Primer set for determining serotype of Salmonella spp., PCR product, microarray for determining serotype of Salmonella spp., kit for determining serotype of Salmonella spp., and method for determining serotype of Salmonella spp.

Abstract

To provide primer sets capable of determining Salmonella Virchow or the like in salmonella bacteria.SOLUTION: Disclosed is a primer set for determining serotypes of salmonella bacteria comprising a primer set shown in (2). (2) A primer set specific for Salmonella Virchow, wherein a forward primer consists of a first specific base sequence and a reverse primer consists of a third specific base sequence, and / or, the forward primer consists of a second specific base sequence and the reverse primer consists of a third specific base sequence.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a primer for specifically detecting the serotype of Salmonella bacteria in the fields of sanitation control of livestock products, food, the environment, and the like, and to a testing technique using the same. [Background technology]

[0002] Salmonella is a type of enterobacteria that inhabit the digestive tracts of various animals, and some of these bacteria are pathogenic to humans and animals. The serotypes of Salmonella are classified into more than 2,600 types based on the combination of the O antigen, a lipopolysaccharide in the cell wall, and the H antigen, a flagellar protein. Identification of these serotypes is important for the hygiene management of livestock products, food, and the environment.

[0003] Of these, Salmonella Infantis, Salmonella Virchow, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Hadar are subject to regulation in the EU (REGULATIONS, COMMISSION REGULATION (EU) No 200 / 2010 of 10 March 2010).

[0004] In order to simultaneously distinguish between multiple serotypes of Salmonella bacteria, a PCR (Polymerase Chain Reaction) discrimination method targeting serotype-specific DNA regions has been reported (see the prior art documents listed below). However, when the previously reported primer sets are used, there is a problem in that erroneous determinations may occur. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5224559 [Patent Document 2] Patent No. 5727255 [Non-patent literature]

[0006] [Non-Patent Document 1] Kardos G et al., Letters in Applied Microbiology 2007, 45:421-425. [Non-Patent Document 2] Ranjbar R et al., Iran J Public Health 2017, 46(1): 103-111. [Non-Patent Document 3] Chiang YC et al., Journal of Food and Drug Analysis 2017, 26:58-66. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0007] For example, although many primer sets for detecting Salmonella infantis have been reported, there was a problem that some strains of Salmonella infantis were erroneously judged as negative even though they were present in the medium. In addition, one primer set for detecting Salmonella Virchow has been reported (Non-Patent Document 3), but there was a problem that when Salmonella Kentucky was present in the medium, it was erroneously determined as a false positive.

[0008] Therefore, the present inventors have conducted extensive research and developed a primer set capable of preventing erroneous determination of some strains in the detection of Salmonella infantis and preventing erroneous determination in the detection of Salmonella Virchow. Furthermore, the present inventors have succeeded in developing a primer set capable of simultaneously distinguishing Salmonella enteritidis, Salmonella typhimurium, and Salmonella harder in addition to these serotypes, and a probe capable of suitable hybridization to the PCR product, thereby completing the present invention.

[0009] Specifically, the present invention aims to provide a primer set for serotyping Salmonella bacteria, which is capable of simultaneously and specifically distinguishing multiple serotypes of Salmonella bacteria, a PCR product, a microarray for serotyping Salmonella bacteria, a kit for serotyping Salmonella bacteria, and a method for serotyping Salmonella bacteria. [Means for solving the problem]

[0010] In order to achieve the above-mentioned object, the primer set for serotyping Salmonella genus bacteria of the present invention is characterized by comprising the primer sets shown in (1) and (2) below: (1) a primer set specific to Salmonella Infantis, in which the forward primer consists of the nucleotide sequence shown in SEQ ID NO: 1 and the reverse primer consists of the nucleotide sequence shown in SEQ ID NO: 3, and / or the forward primer consists of the nucleotide sequence shown in SEQ ID NO: 2 and the reverse primer consists of the nucleotide sequence shown in SEQ ID NO: 3; (2) a primer set specific to Salmonella Virchow, in which the forward primer consists of the nucleotide sequence shown in SEQ ID NO: 4 and the reverse primer consists of the nucleotide sequence shown in SEQ ID NO: 6, and / or the forward primer consists of the nucleotide sequence shown in SEQ ID NO: 5 and the reverse primer consists of the nucleotide sequence shown in SEQ ID NO: 6.

[0011] The primer set for serotyping Salmonella of the present invention is preferably configured as follows: (3) a primer set specific to Salmonella Infantis, in which the forward primer has the nucleotide sequence shown in SEQ ID NO: 7 and the reverse primer has the nucleotide sequence shown in SEQ ID NO: 8; (4) a primer set specific to Salmonella Enteritidis, in which the forward primer has the nucleotide sequence shown in SEQ ID NO: 9 and the reverse primer has the nucleotide sequence shown in SEQ ID NO: 10; or (5) a primer set specific to Salmonella Typhimurium, in which the forward primer has the nucleotide sequence shown in SEQ ID NO: 11 and the reverse primer has the nucleotide sequence shown in SEQ ID NO: 12.

[0012] Furthermore, the primer set for serotyping Salmonella genus bacteria of the present invention comprises, together with the above primer set, at least one primer set selected from the primer sets shown in (6) to (7) below: (6) a primer set specific to Salmonella Hadar, in which the forward primer has the nucleotide sequence shown in SEQ ID NO: 13 and the reverse primer has the nucleotide sequence shown in SEQ ID NO: 14; (7) It is also preferable that the primer set is for amplifying an invA gene region specific to Salmonella spp., the forward primer having the nucleotide sequence shown in SEQ ID NO: 15 and the reverse primer having the nucleotide sequence shown in SEQ ID NO: 16.

[0013] Furthermore, the PCR product of the present invention is configured to be obtained by amplification by PCR using the above primer set.

[0014] The microarray for distinguishing serotypes of bacteria of the genus Salmonella according to the present invention has a configuration in which probes that bind complementarily to the PCR products described above are immobilized.

[0015] It is also preferable that the Salmonella serotyping microarray of the present invention is configured so that the probes include at least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 17 to 21 or their complementary sequences, and at least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 22 to 23 or their complementary sequences.

[0016] Furthermore, it is also preferred that the Salmonella serotyping microarray of the present invention is configured such that the probes include at least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 25 to 29 or a complementary sequence thereof, at least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 30 to 35 or a complementary sequence thereof, at least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 36 to 40 or a complementary sequence thereof, at least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 41 to 44 or a complementary sequence thereof, and at least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 45 to 47 or a complementary sequence thereof.

