JP2019138811A - Microorganism analysis method - Google Patents

Abstract

To heighten sensitivity in a high mass region when a microorganism to be analyzed that includes Salmonella is analyzed using an MALDI-MS.SOLUTION: The present invention is a method for analyzing a microorganism using a matrix-assisted laser desorption-ionization mass spectrometer characterized in that Salmonella raised in a culture medium containing ciprofloxacin and a matrix material are mixed and a matrix-microorganism mixed solution is prepared, the matrix-microorganism mixed solution is dried and a matrix-microorganism mixed crystal is formed, and the matrix-microorganism mixed crystal is used in matrix-assisted laser desorption-ionization.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for analyzing microorganisms using a matrix-assisted laser desorption / ionization mass spectrometer (MALDI-MS).

  Matrix Assisted Laser Desorption / Ionization (MALDI), which is one of the ionization methods in mass spectrometry, analyzes materials that are difficult to absorb laser light and materials that are easily damaged by laser light, such as proteins. Therefore, this is a technique in which a matrix substance that is easily absorbed by laser light and is easily ionized is mixed with an analysis target substance, and this is irradiated with laser light to ionize the analysis target substance. In general, the matrix substance is mixed with the substance to be analyzed as a solution, and the matrix solution takes in the substance to be analyzed. And by making it dry, the solvent in a solution vaporizes and the crystal | crystallization containing the to-be-analyzed substance is formed. When this is irradiated with laser light, the matrix material absorbs the energy of the laser light and is rapidly heated and vaporized. At that time, the analysis target substance is vaporized together with the matrix substance, and the analysis target substance is ionized in the process.

  A mass spectrometer (MALDI-MS) using such a MALDI method can analyze a polymer compound such as a protein without causing significant dissociation, and is also suitable for trace analysis. Widely used in the field. One use of MALDI-MS in the life science field is identification of microorganisms using MALDI-MS. This is a method for identifying microorganisms based on a mass spectrum pattern obtained using a test microorganism. Since analysis results can be obtained in a short time, it is possible to easily and quickly identify microorganisms. .

  Salmonella belongs to the Enterobacteriaceae family of Gram-negative facultative anaerobes and is known as one of the leading causative bacteria for food poisoning. The Salmonella genus includes Salmonella (sometimes abbreviated as “S.”) enterica, S. bongori and S. subterranea, and S. enterica is further classified into six subspecies. Many pathogenic Salmonella that cause food poisoning belong to S. enterica, and its variants are classified into a number of serotypes. Discrimination of Salmonella species, subspecies, and serotypes is important for elucidating the route of infection of food poisoning and preventing infection. In recent years, attempts have been made to identify Salmonella species using MALDI-MS. .

  By the way, when a sample containing microorganisms is analyzed by MALDI-MS, the sensitivity in a high mass region (or high mass to charge ratio region) having a mass to charge ratio of m / z 15000 or more is low, and the reliability and accuracy of the mass spectrum are low. It is known. Many of the marker proteins that are effective for distinguishing Salmonella are in the high-mass region with a mass-to-charge ratio of m / z 10000 or more. Therefore, the current analysis technology for microorganisms using MALDI-MS accurately identifies Salmonella. Can not do.

  On the other hand, research and development for improving the sensitivity of MALDI-MS has been made by various approaches from the past, and one of them is to mix the matrix solution and the substance to be analyzed to mix the matrix / object mixture solution. There is a method in which another substance is added as a matrix additive in the preparation.

  For example, Non-Patent Documents 1 and 2 describe that by using a surfactant as a matrix additive, the sensitivity when a sample containing microorganisms is analyzed by MALDI-MS can be improved. In the methods described in these documents, a solution containing a test microorganism (test microorganism solution) is applied to a sample plate, a surfactant is dropped on the sample plate, and a matrix solution is further dropped on the sample plate. Prepare a sample. The surfactant is used to increase the solubility of the test microorganism solution in the matrix solution.

