Bacteria inspection method, microarray for bacteria inspection, kit for bacteria inspection, probe set for bacteria inspection, and primer set for bacteria inspection

Abstract

To make it possible to detect an existing bacterium of the genus Listeria accurately and specifically in examination for checking the presence of a bacterium.SOLUTION: There is provided a method for examining the presence of a bacterium in a sample in which a target bacterium is selected from bacteria of the genus Listeria described in the following (1) to (10), (1) Listeria floridensis, (2) Listeria weihenstephanensis, (3) Listeria cornellensis, (4) Listeria riparia, (5) Listeria grandensis, (6) Listeria booriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria fleischmannii, (10) Listeria marthii, and the method includes: (a) a preparation process of preparing reaction liquid containing a primer set for amplifying an IGS (intergenic spacer) region of rRNA gene; and (b) an amplification process of amplifying nucleic acid of the target bacterium included in a sample by using the reaction liquid obtained in the preparation process.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a testing technique for Listeria, which is a problem in food manufacturing facilities and the like. [Background technology]

[0002] Currently, 17 species of Listeria have been identified, of which Listeria monocytogenes is the pathogenic species that causes food poisoning. Food poisoning caused by Listeria monocytogenes is likely to occur in pregnant women, newborns, the elderly, and people with weakened immune systems, and in recent years, it has been reported that approximately 1,600 people have been infected and approximately 260 have died each year in the United States.

[0003] The VIDAS (registered trademark, bioMérieux) method is known as a method for detecting Listeria bacteria. However, when Listeria rocourtiae is tested using this method, it is erroneously identified as Listeria monocytogenes, resulting in a problem of misjudgment. For these reasons, it would be desirable to accurately identify all 17 species of Listeria, but to date no such test kits have existed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4621919 specification [Non-patent literature]

[0005] [Non-Patent Document 1] Dmitriy Volokhov. et al., Identification of Listeria Species by Microarray-Based Assaay. J.Clin.Microbiol., 40, 12, (2002),4720-4728 [Non-Patent Document 2] Jun Wang. et al., Rapid identification of Listeria species and screening for variants by melting curve and high-resoulution melting curve analyzes of the intergenic spacer region of the rRNA gene. Can.J. Microbiolo. 56: (2010),676-682 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0006] Here, an example of a prior art document relating to a detection method for Listeria bacteria is the multiplex detection method for microorganisms described in Patent Document 1. This method makes it possible to detect two or more types of microorganisms with different characteristics, including Listeria monocytogenes, in food under certain conditions. However, this method was not able to accurately distinguish all 17 species of Listeria.

[0007] Furthermore, Non-Patent Document 1 describes a method for detecting six bacterial species, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri, using the iap gene region as the test target. In addition, Non-Patent Document 2 describes a method for detecting the same six bacterial species as above, using IGS as the genetic region to be examined. However, none of these documents describes a method for detecting all 17 species of Listeria.

[0008] Furthermore, of the 17 species in the genus Listeria, 10 species, Listeria floridensis, Listeria weihenstephanensis, Listeria cornellensis, Listeria riparia, Listeria grandensis, Listeria booriae, Listeria newyorkensis, Listeria aquatica, Listeria fleischmannii, and Listeria marthii, have not been detected so far.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide a bacterial testing method capable of accurately identifying all 17 species of Listeria, a microarray for bacterial testing, a kit for bacterial testing, a probe set for bacterial testing, and a primer set for bacterial testing. [Means for solving the problem]

[0010] In order to achieve the above-mentioned object, the method for testing bacteria of the present invention is a method for testing the presence of bacteria in a sample, and the target bacteria is selected from the Listeria genus bacteria listed in the following (1) to (10), and is: (1) Listeria floridensis, (2) Listeria weihenstephanensis, (3) Listeria cornellensis, (4) Listeria riparia, (5) Listeria grandensis, (6) Listeria booriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria fleischmannii, (10) Listeria marti, (11) Listeria malti, (12) Listeria malti, (13) Listeria malti, (14) Listeria malti, (15) Listeria malti, (16) Listeria malti, (17) Listeria malti, (18) Listeria malti, (19) Listeria malti, (20) Listeria malti, (21) Listeria malti, (22) Listeria malti, (23) Listeria malti, (24) Listeria malti, (25) Listeria malti, (26) Listeria malti, (27) Listeria malti, (28) Listeria malti, (29) Listeria malti, (30) Listeria malti, (31) Listeria malti, (32) Listeria malti, (33) Listeria malti, (34) Listeria malti, (35) Listeria malti, (36) Listeria malti, (37) Listeria malti, (38) Listeria malti, (39) Listeria malti, (40) Listeria malti, (41) Listeria malti, (42) Listeria malti, (43) Listeria malti, (44) Listeria malti, (45) Listeria mal marthii), the method includes (a) a preparation step of preparing a reaction solution containing a primer set for amplifying the IGS (intergenic spacer) region of the rRNA gene, and (b) an amplification step of amplifying the nucleic acid of the target bacterium contained in the sample using the reaction solution obtained by the preparation step.