[0017] Furthermore, the kit for distinguishing the serotype of bacteria of the genus Salmonella according to the present invention comprises the above-mentioned primer set and the above-mentioned microarray.

[0018] In addition, the method for distinguishing the serotype of Salmonella bacteria of the present invention is a method for distinguishing the serotype of Salmonella bacteria by obtaining a PCR product by a PCR method using the above-mentioned primer set and hybridizing this with the probe immobilized on the above-mentioned microarray. Effect of the Invention

[0019] According to the present invention, it is possible to simultaneously and specifically distinguish a plurality of serotypes of Salmonella bacteria. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows the base sequences of a primer set for determining the serotype of Salmonella bacteria according to an embodiment of the present invention. [Diagram 2] FIG. 1 shows the base sequences of probes (for Salmonella infantis and Salmonella Virchow) in the microarray for serotyping Salmonella bacteria and the kit for serotyping Salmonella bacteria according to an embodiment of the present invention. [Diagram 3] FIG. 1 shows the base sequences of probes (for Salmonella enteritidis, Salmonella typhimurium, Salmonella hardyli, and Salmonella genus) in a microarray for serotyping Salmonella bacteria and a kit for serotyping Salmonella bacteria according to an embodiment of the present invention. [Figure 4] FIG. 1 shows the base sequences of the primer set used in detection of Salmonella infantis in Test 1. [Diagram 5] FIG. 1 shows the electrophoresis results for detection of Salmonella infantis in Test 1. [Figure 6] FIG. 1 shows the base sequences of the primer set used in the detection of Salmonella Virchow in Test 2. [Figure 7] FIG. 1 shows the electrophoretic results of detection of Salmonella Virchow in Test 2. [Figure 8]FIG. 1 shows the base sequences of the primer set used for simultaneous detection of Salmonella Infantis strain 2 and Salmonella Virchow strain 1 in Test 3. [Figure 9] FIG. 13 shows the electrophoretic results of simultaneous detection of two Salmonella Infantis strains and one Salmonella Virchow strain in Test 3. [Figure 10] FIG. 1 shows the base sequences of the primer set used for simultaneous detection of 5 serotypes and 6 strains of Salmonella in Test 4. [Figure 11] FIG. 13 shows the results of simultaneous detection of 5 serotypes and 6 strains of Salmonella by microarray in Test 4 (subject strains, samples 1 to 11). [Figure 12] FIG. 13 shows the results of simultaneous detection of 5 serotypes and 6 strains of Salmonella by microarray in Test 4 (non-target strains, samples 12 to 21). [Figure 13] FIG. 13 shows the results of simultaneous detection of 5 serotypes and 6 strains of Salmonella by microarray in Test 4 (non-target strains, samples 22 to 31). [Figure 14] FIG. 13 shows the results of simultaneous detection of 5 serotypes and 6 strains of Salmonella by microarray in Test 4 (non-target strains, samples 32 to 41). [Figure 15] FIG. 13 shows the results of the microarray for simultaneous detection of 5 serotypes and 6 strains of Salmonella in Test 4 when using other combinations of primer sets specific to Salmonella infantis (A) and Salmonella Virchow (target strain sample 11). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Hereinafter, an embodiment of the primer set for serotyping Salmonella bacteria, the PCR product, the microarray for serotyping Salmonella bacteria, the kit for serotyping Salmonella bacteria, and the method for serotyping Salmonella bacteria of the present invention will be described in detail, however, the present invention is not limited to the specific contents of the following embodiment and the examples described later.

[0022] First, a description will be given of a procedure for designing the primer set for serotyping Salmonella bacteria of this embodiment and the probes immobilized on the microarray for serotyping Salmonella bacteria. Sequence information of the target region amplified by PCR using the primer set for detecting Salmonella Infantis described in Non-Patent Document 3 was obtained from Genbank (Accession No.: CP019202, Region: 201330-201604). Similarly, sequence information of the region amplified by PCR using the primer set for detecting Salmonella Virchow described in Non-Patent Document 3 was obtained from Genbank (Accession No.: AOXL01000191, Region: 67110-67390).

[0023] Furthermore, for Salmonella Infantis strains having sequences different from the amplification target region of the above-mentioned primers for detecting Salmonella Infantis, sequence information of the target region was obtained from Genbank (accession number: AFYI01000003, region: 255560-255834). Also, for Salmonella Kentucky strains that are falsely detected when using the above-mentioned primer set for detecting Salmonella Virchow, sequence information of the target region was obtained from Genbank (accession number: CP026327, region: 241939-242216).

[0024] By comparing these four sequences, we found characteristic regions in each sequence and developed a primer set that can detect Salmonella infantis and Salmonella Virchow without misjudgment. We also designed a probe from the same target region to hybridize with the PCR product amplified by PCR using the developed primer set.

[0025] In addition, we developed primer sets that can be used together with the above primer sets to specifically distinguish the serotypes of Salmonella Enteritidis and Salmonella Typhimurium simultaneously, and designed probes that hybridize to the PCR products amplified by PCR using these primer sets. Furthermore, by combining existing primer sets for detecting Salmonella hardylii and Salmonella spp., and designing probes that hybridize to the PCR products amplified by PCR using these primer sets, it became possible to specifically distinguish six strains of five serotypes of Salmonella spp. simultaneously.

[0026] The primer set (first primer set) for serotyping Salmonella bacteria of this embodiment is characterized by including the primer set shown in (1) and (2) below. (1) A primer set specific to Salmonella Infantis, The forward primer consists of the nucleotide sequence shown in SEQ ID NO: 1, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO: 3, and / or The forward primer consists of the nucleotide sequence shown in SEQ ID NO: 2, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO:3 (2) a primer set specific to Salmonella Virchow, The forward primer consists of the nucleotide sequence shown in SEQ ID NO: 4, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO: 6, and / or the forward primer consists of the nucleotide sequence shown in SEQ ID NO:5, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO:6

[0027] In addition, it is preferable that the primer set for distinguishing the serotype of Salmonella genus bacteria (second primer set) of this embodiment is characterized in that it contains, in addition to the above-mentioned primer set, at least one primer set selected from the primer sets shown in (3) to (5) below. (3) A primer set specific to Salmonella Infantis, The forward primer consists of the nucleotide sequence shown in SEQ ID NO: 7, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO:8 (4) A primer set specific to Salmonella Enteritidis, The forward primer consists of the nucleotide sequence shown in SEQ ID NO:9, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO: 10 (5) A primer set specific to Salmonella Typhimurium, The forward primer consists of the nucleotide sequence shown in SEQ ID NO: 11, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO: 12