  For example, in Patent Document 1 and Non-Patent Document 3, by adding methylene diphosphonic acid (MDPNA) as a matrix additive to 2,5-dihydroxybenzoic acid (DHB) which is a matrix substance, It is described that a certain phosphorylated peptide can be detected with high sensitivity. In the method described in the above document, a solution containing a phosphorylated peptide, a matrix additive solution, and a matrix solution are dropped onto a sample plate in this order, and a sample is prepared by drying them, and then the sample is MALDI. -Analyze using MS.

  Furthermore, Non-Patent Document 4 uses MDPNA, which is a matrix additive, together with DHB, which is a matrix substance, in peptide analysis using LC-MALDI-MS, which is a combination of liquid chromatograph (LC) and MALDI-MS. It is described to do. According to this document, by using MDPNA, the signal suppression effect of the peptide peak is reduced by the desalting effect, so that the peptide-derived peak can be detected with high sensitivity.

JP 2009-121857 JP

J. Am. Soc. Mass. Spectrom., 2005, Vol.16, pp.1422-1426 Rapid Commun. Mass Spectrom., 1999, Vol.13, pp.1067-1071 Rapid Commun. Mass Spectrom., 2008, Vol.22, pp.1109-1116 Mass Spectrometry, 2014, A0031 Journal of Applied Microbiology, 2012, 113, 192-199 Micobiology, 2013, 159, 2049-2057 Antimicrobial Agents and Chemotherapy, 2006, 50, 949-954 J. Bacteriol. March 2005 vol. 187 no. 5 1763-1772 Applied and Environmental Microbiology, 2014, 80, 2928-2940 J. Biomed. Biotechnol., 2010, 123460

  Although all of the conventional methods described above have improved the detection sensitivity of the analyte, the sensitivity in the high mass region with a mass-to-charge ratio of m / z 10000 or more is still low, and the species and subtype of Salmonella spp. It was insufficient as a technique for accurately identifying species and serotypes.

  The problem to be solved by the present invention is to increase the sensitivity of a high mass region when a test microorganism sample containing Salmonella is analyzed using MALDI-MS, and in particular, the sensitivity is improved over the conventional method. That is the purpose.

The present invention made to solve the above problems is a method for analyzing microorganisms using a matrix-assisted laser desorption / ionization mass spectrometer,
Mixing Salmonella spp., Grown in a medium containing ciprofloxacin, with a matrix substance to produce a matrix / microbe mixed solution,
The matrix / microbe mixed solution is dried to form a matrix / microbe mixed crystal,
The matrix / microorganism mixed crystal is used in matrix-assisted laser desorption / ionization.

  Ciprofloxacin (CP) is known as an antibiotic and is active against both gram-negative and gram-positive bacteria. CP inhibits bacterial cell division by inhibiting DNA replication, thereby exhibiting antibacterial action. For this reason, CP is used to cope with infection with mycoplasma in cell culture. On the other hand, it has been reported that CP induces the gene expression of bacterial SodA (Superoxide dismutase). For example, Non-Patent Document 5 shows that CP induces gene expression of SodA in E. coli. Non-Patent Document 6 shows that the BaeSR control mechanism (Non-patent Document 8) important for the control of gene expression involved in drug transporters is involved in the reaction of Salmonella SodA to CP. ing. Furthermore, in Non-Patent Document 7, the mechanism by which reactive oxygen in bacteria cells increases when reacted with CP has been elucidated using E. coli. Furthermore, in Non-Patent Document 9, Shiga toxin 2 (Stx2) is excessively expressed by culturing Shiga toxin-producing Escherichia coli (STEC) strain on an agar medium supplemented with CP, and the STEC strain may proliferate. It is shown.

  As a result of the present inventor's extensive research with reference to the findings of Non-Patent Documents 5 to 7 and 9 described above, the present invention has been achieved, and Salmonella spp. Grown on a medium containing CP are tested microorganisms. As a result, a matrix / microorganism mixed crystal containing a large amount of SodA, which is one of the marker proteins of the genus Salmonella, can be obtained. And the sensitivity of a high mass area | region where the peak derived from the marker protein of Salmonella genus bacteria is contained can be improved by analyzing such a matrix and microorganism mixed crystal using MALDI-MS.

