamae, indica, dializonae, arizonae, enterica). The theoretical m / z value of YibT, L15, YaiA, Gns) is shown. Hyphens in the table indicate that they are not listed in the database. The underlined numbers indicate the theoretical m / z values confirmed only for that variant. However, if there is an m / z value within 500 ppm between species, it is not underlined. When there are multiple m / z values, the values other than the representative values are shown as "etc.". In that case, as the representative value, the representative m / z value having a difference between the species and the m / z value that is close to the species are given priority and exemplified.
The four exemplified proteins are four of the 12 markers for serotyping reported in Non-Patent Document 1, and are not known as the markers for discriminating the subspecies exemplified in Table 5.
If a peak is confirmed at the underlined value of m / z, it indicates that the species may be identified only by the peak. Further, even when it is difficult to distinguish one protein, it can be understood that it may be possible to distinguish by checking the m / z values of a plurality of proteins.
In addition, by combining with the detection status of the m / z value of the new marker protein as shown in Table 4 above, there is a possibility that identification that was difficult with the known marker protein alone can be performed.

Table 6 shows 26 novel marker proteins for 14 salmonella enterica subsp. Enterica serotypes (Adelaide, Agama, Agona, Alachua, Albany, Altona, Anatum, Barreilly, Berta, Bovismorbificans, Braenderup, Brancaster, Bredeney, Cerro). The theoretical m / z values of 7 representative species (YjbJ, ChaB, YeiS, SsaG, YgaM, RaiA, Endolysin) are shown. Hyphens in the table indicate no database. It can be seen that there is a possibility of identification by combining the m / z values of a plurality of proteins. At the same time, by combining with the detection status of the m / z value of the known marker protein as shown in Table 7 described later, there is a possibility that identification that was difficult with the known marker protein alone can be performed.

Table 7 shows representatives of novel marker proteins for 14 salmonella enterica subsp. Enterica serotypes (Adelaide, Agama, Agona, Alachua, Albany, Altona, Anatum, Barreilly, Berta, Bovismorbificans, Braenderup, Brancaster, Bredeney, Cerro). The theoretical m / z values of species (SodA, YibT, L15, PPlase, L25, Gns) are shown. Hyphens in the table indicate no database. It can be seen that it may be possible to identify by combining the m / z values of a plurality of proteins. Since SodA is a high-mass protein, it is less sensitive than other proteins, and the peak shape confirmed in the high-mass range is liable to mutate, so the accuracy of the m / z value tends to decrease. Are known.
Strictly speaking, the 6 kinds of proteins exemplified are 6 of the 12 markers for serotype identification of 22 kinds reported in Non-Patent Document 1, and are effective as identification markers for serotypes other than 22 kinds. unknown. In Table 7, serotypes other than 22 species are classified except S. Altona and S. Braenderup.
In addition, by combining with the detection status of the m / z value of the new marker protein as shown in Table 6, there is a possibility that identification that was difficult with the known marker protein alone can be performed.

From the results in Tables 2 to 7, it was found that the 26 novel marker proteins confirmed by this protocol are effective in distinguishing the species, subspecies, and serotypes of Salmonella. At the same time, it was suggested that these marker proteins may enable difficult identification with known marker proteins alone.

[Aspect]
It will be understood by those skilled in the art that the above-mentioned exemplary embodiments are specific examples of the following embodiments.

[1] A method for identifying Salmonella using at least one protein selected from the group consisting of the following proteins as a marker.
S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5

According to the invention of [1] above, by using a specific protein as a marker for distinguishing Salmonella, it is possible to more quickly and easily identify the species, subspecies, and serotype of Salmonella. In addition, by combining with other markers, proteins, Salmonella species, subspecies, and serotypes can be identified more efficiently.

[2] The method for identifying Salmonella according to the above [1], wherein at least one protein selected from the group consisting of the following proteins is used as a marker.
Gns, YaiA, YibT, PPIase, L25, L21, S8, L17,
L15, S7, YciF, SodA

According to the invention of the above [2], by combining the identification marker described in the above [1] with a protein which is another serotype identification marker described in [2], the species of Salmonella, subspecies, can be more effectively used. Species and serotypes can be identified. In addition, identification that is difficult with the other marker proteins described in [2] alone may be possible in combination with [1].

[3] The method for identifying Salmonella according to the above [1] or [2], which analyzes the salmonella to be identified by mass spectrometry.
[4] The method for identifying Salmonella according to the above [3], wherein the mass spectrometry is MALDI-MS.

According to the inventions of [3] and [4] above, an analysis result can be obtained in a short time by using a very small amount of sample, and continuous analysis of a large number of samples is easy.

[5] A database consisting of m / z values of the following proteins and at least one of Salmonella species, subspecies and serotypes.
S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5
[6] The database according to the above [5], which further contains m / z values of the following proteins.
Gns, YaiA, YibT, PPIase, L25, L21, S8, L17,
L15, S7, YciF, SodA
[7] The database according to [5] or [6] above, which comprises Salmonella species, variants and serotypes.

According to the inventions of [5], [6] and [7] above, the species, subspecies and serotype can be immediately identified from the result of mass spectrometry of the Salmonella to be identified.

Claims (7)

A method for identifying Salmonella using at least one protein selected from the group consisting of the following proteins as a marker.
S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5 The method for identifying Salmonella according to claim 1, wherein at least one protein selected from the group consisting of the following proteins is used as a marker.
Gns, YaiA, YibT, PPIase, L25, L21, S8, L17,
L15, S7, YciF, SodA The method for identifying Salmonella according to claim 1 or 2, wherein the salmonella to be identified is mass-analyzed. The method for identifying Salmonella according to claim 3, wherein the mass spectrometry is MALDI-MS. A database consisting of m / z values of the following proteins and at least one of Salmonella species, variants and serotypes.
S22, YcaR, L35, BcsR, SsaG,
Nucletidyl transferase, YibJ, OadG,
ChaB, ZapB, HU-1, YeiS, HU-2, IraP, S15,
rpoZ, IHFb, IHFa, YgaM, RaiA, YifE,
YeX, Endrysin, RNaseP, CheY, S5 The database according to claim 5, further comprising m / z values of the following proteins.
Gns, YaiA, YibT, PPIase, L25, L21, S8, L17,
L15, S7, YciF, SodA The database of claim 5 or 6, comprising Salmonella species, variants and serotypes.

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