[0028] In addition, the primer set for distinguishing the serotype of Salmonella bacteria of this embodiment (third primer set) is preferably characterized by including, in addition to any one of the primer sets described above, at least one primer set selected from the primer sets shown in (6) to (7) below. (6) A primer set specific to Salmonella Hadar, The forward primer consists of the nucleotide sequence shown in SEQ ID NO: 13, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO: 14 (7) A primer set for amplifying an invA gene region specific to Salmonella spp., The forward primer consists of the nucleotide sequence shown in SEQ ID NO: 15, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO: 16

[0029] Furthermore, the PCR product of this embodiment is characterized in that it is obtained by amplification by PCR using any one of the first to third primer sets described above. The specific base sequences of the above primer sets and the lengths of the amplified PCR products are shown in Figure 1. In the figure, "F / R" indicates that F is the forward primer and R is the reverse primer.

[0030] The microarray for distinguishing serotypes of bacteria of the genus Salmonella in this embodiment is characterized in that probes that bind complementarily to the PCR products described above are immobilized thereon. Specific base sequences of these probes are shown in FIG. 2 and FIG.

[0031] The microarray for distinguishing serotypes of Salmonella bacteria of this embodiment preferably has immobilized thereon at least one probe selected from a group of probes having the base sequences shown in SEQ ID NOs: 17 to 21 or their complementary sequences, and at least one probe selected from a group of probes having the base sequences shown in SEQ ID NOs: 22 to 24 or their complementary sequences.

[0032] At least one of the probes selected from the group of probes having the base sequences shown in SEQ ID NOs: 17 to 21 can complementarily bind by hybridization to one of the target strands of the double stranded PCR product obtained by PCR amplification using the above (1) primer set specific to Salmonella infantis (the primer set of SEQ ID NOs: 1 and 3, and / or the primer set of SEQ ID NOs: 2 and 3). Furthermore, at least one probe selected from the group of probes having complementary sequences to the base sequences shown in SEQ ID NOs: 17 to 21 can complementarily bind to the other target strand of the double strand of the PCR product by hybridization.

[0033] At least one of the probes selected from the group of probes having the base sequences shown in SEQ ID NOs: 22 to 24 can complementarily bind by hybridization to one of the target strands of the double stranded PCR product obtained by amplifying by PCR using the above-mentioned (2) Salmonella Virchow-specific primer set (the primer set of SEQ ID NOs: 4 and 6, and / or the primer set of SEQ ID NOs: 5 and 6). Furthermore, at least one probe selected from a group of probes having a complementary sequence to the base sequences shown in SEQ ID NOs: 22 to 24 can complementarily bind to the other target strand of the double strand of the PCR product by hybridization.

[0034] Furthermore, the microarray for distinguishing Salmonella serotypes of this embodiment preferably has immobilized thereon at least one probe selected from a group of probes having the base sequences shown in SEQ ID NOs: 25 to 29, at least one probe selected from a group of probes having the base sequences shown in SEQ ID NOs: 30 to 35 or a complementary sequence thereof, at least one probe selected from a group of probes having the base sequences shown in SEQ ID NOs: 36 to 40 or a complementary sequence thereof, at least one probe selected from a group of probes having the base sequences shown in SEQ ID NOs: 41 to 44 or a complementary sequence thereof, and at least one probe selected from a group of probes having the base sequences shown in SEQ ID NOs: 45 to 47 or a complementary sequence thereof.

[0035] At least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 25 to 29 can complementarily bind by hybridization to one target strand of the double strand of a PCR product obtained by PCR amplification using the above (3) primer set specific to Salmonella infantis (primer set of SEQ ID NOs: 7 and 8). Furthermore, at least one probe selected from a group of probes having a complementary sequence to the base sequences shown in SEQ ID NOs: 25 to 29 can complementarily bind to the other target strand of the double strand of the PCR product by hybridization.

[0036] At least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 30 to 35 can complementarily bind by hybridization to one target strand of the double strand of a PCR product obtained by amplification by PCR using the primer set (primer set of SEQ ID NOs: 9 and 10) specific to Salmonella Enteritidis (4). Furthermore, at least one probe selected from the group of probes having a complementary sequence to the base sequence shown in SEQ ID NOs: 30 to 35 can complementarily bind to the other target strand of the double strand of the PCR product by hybridization.

[0037] At least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 36 to 40 can complementarily bind by hybridization to one target strand of the double strand of a PCR product obtained by PCR amplification using the above-mentioned (5) primer set specific to Salmonella Typhimurium (primer set of SEQ ID NOs: 11 and 12). Furthermore, at least one probe selected from the group of probes having complementary sequences to the base sequences shown in SEQ ID NOs: 36 to 40 can complementarily bind to the other target strand of the double strand of the PCR product by hybridization.

[0038] At least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 41 to 44 can complementarily bind by hybridization to one of the target strands of the double stranded PCR product obtained by PCR amplification using the primer set (primer set of SEQ ID NOs: 13 and 14) specific to Salmonella hardylii (6). Furthermore, at least one probe selected from the group of probes having a complementary sequence to the base sequence shown in SEQ ID NOs: 41 to 44 can complementarily bind to the other target strand of the double strand of the PCR product by hybridization.

[0039] At least one probe selected from the group of probes having the base sequences shown in SEQ ID NOs: 45 to 47 can complementarily bind by hybridization to one target strand of the double strand of a PCR product obtained by PCR amplification using the primer set (primer set of SEQ ID NOs: 15 and 16) for amplifying the invA gene region specific to Salmonella genus bacteria (7). Furthermore, at least one probe selected from the group of probes having a complementary sequence to the base sequence shown in SEQ ID NOs: 45 to 47 can complementarily bind to the other target strand of the double strand of the PCR product by hybridization.