In Non-Patent Document 9 described above, evaluation of culturing a STEC strain using a CP-added medium is performed using the results of analysis by MALDI-MS, but MALDI-MS described in the same document is performed. The method for preparing the sample differs between the analysis method using the method and the present invention. That is, in Non-Patent Document 9, 0.5 μL of a sample suspension is dropped on a sample plate and dried, and then 0.5 μL of a matrix solution is dropped and further dried, followed by MALDI-MS measurement. ing. The matrix solution is an aqueous solution containing 33% acetonitrile (ACN) and 0.2% trifluoroacetic acid (TFA) (Non-Patent Document 10), and sinapinic acid (SA), which is a matrix substance, at a concentration of 10 mg / mL. What was melt | dissolved so that it may become is used. Moreover, it is shown that the suitable concentration range of CP contained in an agar medium is 10-60 ng / mL.
However, in Non-Patent Document 9, MS / MS measurement using MALDI-TOF / TOF is performed, and peptides in the area of mass / charge ratio m / z 10000 or less, such as m / z 7821, m / z 5291, etc. Shiga toxin 2 (Stx2) has been identified using a marker. That is, the method described in Non-Patent Document 9 and the present invention have different types of microorganisms (bacterial species) to be analyzed and mass-to-charge ratio regions to be analyzed using MALDI-MS. The sample preparation method is also different.

In addition, CP induces Salmonella SodA gene expression, while it exhibits antibacterial activity against Salmonella spp., So the amount of CP added to the medium has the effect of inducing SodA gene expression and antibacterial activity. It is important to make comparative decisions.
The present inventor changed the amount of CP added to the medium stepwise in the range of 1 to 1000 ng / mL, and tested six strains for the test of Salmonella (Typhimurium, NBRC 13245, Enteritidis, NBRC 3313, Thompson, ATCC). BAA-1738, Choleraesuis, NBRC 105684, Infantis, jfrlSe 1402-4, Orion, jfrlSe 1402-15) were subjected to culture experiments, and growth inhibition was observed at CP concentrations of 100 ng / mL or higher. Moreover, it was confirmed that some serotypes (Choleraesuis, Thompson, etc.) out of 6 Salmonella test strains inhibit growth even when the CP concentration in the medium is 10 ng / mL.

Therefore, in the present invention, the Salmonella spp. Preferably grows in a medium containing 1 ng / mL to 10 ng / mL CP.
If it is a culture medium containing CP of the said density | concentration, at least 6 typical strains of Salmonella genus bacteria can be grown without a problem.

  In this case, a matrix / additive mixed solution obtained by mixing Salmonella spp. Grown on a medium containing ciprofloxacin with an additive that is at least one of alkylphosphonic acid and a surfactant and the matrix substance is mixed. When the matrix / microbe mixed solution is prepared, the sensitivity of MALDI-MS is further improved.

That is, alkylphosphonic acid can suppress the formation of alkali metal addition ions ([M + Na] ⁺ , [M + K] ^+, etc.) when a sample containing a protein is analyzed using MALDI-MS. Therefore, it can be expected that the S / N ratio of the peak (marker peak) derived from the marker protein of Salmonella is improved. Examples of the alkylphosphonic acid include one phosphonic acid group, phosphonic acid, methylphosphonic acid, phenylphosphonic acid, 1-naphthylmethylphosphonic acid, and the like. As compounds containing two or more phosphonic acid groups, methylenediphosphonic acid, ethylene diphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid (Ethane-1-hydroxy-) 1,1-diphosphonic acid), nitrilotriphosphonic acid, ethylenediaminotetraphosphonic acid, and the like. Preferred embodiments of the present invention include methylenediphosphonic acid (MDPNA) as alkylphosphonic acid. Can be used.