[0040] Moreover, the microarray for distinguishing serotypes of bacteria of the genus Salmonella in this embodiment can be produced by an existing general method using probes having the base sequences shown in SEQ ID NOs: 17 to 47 or complementary sequences thereto. For example, when preparing a stick-on DNA chip as this microarray, it can be prepared by immobilizing probes on a glass substrate using a DNA spotter and forming spots corresponding to each probe. When preparing a synthetic DNA chip, it can be prepared by synthesizing single-stranded oligo DNA having the above sequence on a glass substrate using photolithography technology. Furthermore, the substrate is not limited to being made of glass, and plastic substrates, silicon wafers, etc. can also be used. Furthermore, the shape of the substrate is not limited to being flat, and various three-dimensional shapes can be used, and functional groups can be introduced onto the surface to enable chemical reactions.

[0041] The probes in this embodiment are all 20 to 60 bases long and can be synthesized by a general DNA synthesizer. The probes having the base sequences shown in SEQ ID NOs: 17 to 47 used in the examples described later were all synthesized by a DNA synthesizer. In addition, the primer set for distinguishing the serotype of Salmonella in this embodiment is also 18 to 30 bases in length, and the primers consisting of the base sequences shown in SEQ ID NOs: 1 to 16 used in PCR in the examples were synthesized using a DNA synthesizer.

[0042] In this embodiment, the probes consisting of the base sequences shown in SEQ ID NOs: 17 to 47 are not limited to the sequences themselves, and probes in which one or several bases are deleted, substituted, or added in the base sequences shown in SEQ ID NOs: 17 to 47 can be used. Also, probes that can hybridize under stringent conditions to nucleic acid fragments consisting of base sequences complementary to the base sequences shown in SEQ ID NOs: 17 to 47 can be used. Also, probes having base sequences complementary to these probes can be used.

[0043] The stringent conditions refer to conditions under which a specific hybrid is formed and a non-specific hybrid is not formed. For example, the conditions include conditions under which a DNA having high homology (homology of 90% or more, preferably 95% or more) to a DNA consisting of the nucleotide sequences shown in SEQ ID NOs: 17 to 47 hybridizes with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequences shown in SEQ ID NOs: 17 to 47. Usually, the conditions refer to a case in which hybridization occurs at a temperature about 5°C to about 30°C, preferably about 10°C to about 25°C, lower than the melting temperature (Tm) of a complete hybrid. As stringent conditions, conditions described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), especially in section 11.45 "Conditions for Hybridization of Oligonucleotide Probes", can be used.

[0044] Furthermore, the Salmonella serotype discrimination kit of this embodiment is characterized by including a first primer set and a microarray on which a first probe group is immobilized that complementarily binds to PCR products obtained by amplification by PCR using the primer set. The first probe group consists of at least one probe selected from a probe group consisting of probes having the base sequences shown in SEQ ID NOs: 17 to 21 or their complementary sequences shown in Figure 2, and at least one probe selected from a probe group consisting of probes having the base sequences shown in SEQ ID NOs: 22 to 24 or their complementary sequences.

[0045] In addition, the Salmonella serotype discrimination kit of this embodiment is also preferably configured to include a second primer set and a microarray on which a second probe group is immobilized that complementarily binds to the PCR products obtained by amplifying the PCR products by PCR using the second primer set. The second probe group consists of, in addition to the first probe group, at least one probe selected from the probe group consisting of probes having the base sequences shown in SEQ ID NOs: 25 to 29 shown in Figure 2 or their complementary sequences, at least one probe selected from the probe group consisting of probes having the base sequences shown in SEQ ID NOs: 30 to 35 shown in Figure 3 or their complementary sequences, and at least one probe selected from the probe group consisting of probes having the base sequences shown in SEQ ID NOs: 36 to 40 shown in Figure 3 or their complementary sequences.

[0046] In addition, the Salmonella serotype discrimination kit of this embodiment is also preferably configured to include a third primer set and a microarray on which a third probe group is immobilized that complementarily binds to the PCR products obtained by amplifying the primer set by PCR. The third probe group consists of, in addition to the second probe group, at least one probe selected from the probe group consisting of probes having the base sequences shown in SEQ ID NOs: 41 to 44 in Figure 3 or their complementary sequences, and at least one probe selected from the probe group consisting of probes having the base sequences shown in SEQ ID NOs: 45 to 47 or their complementary sequences.

[0047] In addition, the method for distinguishing the serotype of Salmonella bacteria of this embodiment is characterized in that a PCR product is obtained by a PCR method using a first primer set, and the PCR product is hybridized with a first probe group immobilized on a microarray, thereby distinguishing the serotype of Salmonella bacteria.

[0048] In addition, the method for discriminating the serotype of Salmonella bacteria of this embodiment is also preferably a method for discriminating the serotype of Salmonella bacteria by obtaining a PCR product by a PCR method using a second primer set and hybridizing the PCR product with a second group of probes immobilized on a microarray.

[0049] In addition, the method for discriminating the serotype of Salmonella bacteria of this embodiment is also preferably a method for discriminating the serotype of Salmonella bacteria by obtaining a PCR product by a PCR method using a third primer set and hybridizing the PCR product with a third group of probes immobilized on a microarray.

[0050] The extraction of genomic DNA from a strain of Salmonella genus bacteria, which is performed prior to the method for distinguishing the serotype of Salmonella genus bacteria of this embodiment, can be performed by a general method such as a method using a kit or a method using a DNA extraction device.

[0051] Then, the target region (region to be amplified) in the extracted genomic DNA is amplified. That is, a DNA fragment containing the target region in the genomic DNA is amplified. The method for amplifying the target region is not particularly limited, but the PCR method can be preferably used. A general thermal cycler or the like can be used as the PCR device.

[0052] In this embodiment, the target region in the genomic DNA can be suitably amplified by carrying out PCR under the following reaction conditions, for example. (a) 95°C for 15 min, (b) 94°C (DNA denaturation step) for 1 min, (c) 60°C (annealing step) for 1 min, (d) 72°C (DNA synthesis step) for 1 min (35 cycles of (b) to (d)), (e) 72°C for 10 min.

[0053] As the reaction solution for PCR, it is preferable to use, for example, one having the following composition. That is, a PCR reaction solution containing a nucleic acid synthesis substrate (dNTP mixture (dCTP, dATP, dTTP, dGTP)), a primer set, a nucleic acid synthesis enzyme (HotStart DNA polymerase, etc.), genomic DNA of the sample, a buffer solution, and sterile water as the remaining component can be preferably used.

[0054] In this embodiment, hybridization can be carried out as follows. That is, the presence or absence of the amplification product is confirmed by dropping an amplification product obtained by a PCR method or the like onto the microarray for serotyping Salmonella of this embodiment and detecting the label of the amplification product that has hybridized to the probe immobilized thereon, thereby making it possible to identify Salmonella present in the livestock products, food, and environment to be inspected.