  The surfactant is mainly used to improve the solubility of the constituent components of the microorganism. Therefore, since the amount of marker protein contained in the matrix / microorganism mixed crystal can be increased, it can be expected that the detection intensity of the marker peak is improved. Examples of the surfactant include surfactants used for extraction of proteins in cells such as membrane proteins, and it is particularly preferable to use surfactants that can extract proteins without denaturing them. Examples of such surfactants include decyl-β-D-maltopyranoside (DMP), N-decyl-β-D-maltopyranoside (N—), which are nonionic surfactants. decyl-β-D-maltopyranoside), N-octyl-β-D-gractopyranoside (N-octyl-β-D-glactopyranoside), N-dodecyl-β-D-maltoside (N-dodecyl-β- D-maltoside), N-octyl-β-D-glucopyranoside, and the like, zwitterionic surfactants, N, N-dimethyldodecylamine, which are amphoteric surfactants -N-oxide (N, N-dimethyldodecylamine-N-oxide), zwittergent 3-12, etc. are mentioned, As a preferable form of the present invention, DMP can be used as a surfactant.

  In addition, the matrix substance that constitutes the matrix / microbe mixed solution is appropriately selected according to the type of microorganism to be analyzed and the identification level of the microorganism (family, genus, species, subspecies, pathogenic type, serotype, strain, etc.). A substance can be selected. As such matrix substances, sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), ferulic acid (FA) In a preferred embodiment of the present invention, SA can be used as the matrix material. In general, SA is known to be effective for analysis of high-mass molecules such as proteins in MALDI analysis.

  Further, the matrix / additive mixture solution is prepared by adding 25 mg / mL of sinapinic acid as a matrix substance and methylene diphosphonic acid as an alkylphosphonic acid to a solution containing 50% acetonitrile and 0.6% trifluoroacetic acid. It can be prepared by mixing so as to contain 1% of decyl-β-D-maltopyranoside which is 1% of a surfactant.

  Furthermore, the matrix / microbe mixed solution may be a matrix / microbe suspension obtained by suspending Salmonella colonies grown in a medium containing the ciprofloxacin in the matrix / additive mixed solution. .

  In this case, 0.5 μL of an ethanol solution containing 25 mg / mL of sinapinic acid as a matrix substance or a saturated ethanolic solution of sinapinic acid was dropped on the sample plate and dried, and then, It is preferable that 1.2 μL of the matrix microorganism suspension is dropped and dried to form the matrix / microorganism mixed crystal.

  Further, the microorganism analysis method of the present invention uses a mass spectrum pattern of a mass region including a high mass region having a mass-to-charge ratio of m / z 10,000 or more among mass spectra obtained for Salmonella spp. It is preferable to identify the Salmonella genus based on the result of comparison with the stored mass spectrum pattern.

  According to the microorganism analysis method of the present invention, the sensitivity in the high mass region when Salmonella is analyzed by MALDI-MS is improved.

The figure which shows the mass spectrum of the area | region containing the peak derived from the marker protein SodA obtained in Example 1 which used Salmonella enterica (serotype Orion, jfrlSe 1402-15) as a test microorganism. The figure which shows the mass spectrum of the area | region containing the peak derived from marker protein S7 obtained in Example 1 which used Salmonella enterica (serotype Infantis, jfrlSe 1402-4) as a test microorganism. The figure which shows the mass spectrum of the area | region containing the peak derived from the marker protein L17 obtained in Example 1 which used Salmonella enterica (serotype Infantis, jfrlSe 1402-4) as a test microorganism. The figure which shows the mass spectrum of the area | region containing the peak derived from marker protein SodA obtained in Example 2 which used Salmonella enterica (serotype Orion, jfrlSe 1402-15) as a test microorganism. The figure which shows the mass spectrum of the area | region containing the peak derived from the marker protein SodA obtained in Example 2 using Salmonella enterica (serotype Enteritidis, NBRC 3313) as a test microorganism.

  In the microorganism analysis method according to the present invention, a matrix / microbe mixed solution is prepared by mixing Salmonella spp. Grown on a medium containing CP and a matrix substance, and the matrix / microbe mixed solution is dried to mix the matrix / microorganism. A mass spectrum is obtained by forming a crystal and setting the matrix / microorganism mixed crystal in MALDI-MS to perform mass spectrometry. Then, by comparing the obtained mass spectrum with the mass spectrum pattern stored in the database, the type (species, subspecies, pathogenic type, serotype, strain, etc.) of the test microorganism that is a genus Salmonella is identified. can do. As will be described in detail later, in the microorganism analysis method according to the present invention, it is possible to obtain the molecular weight information of microorganisms with high sensitivity, particularly in a high mass region having a mass-to-charge ratio of m / z 10,000 or more, among the obtained mass spectra. Therefore, the marker peak existing in the high mass region can be detected with high sensitivity.

MALDI-MS includes a MALDI time-of-flight mass spectrometer (MALDI-TOF
MS) is preferably used. Since MALDI-TOFMS has a very wide range of mass-to-charge ratios that can be measured, a mass spectrum suitable for analysis of high-mass molecules such as proteins that are constituents of microorganisms can be obtained.

  Hereinafter, although the microorganisms analysis method concerning the present invention is explained by Examples 1-12, these are only illustrations and the present invention is not limited to these.

<1. Culture of Salmonella spp.>
(1) An ethanol solution containing 10 mg / mL of ciprofloxacin (CP, Sigma-Aldrich Japan GK) in ethanol is prepared, and this ethanol solution is diluted 1000 times with water (dilution ratio: 1/1000). ), And a solution (CP addition solution) containing 10 μg / mL of CP. Moreover, a solution (CP non-added solution) in which ethanol was diluted 1000 times with water (dilution ratio: 1/1000) was prepared.
(2) A medium (CP-added medium) prepared by adding 10 μL of CP-added solution to 100 mL of LB agar medium, and a medium (CP-free medium) prepared by adding 10 μL of CP-free solution to 100 mL of LB agar medium. Each medium was prepared, and each medium was aseptically seeded in a plurality of petri dishes to prepare agar medium for growing Salmonella. The CP concentration of this medium is about 1 ng / mL.
(3) Apply one of the two Salmonella species (serotype Orion, jfrlSe 1402-15, and serotype Infantis, jfrlSe 1402-4) to each petri dish of (2) at 20 ° C for 20 hours. Cultured.

<2. Preparation of matrix solution>
The following two types of matrix solutions were prepared.
(1) A matrix solution (saturated solution) was prepared by dissolving 25 mg / mL of sinapinic acid (SA, Wako Pure Chemical Industries, Ltd.), which is a matrix substance, in ethanol. This matrix solution is designated as “SA-1.”
(2) A matrix solution was prepared by dissolving 25 mg / mL of SA in an aqueous solution containing 50% acetonitrile (ACN) and 0.6% trifluoroacetic acid (TFA). This matrix solution is designated as “SA-2”.

<3. Preparation of matrix / microbe suspension>
10 μL of the matrix solution SA-2 was placed in a tube, and 2 to 3 Salmonella colonies cultured in a petri dish were suspended therein to prepare a matrix / sample suspension.

<4. Analysis by MALDI-MS>
(1) 0.5 μL of the matrix solution SA-1 was dropped on a MALDI sample plate and dried.
(2) Subsequently, 1.2 μL of the matrix / sample suspension was dropped onto the sample plate and dried.
(3) The matrix / microorganism mixed crystal obtained in (2) was set in MALDI-MS and analyzed.

  As MALDI-MS, a MALDI-time-of-flight mass spectrometer (MALDI-TOFMS, manufactured by Shimadzu Corporation, trade name: AXIMA-Performance) was used, and measurement was performed in a linear mode (positive ion mode). All data were obtained by raster analysis. Raster analysis is an automatic measurement function provided in the above-described mass spectrometer, and is a method for obtaining mass spectrum data by irradiating a sample in each well of a sample plate with laser at a preset number of points and shots It is.