[0055] The detection of the label can be performed using a general label detection device such as a fluorescent scanning device, for example, by measuring the fluorescence intensity of the amplified product using a GENOGATE(R) reader from Toyo Seikan Group Engineering Co., Ltd. The measurement result is preferably obtained as an S / N ratio value (signal to noise ratio), because whether the measurement result is positive or negative can be determined with high accuracy based on the S / N ratio value. In the case of the microarray used in the examples described below, a positive result can be obtained when the S / N ratio is 3.0 or more. The S / N ratio described in this specification is calculated by (median fluorescence intensity value-background value)÷background value. Note that the label is not limited to fluorescence, and other labels can also be used.

[0056] The primer set for serotyping Salmonella bacteria, the PCR product, the microarray for serotyping Salmonella bacteria, the kit for serotyping Salmonella bacteria, and the method for serotyping Salmonella bacteria of the present embodiment described above can prevent erroneous determination of some bacterial strains in the detection of Salmonella infantis, and can prevent erroneous determination in the detection of Salmonella Virchow. In addition to these serotypes, it is possible to simultaneously and specifically determine multiple serotypes including Salmonella enteritidis, Salmonella typhimurium, and Salmonella hardyli. EXAMPLES

[0057] Below, we will specifically describe tests that were conducted to confirm the effects of the primer set for serotyping Salmonella bacteria, the PCR product, the microarray for serotyping Salmonella bacteria, the kit for serotyping Salmonella bacteria, and the method for serotyping Salmonella bacteria according to the embodiments of the present invention.

[0058] [Test 1] In this test, it was confirmed that some strains of Salmonella Infantis that could not be detected in the past (Salmonella Infantis ATCC BAA-1675 strain, hereinafter sometimes referred to as Salmonella Infantis (A)) can be detected by using the primer set for distinguishing the serotype of Salmonella of this embodiment.

[0059] Specifically, as shown in Figure 4, an experiment using a primer set of sequence numbers 1 and 3 specific to Salmonella infantis (A) was performed as Example 1, and an experiment using a primer set of sequence numbers 2 and 3 specific to Salmonella infantis (A) was performed as Example 2. Furthermore, Comparative Examples 1 to 9 were prepared as conventional primer sets specific to Salmonella infantis.

[0060] The primer sets of SEQ ID NOs: 48 and 49, 50 and 51, and 52 and 53 are described in Patent Document 1, and experiments using these primer sets were performed as Comparative Examples 1, 2, and 3, respectively. The primer set of SEQ ID NOs: 54 and 55 is described in Patent Document 2, and an experiment using this primer set was performed as Comparative Example 4.

[0061] The primer set of SEQ ID NOs: 56 and 58 and the primer set of SEQ ID NOs: 57 and 58 are those described in Non-Patent Document 1, and experiments using these primers were performed as Comparative Examples 5 and 6, respectively. The primer set of SEQ ID NOs: 59 and 60 is described in Non-Patent Document 2, and an experiment using this primer set was performed as Comparative Example 7. The primer set of SEQ ID NOs: 61 and 63 and the primer set of SEQ ID NOs: 62 and 63 are described in Non-Patent Document 3, and experiments using these primers were performed as Comparative Examples 8 and 9, respectively.

[0062] First, 802 medium was prepared. Specifically, the following reagents were mixed, then 10 mL was dispensed into test tubes and sterilized at 121° C. for 15 minutes. The pH was adjusted to 7.0. (1) Hypopolypeptone (manufactured by Nissui Pharmaceutical Co., Ltd.) 10g (2) Yeast extract (Difco) 2g (3) MgSO47H2O (Fujifilm Wako Pure Chemical Industries, Ltd.) 1g (4) Distilled water, 1L

[0063] Next, genomic DNA was extracted from the Salmonella strains. The following strains were used. The strains were obtained from the following institutions: ATCC: American Type Culture Collection Sample 1: Salmonella Infantis ATCC BAA-1675 Sample 2: Negative control (sterile water was added instead of template)

[0064] This strain was cultured in 802 medium at 30° C. with shaking at 200 rpm for 24 hours. Next, genomic DNA was extracted according to the instructions of the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (manufactured by Cytrop).

[0065] For each sample, 20 μL of PCR reaction solution containing the primer set for detecting Salmonella Infantis was prepared. The primers were synthesized by Sigma-Aldrich Japan LLC or FASMAC Corporation. (1) PCR buffer (QIAGEN) 2.0 μL (2) dNTP mix (Cat BR0600503, manufactured by biotechrabbit) 0.4μL (3) 10 μM primer, 0.4 μL each (4) HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μL (5) Template DNA (10 ng / PCR reaction solution) 2.0 μl each (6) Sterile water (add water until the total volume is 20.0 μL)

[0066] Using each of the above PCR reaction solutions, DNA was amplified under the following conditions using a nucleic acid amplifier (Master cycler ep, Eppendorff). (a) 95℃ 15 minutes (b)94℃ 1 minute (c)60℃ 1 minute (d) 72°C for 1 minute (35 cycles of (b) to (d)) (e) 72℃ 10 minutes

[0067] Then, electrophoresis was performed using an electrophoresis apparatus (MultiNA, Shimadzu Corporation) to confirm the presence or absence of an amplification product, and the results are shown in FIG. In the figure, Comparative Examples 1 to 3 are shown in lane 1, Comparative Example 4 in lane 2, Comparative Example 5 in lane 3, Comparative Example 6 in lane 4, Comparative Example 7 in lane 5, Comparative Example 8 in lane 6, Comparative Example 9 in lane 7, Example 1 in lane 8, and Example 2 in lane 9.

[0068] As is clear from these results, Salmonella Infantis ATCC BAA-1675 was not detected in the lanes of Comparative Examples 1 to 9. In contrast, it was confirmed that the same strain could be detected in Examples 1 and 2.

[0069] [Test 2] In this test, it was confirmed that the use of the primer set for serotyping Salmonella of the present embodiment can prevent false positive determination of Salmonella Kentuckyi in detection of Salmonella Virchow.

[0070] Specifically, as shown in FIG. 6, an experiment using a primer set of SEQ ID NOs: 4 and 6 specific to Salmonella Virchow was performed as Example 3, and an experiment using a primer set of SEQ ID NOs: 5 and 6 specific to Salmonella Virchow was performed as Example 4. In addition, as conventional primer sets specific to Salmonella Virchow, a primer set of SEQ ID NOs: 4 and 63 and a primer set of SEQ ID NOs: 5 and 63 were prepared. The primer set of SEQ ID NOs: 4 and 63 and the primer set of SEQ ID NOs: 5 and 63 are those described in Non-Patent Document 3, and experiments using these primers were performed as Comparative Examples 10 and 11, respectively.

[0071] First, 802 medium was prepared in the same manner as in Test 1. Next, genomic DNA was extracted from the Salmonella strains listed below. These strains were obtained from the following institutions: ATCC: American Type Culture Collection GTC: Gifu University Sample 1: Salmonella Virchow ATCC 51955 Sample 2: Salmonella Kentucky GTC 12786 Sample 3: Negative control (sterile water was added instead of template)

[0072] These strains were cultured in 802 medium at 30° C. with shaking at 200 rpm for 24 hours. Next, genomic DNA was extracted according to the instructions of the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (manufactured by Cytrop).

[0073] For each sample, 20 μL of PCR reaction solution containing the primer set for detecting Salmonella Virchow was prepared. The primers were synthesized by Sigma-Aldrich Japan LLC. (1) PCR buffer (QIAGEN) 2.0 μL (2) dNTP mix (Cat BR0600503, manufactured by biotechrabbit) 0.4μL (3) 10 μM primer, 0.4 μL each (4) HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μL (5) Template DNA (10 ng / PCR reaction solution) 2.0 μl each (6) Sterile water (add water until the total volume is 20.0 μL)

[0074] Using each of the above PCR reaction solutions, DNA was amplified under the following conditions using a nucleic acid amplifier (Master cycler ep, Eppendorff). (a) 95℃ 15 minutes (b)94℃ 1 minute (c)60℃ 1 minute (d) 72°C for 1 minute (35 cycles of (b) to (d)) (e) 72℃ 10 minutes

[0075] Then, electrophoresis was performed using an electrophoresis apparatus (MultiNA, Shimadzu Corporation) to confirm the presence or absence of an amplification product, and the results are shown in FIG. In the figure, Comparative Example 10 is shown in lane 1, Comparative Example 11 in lane 2, Example 3 in lane 3, and Example 4 in lane 4.

[0076] As is clear from these results, in the lanes of Comparative Examples 10 and 11, Salmonella Virchow was detected, but Salmonella Kentuckyi was also detected, indicating the occurrence of false positives. In contrast, in the lanes of Examples 3 and 4, Salmonella Virchow was detected, but Salmonella Kentuckyi was not detected. Thus, it was confirmed that the primer set for determining the serotype of Salmonella genus bacteria of this embodiment can prevent erroneous determination of Salmonella Virchow.

[0077] [Test 3] In this test, two strains of Salmonella infantis and one strain of Salmonella Virchow were targeted, and it was confirmed that these can be simultaneously and specifically detected by using the primer set for distinguishing the serotype of Salmonella of this embodiment.

[0078] Specifically, as shown in FIG. 8, a primer set of sequence numbers 2 and 3 specific to a previously undetectable Salmonella Infantis strain (Salmonella Infantis (A)), a primer set of sequence numbers 7 and 8 specific to a previously detectable Salmonella Infantis strain (hereinafter sometimes referred to as Salmonella Infantis (B)), and a primer set of sequence numbers 4 and 6 specific to Salmonella Virchow were prepared for the purposes of the present invention. In addition, a conventional primer set of SEQ ID NOs: 61 and 63 specific to Salmonella infantis and a conventional primer set of SEQ ID NOs: 4 and 63 specific to Salmonella Virchow were prepared for comparison. These were described in Non-Patent Document 3.

[0079] First, 802 medium was prepared in the same manner as in Test 1. Next, genomic DNA was extracted from the Salmonella strains listed below. These strains were obtained from the following institutions: ATCC: American Type Culture Collection GTC: Gifu University Sample 1: Salmonella Infantis ATCC 51741 Sample 2: Salmonella Infantis ATCC BAA-1675 Sample 3: Salmonella Virchow ATCC 51955 Sample 4: Salmonella Kentucky GTC 12786 Sample 5: All strains from samples 1 to 3 Sample 6: Negative control (sterile water was added instead of template)

[0080] These strains were cultured in 802 medium at 30° C. with shaking at 200 rpm for 24 hours. Next, genomic DNA was extracted according to the instructions of the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (manufactured by Cytrop).

[0081] As a PCR reaction solution, 20 μL of the following composition containing each of the above primer sets was prepared for each sample of each Example and Comparative Example. The primers were synthesized by Sigma-Aldrich Japan LLC. (1) PCR buffer (QIAGEN) 2.0 μL (2) dNTP mix (Cat BR0600503, manufactured by biotechrabbit) 0.4μL (3) 10 μM primer, 0.4 μL each (4) HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μL (5) Template DNA (10 ng / PCR reaction solution) 2.0 μl each (6) Sterile water (add water until the total volume is 20.0 μL)

[0082] Using each of the above PCR reaction solutions, DNA was amplified under the following conditions using a nucleic acid amplifier (Master cycler ep, Eppendorff). (a) 95℃ 15 minutes (b)94℃ 1 minute (c)60℃ 1 minute (d) 72°C for 1 minute (35 cycles of (b) to (d)) (e) 72℃ 10 minutes

[0083] Then, electrophoresis was performed using an electrophoresis apparatus (MultiNA, Shimadzu Corporation) to confirm the presence or absence of an amplification product, and the results are shown in FIG. In the figure, comparative examples are shown in lanes 1 to 6 on the left (Comparative Examples 12 to 17, respectively), and examples are shown in lanes 1 to 6 on the right (Examples 5 to 10, respectively).

[0084] In the comparative example, it can be seen that Salmonella Infantis ATCC BAA-1675 in sample 2 was not detected as a test strain in lane 2. It can also be seen that Salmonella Kentucky GTC 12786 in sample 4 was erroneously detected as a test strain in lane 4.

[0085] In contrast, in the Example, it can be seen that Salmonella Infantis ATCC BAA-1675 in Sample 2, which is the test strain, is detected in lane 2. Also, in Sample 4, Salmonella Kentucky GTC 12786, which is the test strain, is not erroneously detected in lane 4. Furthermore, in lane 5, in which all of the strains in samples 1 to 3 of sample 5 are used as test strains, it is possible to distinguish between two Salmonella infantis strains and one Salmonella Virchow strain.