<5. Result>
1 to 3 show mass spectra obtained in Example 1. FIG.
FIG. 1 shows a mass spectrum of a region including a peak derived from SodA obtained for a matrix / microorganism mixed crystal containing Salmonella serotype Orion, jfrlSe 1402-15. 1A is a mass spectrum of Salmonella cultured in a CP-added medium, and FIG. 1B is a mass spectrum of Salmonella cultured in a CP-free medium. When focusing on the SodA-derived peak (the peak indicated by the black arrow), the intensity of the SodA-derived peak of Salmonella cultured in CP-added medium (0.6 mV) is the intensity of the SodA-derived peak of Salmonella cultured in CP-free medium (0). .2 mV) and there was little background noise in the mass spectrum. The S / N ratio (= 15) of the SodA-derived peak of Salmonella cultured in the CP-added medium is about 3 times the S / N ratio (= 4) of the SodA-derived peak of Salmonella cultured in the CP-free medium. there were. That is, the detection sensitivity of the SodA-derived peak was improved by adding CP to the culture medium.

FIG. 2 shows a mass spectrum of a region including a peak derived from ribosomal protein S7 obtained for a matrix / microorganism mixed crystal containing Salmonella serotype Infantis, jfrlSe 1402-4. 1A is a mass spectrum when cultured in a CP-added medium, and FIG. 1B is a mass spectrum when cultured in a CP-free medium. Ribosomal protein S7 is known as a marker protein for distinguishing Salmonella serotypes.
Paying attention to the S7-derived peak, the intensity (5.2 mV) of the S7-derived peak of the Salmonella cultured in the CP-added medium is larger than the intensity (0.8 mV) of the S7-derived peak of the Salmonella cultured in the CP-free medium, and There was little background noise in the mass spectrum. Moreover, the S / N ratio (100) of the S7-derived peak of Salmonella cultured in the CP-added medium was about 7 times the S / N ratio (15) of the S7-derived peak of Salmonella cultured in the CP-free medium. . That is, the detection sensitivity of the S7-derived peak was improved by adding CP to the culture medium.

FIG. 3 shows a mass spectrum of a region including a peak derived from the ribosomal protein L17, obtained for a matrix / microorganism mixed crystal containing Salmonella serotype Infantis, jfrlSe 1402-4. 1A is a mass spectrum when cultured in a CP-added medium, and FIG. 1B is a mass spectrum when cultured in a CP-free medium. The ribosomal protein L17 is known as a marker protein for distinguishing Salmonella serotypes.
Paying attention to the L17-derived peak, the intensity (7.4 mV) of the L17-derived peak of Salmonella cultured in the CP-added medium is greater than the intensity (0.6 mV) of the L17-derived peak of Salmonella cultured in the CP-free medium, and There was little background noise in the mass spectrum. Moreover, the S / N ratio (100) of the L17-derived peak of Salmonella cultured in the CP-added medium was about 10 times the S / N ratio (10) of the L17-derived peak of Salmonella cultured in the CP-free medium. . That is, the detection sensitivity of the L17-derived peak was improved by adding CP to the culture medium.

  As can be seen from FIGS. 1 to 3, the peaks (marker peaks) derived from SodA, S7, and L17, which are marker proteins for distinguishing Salmonella serotypes, all have a high mass with a mass-to-charge ratio of m / z 10,000 or more. Present in the area. Therefore, it was confirmed that the addition of CP to the Salmonella culture medium contributes to an improvement in sensitivity in a high mass region having a mass-to-charge ratio of MALDI-MS with Salmonella as a test microorganism of m / z 10,000 or more.

<1. Culture of Salmonella spp.>
(1) A CP-added solution and a non-CP-added solution were prepared in the same manner as in Example 1.
(2) A CP-added medium and a non-CP-added medium were prepared in the same manner as in Example 1, and appropriate amounts of each of these media were seeded in a plurality of dishes.
(3) Two types of Salmonella (serotype Orion, jfrlSe 1402-15, and serotype Enteritidis, NBRC 3313) were applied to each petri dish of (2) and cultured at 37 ° C. for 20 hours.

<2. Preparation of matrix solution>
The following three types of matrix solutions were prepared.
(1) A matrix solution SA-1 having the same composition as in Example 1 was prepared.
(2) A matrix solution SA-2 having the same composition as in Example 1 was prepared.
(3) In an aqueous solution containing 50% acetonitrile (ACN) and 0.6% trifluoroacetic acid (TFA), 25 mg / mL SA, 1% methylenediphosphonic acid (MDPNA), decyl, a surfactant A matrix solution was prepared by dissolving -β-D-maltopyranoside (decyl-β-D-maltopyranoside, DMP) so as to contain 1 mM each. This matrix solution is designated as “SA-3”.

<3. Preparation of matrix / microbe suspension>
10 μL of the matrix solution SA-2 or the matrix solution SA-3 was placed in a tube, and 2 to 3 Salmonella colonies cultured in a petri dish were suspended therein to prepare a matrix / sample suspension.
<4. Analysis by MALDI-MS>
(1) 0.5 μL of the matrix solution SA-1 was dropped on a MALDI sample plate and dried.
(2) Subsequently, 1.2 μL of the matrix / sample suspension was dropped onto the sample plate and dried.
(3) The matrix / microorganism mixed crystal obtained in (2) was set in MALDI-MS and analyzed.

  The MALDI-MS and measurement mode were the same as in Example 1, and data was collected by raster analysis.

<5. Result>
4 and 5 show the mass spectrum obtained in Example 2. FIG.
FIG. 4 shows a mass spectrum of a region including a peak derived from SodA obtained for a matrix / microorganism mixed crystal containing Salmonella serotype Orion, jfrlSe 1402-15. 4A shows a matrix / microorganism suspension obtained by suspending a Salmonella colony cultured in a CP-added medium in a matrix solution SA-3, and FIG. 4B shows a Salmonella cultured in a CP-added medium in a matrix solution SA. -C suspension of matrix and microorganisms suspended in -2, (C) is a matrix / microorganism suspension obtained by suspending Salmonella cultured in CP-free medium in matrix solution SA-3, and (D) is CP-free It is the mass spectrum obtained by analyzing the matrix and microbial suspension which suspended Salmonella cultured by the culture medium in matrix solution SA-2, and the matrix and microorganism mixed crystal produced using each, by MALDI-MS.

As shown in FIG. 4, the combination of the CP non-added medium and the matrix solution SA-3 (C) has a peak derived from SodA as compared to the combination of the CP non-added medium and the matrix solution SA-2 (D). The / N ratio improved about twice.
In addition, the combination of CP-added medium and matrix solution SA-2 (B) has an S / N ratio of about 3 peaks compared to the combination of CP non-added medium and matrix solution SA-2 (D). Doubled.
Further, the combination (A) of the CP-added medium and the matrix solution SA-3 has a peak of SodA derived from the S / N ratio of about 10 in comparison with the combination (D) of the CP-free medium and the matrix solution SA-2. Doubled.

  FIG. 5 shows a mass spectrum of a region including a peak derived from SodA obtained for a matrix / microorganism mixed crystal containing Salmonella serotype Enteritidis, NBRC 3313. 5A shows a matrix / microorganism suspension obtained by suspending a Salmonella colony cultured in a CP-added medium in a matrix solution SA-3, and FIG. 5B shows a Salmonella cultured in a CP-added medium in a matrix solution SA. -C suspension of matrix and microorganisms suspended in -2, (C) is a matrix / microorganism suspension obtained by suspending Salmonella cultured in CP-free medium in matrix solution SA-3, and (D) is CP-free It is the mass spectrum obtained by analyzing the matrix and microbial suspension which suspended Salmonella cultured by the culture medium in matrix solution SA-2, and the matrix and microorganism mixed crystal produced using each, by MALDI-MS.