[0086] As described above, the primer set for distinguishing serotypes of bacteria of the genus Salmonella according to the present embodiment can detect Salmonella infantis 2 strain and Salmonella Virchow 1 strain without producing false negatives or false positives, and can further distinguish between and specifically detect these strains.

[0087] [Test 4] In this test, five serotypes and six strains of Salmonella were targeted, and it was confirmed that they can be simultaneously and specifically detected by using the primer set for discriminating the serotype of Salmonella of this embodiment.

[0088] Specifically, as shown in FIG. 10, a primer set of SEQ ID NOs: 2 and 3 specific to Salmonella infantis (A), a primer set of SEQ ID NOs: 4 and 6 specific to Salmonella Virchow, a primer set of SEQ ID NOs: 7 and 8 specific to Salmonella infantis (B), a primer set of SEQ ID NOs: 9 and 10 specific to Salmonella enteritidis, a primer set of SEQ ID NOs: 11 and 12 specific to Salmonella typhimurium, a primer set of SEQ ID NOs: 13 and 14 specific to Salmonella harderii, and a primer set of SEQ ID NOs: 15 and 16 specific to the Salmonella genus were prepared.

[0089] In addition, instead of the above combination of the primer set specific to Salmonella infantis (A) and the primer set specific to Salmonella Virchow, other combinations of primer sets were prepared. Specifically, as shown in the primer column of Figure 15, a combination of a primer set of SEQ ID NOs: 1 and 3 specific to Salmonella infantis (A) and a primer set of SEQ ID NOs: 4 and 6 specific to Salmonella Virchow, a combination of a primer set of SEQ ID NOs: 1 and 3 specific to Salmonella infantis (A) and a primer set of SEQ ID NOs: 5 and 6 specific to Salmonella Virchow, and a combination of a primer set of SEQ ID NOs: 2 and 3 specific to Salmonella infantis (A) and a primer set of SEQ ID NOs: 5 and 6 specific to Salmonella Virchow were prepared.

[0090] First, 802 medium was prepared in the same manner as in Test 1. Brain heart infusion (BHI) medium (Difco) was prepared according to a standard method, dispensed in 10 mL portions into test tubes, and sterilized at 121° C. for 15 minutes.

[0091] Next, genomic DNA was extracted from the Salmonella strains listed below. These strains were obtained from the following institutions: ATCC: American Type Culture Collection NCTC: National Collection of Type Cultures GTC: Gifu University

[0092] Sample 1: Salmonella Infantis ATCC BAA-1675 Sample 2: Salmonella Virchow ATCC 51955 Sample 3: Salmonella Infantis ATCC 51741 Sample 4: Salmonella Enteritidis GTC 03838 Samples 5 to 9: Salmonella Typhimurium, NBRC 13245, GTC 02577, GTC 11393, GTC 11566, and GTC 14142, respectively Sample 10: Salmonella Hadar ATCC 51956 Sample 11: All strains from samples 1 to 5 and 10

[0093] Sample 12: Salmonella Abaetetuba ATCC 35640 Sample 13: Salmonella Agona ATCC BAA-707 Sample 14: Salmonella Albany ATCC 51960 Sample 15: Salmonella Anatum ATCC 9270 Sample 16: Salmonella Bareilly ATCC 9115 Sample 17: Salmonella Bredney ATCC 10728 Sample 18: Salmonella Cerro GTC 02554 Sample 19: Salmonella Derby ATCC 6960 Sample 20: Salmonella Essen GTC 12814 Sample 21: Salmonella Kentucky GTC 12786

[0094] Sample 22: Salmonella Mbandaka ATCC 51958 Samples 23 to 26: Salmonella Montevideo, ATCC BAA-710, ATCC BAA-1735, ATCC 8387, GTC 02525 Samples 27-28: Salmonella Newport, ATCC 5962 and ATCC 27869 Sample 29: Salmonella Paratyphi C ATCC 9068 Sample 30: Salmonella Poona NCTC 4840 Sample 31: Salmonella Saintpaul ATCC 9712

[0095] Sample 32: Salmonella Schwarzengrund GTC 12055 Sample 33: Salmonella Senftenberg ATCC 8400 Sample 34: Salmonella Tallahassee ATCC 12002 Sample 35: Salmonella Tennessee ATCC 10722 Samples 36-37: Salmonella Thompson, ATCC BAA-1604, ATCC 8391 Sample 38: Salmonella Vellore ATCC 15611 Sample 39: Salmonella enterica subsp. Salamae ATCC43972 Sample 40: Salmonella bongori ATCC 43975 Sample 41: Negative control (sterile water was added instead of template) *Except for 39 to 41, Salmonalla enterica subsp. enterica

[0096] These strains were cultured in 802 medium or BHI medium with shaking at 30° C. and 200 rpm for 24 hours. Next, genomic DNA was extracted according to the instructions of the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (manufactured by Cytrop).

[0097] For each sample, 20 μL of a PCR reaction solution containing the above primer set was prepared with the following composition: The primers were synthesized by Sigma-Aldrich Japan LLC. (1) PCR buffer (QIAGEN) 2.0 μL (2) dNTP mix (Cat BR0600503, manufactured by biotechrabbit) 0.4μL (3) 10 μM Infantis (A) primer (F) 0.04 μL (4) 10 μM Infantis (A) primer (R) (Cyanine 5 modified at the 5' end) 0.2 μL (5) 10 μM Virchow primer (F) 0.04 μL (6) 10 μM Virchow primer (R) (Cyanine 5 modified at the 5' end) 0.2 μL (7) 10 μM Infantis (B) primer (F) 0.04 μL (8) 10 μM Infantis (B) primer (R) (Cyanine 5 modified at the 5' end) 0.2 μL (9) 10 μM Enteritidis primer (F) (Cyanine 5 modified at the 5' end) 1.0 μL (10) 10 μM Enteritidis primer(R) 0.2 μL (11) 10 μM Typhimurium primer (F) (Cyanine 5 modified at the 5' end) 0.2 μL (12) 10 μM Typhimurium primer(R) 0.04 μL (13) 10 μM Hardener primer (F) (Cyanine 5 modified at the 5' end) 0.5 μL (14) 10 μM Hardener primer(R) 0.1 μL (15) 10 μM Salmonella primer (F) 0.04 μL (16) 10 μM Salmonella primer (R) (Cyanine 5 modified at the 5' end) 0.2 μL (17) HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μL (18) Template DNA (10 ng / PCR reaction solution) 2.0 μl each (19) Sterile water (add water until the total volume is 20.0 μL)

[0098] Using each of the above PCR reaction solutions, DNA was amplified under the following conditions using a nucleic acid amplifier (Master cycler ep, Eppendorff). (a) 95℃ 15 minutes (b)94℃ 1 minute (c)60℃ 1 minute (d) 72°C for 1 minute (35 cycles of (b) to (d)) (e) 72℃ 10 minutes

[0099] A DNA chip (manufactured by Toyo Seikan Group Holdings Co., Ltd.) was used as a microarray for serotyping Salmonella bacteria, and the probes of SEQ ID NOs: 17 to 47 were arranged on the chip. Each probe was synthesized by Sigma-Aldrich Japan LLC. Each probe was immobilized on the substrate using a DNA spotter.