As shown in FIG. 5, the combination of CP-free medium and matrix solution SA-3 (C) has a peak derived from SodA compared to the combination of CP-free medium and matrix solution SA-2 (D). The / N ratio was improved about 1.5 times.
In addition, the combination of CP-added medium and matrix solution SA-2 (B) has an S / N ratio of about 3 peaks compared to the combination of CP non-added medium and matrix solution SA-2 (D). Doubled.
Furthermore, the combination of CP-added medium and matrix solution SA-3 (A) has a peak of SodA in an S / N ratio of about 6 compared to the combination (D) of CP-free medium and matrix solution SA-2. Doubled.

  From the above results, the matrix-microbe mixed crystals obtained from the matrix-microbe suspension prepared using the matrix solution containing MDPNA and DMP for two types of Salmonella spp. Was analyzed by MALDI-MS, and it was confirmed that the sensitivity of a high mass region including a peak derived from SodA and having a mass to charge ratio of m / z 10,000 or more was improved. Moreover, the sensitivity improvement effect by adding CP to the culture medium and adding MDPNA and DMP to the matrix solution was synergistic.

In Examples 1 and 2, the CP concentration of the CP-added medium was set to about 1 ng / mL. However, as long as the CP concentration is in the range of 1 ng / mL to 10 ng / mL, the same as in Examples 1 and 2 above. Result is obtained.
Moreover, in the said Example 2, although matrix solution SA-3 contains both MDPNA and DMP, you may include only one of MDPNA and DMP in a matrix solution.

Claims (10)

A method for analyzing microorganisms using a matrix-assisted laser desorption / ionization mass spectrometer comprising:
Mixing Salmonella spp., Grown in a medium containing ciprofloxacin, with a matrix substance to produce a matrix / microbe mixed solution,
The matrix / microbe mixed solution is dried to form a matrix / microbe mixed crystal,
A method for analyzing a microorganism, characterized in that the matrix-microorganism mixed crystal is used for matrix-assisted laser desorption / ionization.   The microorganism analysis method according to claim 1, wherein the Salmonella spp. Are grown on a medium containing 1 ng / mL to 10 ng / mL of the ciprofloxacin.   A matrix / additive in which the matrix / microorganism mixed solution is a mixture of Salmonella spp. Grown on a medium containing ciprofloxacin, an additive that is at least one of alkylphosphonic acid and surfactant, and the matrix substance. The method for analyzing microorganisms according to claim 1 or 2, wherein the method is prepared by mixing a mixed solution.   The microbial analysis method according to claim 3, wherein the alkylphosphonic acid is methylenediphosphonic acid.   The microbial analysis method according to claim 3 or 4, wherein the surfactant is decyl-β-D-maltopyranoside.   The microbial analysis method according to claim 1, wherein the matrix substance is sinapinic acid.   The matrix / additive mixture solution is a solution containing 50% acetonitrile and 0.6% trifluoroacetic acid, 25 mg / mL of sinapinic acid as a matrix material, and 1% of methylenediphosphonic acid as an alkylphosphonic acid. The microorganism analysis method according to claim 3, wherein the surfactant is mixed so as to contain 1 mM of decyl-β-D-maltopyranoside as a surfactant.   The matrix / microorganism mixed solution is a matrix / microorganism suspension obtained by suspending a colony of Salmonella spp. Grown in a medium containing the ciprofloxacin in the matrix / additive mixed solution. The microorganism analysis method according to claim 7.   On the sample plate, 0.5 μL of an ethanol solution containing 25 mg / mL of the matrix substance sinapinic acid or a saturated ethanolic solution of sinapinic acid is dropped and dried, and then the matrix / microorganism is added thereto. The microorganism analysis method according to claim 8, wherein 1.2 μL of the suspension is dropped and dried to form the matrix / microorganism mixed crystal.   Among the mass spectra obtained for the test microorganism Salmonella, the mass spectrum pattern of the mass region including the high mass region with a mass-to-charge ratio of m / z 10,000 or higher was compared with the mass spectrum pattern stored in the database. The microorganism analysis method according to any one of claims 1 to 9, wherein the Salmonella bacterium is identified based on a result.

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