[0100] SEQ ID NOs: 17 to 21 show the base sequences of a probe for detecting Salmonella infantis (A). SEQ ID NOs: 22 to 24 show the base sequences of a probe for detecting Salmonella Virchow. SEQ ID NOs: 25 to 29 show the base sequences of a probe for detecting Salmonella infantis (B). SEQ ID NOs: 30 to 35 show the base sequences of a probe for detecting Salmonella enteritidis. SEQ ID NOs: 36 to 40 show the base sequences of a probe for detecting Salmonella Typhimurium. SEQ ID NOs: 41 to 44 show the base sequences of a probe for detecting Salmonella hardyli. SEQ ID NOs: 45 to 47 show the base sequences of a probe for detecting the genus Salmonella.

[0101] The reaction solution for hybridization was prepared as follows. The PCR product was treated at 95°C for 5 minutes and then rapidly cooled. 2 μL of hybridization buffer and 4 μL of the PCR product were mixed on the cover of the DNA chip. The buffer composition was 3×SSC / 0.3% Tween 20 and 20 nM Cy5-labeled oligonucleotide. Then, this cover was placed on the DNA chip, and hybridization was carried out in a hybridizer (manufactured by Dako) under hybridization conditions of 45° C. / 1 hour.

[0102] Next, the DNA chip was washed with a washing solution (0.5×SSC / 0.2% SDS, 0.5×SSC) to wash away unhybridized PCR products. Then, using a label detection device (GENOGATE(R)reader, Toyo Seikan Group Engineering Co., Ltd.), the fluorescence intensity (fluorescence intensity of the amplified product bound to the probe) of each probe placed on the DNA chip was measured, and the S / N ratio value ((median fluorescence intensity value-background value)÷background value) of each probe was obtained. A S / N ratio value of 3 or more was determined to be positive. The results are shown in Figures 11 to 15.

[0103] As shown in FIG. 11, it was confirmed that the primer set for serotyping Salmonella and the microarray for serotyping Salmonella of this embodiment can specifically detect Salmonella infantis (A), Salmonella Virchow, Salmonella infantis (B), Salmonella enteritidis, Salmonella typhimurium, Salmonella hardier, and all of the genus Salmonella simultaneously without generating false negatives. Furthermore, as shown in Figures 12 to 14, it was confirmed that the primer set for serotyping Salmonella bacteria and the microarray for serotyping Salmonella bacteria of this embodiment did not cause false positives for other serotypes of the Salmonella genus.

[0104] Furthermore, as shown in FIG. 15, it was confirmed that the primer set for serotyping Salmonella spp. and the microarray for serotyping Salmonella spp. of this embodiment enable simultaneous and specific detection with almost no false negatives, even when all combinations of two primer sets specific to Salmonella infantis (A) and two primer sets specific to Salmonella Virchow are used. Specifically, when the combination of SEQ ID NO:5 and SEQ ID NO:6 was used as the Virchow primer set, the Virchow probe of SEQ ID NO:24 gave a false negative result, but other combinations were able to specifically detect six strains of five serotypes of Salmonella simultaneously.

[0105] The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications and variations are possible within the scope of the present invention. For example, the number of probes arranged on the microarray for serotyping Salmonella of this embodiment is not limited to one spot per type, and each probe may be arranged on multiple spots. In addition, other probes for detecting bacteria other than the Salmonella bacteria that are the detection target of this embodiment may be arranged on the microarray together with the probe of this embodiment, and appropriate changes can be made. [Industrial Applicability]

[0106] INDUSTRIAL APPLICABILITY The present invention can be suitably used for specifically detecting the serotype of Salmonella in the fields of sanitation control of livestock products, food, the environment, and the like.

Claims

1. A primer set for distinguishing serotypes of Salmonella bacteria, comprising the primer set shown in (2) below: (2) A primer set specific to Salmonella Virchow, The forward primer consists of the base sequence shown in SEQ ID NO: 4, A primer set in which the reverse primer consists of the base sequence shown in SEQ ID NO: 6, and / or The forward primer consists of the base sequence shown in SEQ ID NO:5, The reverse primer is a primer set consisting of the base sequence shown in SEQ ID NO:

6.

2. A primer set for distinguishing the serotype of Salmonella spp., comprising the primer set according to claim 1 and at least one primer set selected from the primer sets shown in (3) to (5) below: (3) A primer set specific to Salmonella Infantis, The forward primer consists of the base sequence shown in SEQ ID NO: 7, A primer set in which the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:8; (4) A primer set specific to Salmonella Enteritidis, The forward primer consists of the base sequence shown in SEQ ID NO:9, A primer set in which the reverse primer consists of the nucleotide sequence shown in SEQ ID NO: 10; (5) A primer set specific to Salmonella Typhimurium, The forward primer consists of the base sequence shown in SEQ ID NO: 11, The reverse primer is a primer set consisting of the base sequence shown in SEQ ID NO:

12.

3. A primer set for distinguishing the serotype of Salmonella spp., comprising the primer set according to claim 1 or 2 and at least one primer set selected from the primer sets shown in (6) to (7) below: (6) A primer set specific to Salmonella Hadar, The forward primer consists of the base sequence shown in SEQ ID NO: 13, A primer set in which the reverse primer consists of the nucleotide sequence shown in SEQ ID NO: 14; (7) A primer set for amplifying an invA gene region specific to Salmonella spp., comprising: The forward primer consists of the base sequence shown in SEQ ID NO: 15, The reverse primer is a primer set consisting of the base sequence shown in SEQ ID NO:16.

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