[0011] Furthermore, the microarray for bacterial testing of the present invention is a microarray for bacterial testing that detects the presence of bacteria in a sample, and is configured to have at least one probe having a base sequence shown in SEQ ID NO: 32 to 43, or 47 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria genus bacteria described in (1) to (10) above immobilized thereon.

[0012] Furthermore, the bacteria testing kit of the present invention is a bacteria testing kit for detecting the presence of bacteria in a sample, and is configured to include a microarray on which at least one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43, and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria genus bacteria described in (1) to (10) above is immobilized, and a primer set consisting of primers having the base sequences shown in SEQ ID NOs: 16 and 17 for amplifying nucleic acid that hybridizes (complementarily binds) with the probe.

[0013] Furthermore, the probe set for testing bacteria of the present invention is a probe set for testing bacteria for detecting the presence of bacteria in a sample, and is configured to include at least any one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria genus bacteria described in (1) to (10) above.

[0014] Furthermore, the primer set for testing bacteria of the present invention is a primer set for testing the presence of bacteria in a sample, and is composed of primers having the base sequences shown in SEQ ID NOs: 16 and 17 for amplifying nucleic acid that hybridizes with at least any one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria described in (1) to (10) above. Effect of the Invention

[0015] According to the present invention, it is possible to accurately distinguish all 17 species of Listeria. [Brief description of the drawings]

[0016] [Figure 1] FIG. 2 shows the base sequences of primers used in the examples to analyze the base sequences of the IGS regions of various Listeria species that are the subject of the bacterial testing method according to an embodiment of the present invention. [Diagram 2] FIG. 1 shows the base sequences of the IGS regions of various Listeria species (Listeria floridensis, Listeria wehenstephanensis, Listeria cornelensis, Listeria riparia, and Listeria grandensis) analyzed in the examples and targeted in the bacterial testing method according to an embodiment of the present invention. [Diagram 3] FIG. 1 shows the base sequences of the IGS regions of various Listeria genus bacteria (Listeria boreoliae, Listeria newyorkensis, Listeria aquatica, Listeria freshmannii, and Listeria marti) analyzed in the examples and targeted by the bacterial testing method according to an embodiment of the present invention. [Figure 4] FIG. 1 is a diagram showing the sample numbers and test strains of the samples (Listeria greyi, Listeria innocua, Listeria ivanovii, and Listeria monocytogenes) used in Test 1 for the bacterial testing method according to an embodiment of the present invention. [Diagram 5] FIG. 1 shows the sample numbers and test strains of samples (Listeria locoutiae, Listeria siligeri, Listeria welshimeri, Listeria floridensis, Listeria wehenstephanensis, Listeria cornelensis, Listeria riparia, Listeria grandensis, Listeria booriae, Listeria newyorkensis, Listeria aquatica, Listeria freshmannii, and Listeria marti) used in Test 1 for the bacterial testing method according to an embodiment of the present invention. [Figure 6] FIG. 2 is a diagram showing primers for amplifying a plasmid as an internal control used in each test of the bacterial testing method according to the embodiment of the present invention, and the base sequence of the DNA to be amplified. [Figure 7] FIG. 1 shows the base sequences of primers for amplifying the IGS regions of various Listeria species that are the subject of a bacterial testing method according to an embodiment of the present invention. [Figure 8]FIG. 1 shows the base sequence of a probe for detecting various Listeria species (Listeria genus, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria locoutiae, Listeria siligeri, and Listeria welshimeri) that are the subject of a bacterial testing method according to an embodiment of the present invention. [Figure 9] FIG. 1 shows the base sequences of a probe for detecting various Listeria genus bacteria (Listeria floridensis, Listeria wehenstephanensis, Listeria cornelensis, Listeria riparia, Listeria grandensis, Listeria booriae, Listeria newyorkensis, Listeria aquatica, Listeria freshmannii, and Listeria marti) that are the subject of a bacterial testing method according to an embodiment of the present invention, and a probe for detecting a plasmid as an internal control. [Figure 10] FIG. 1 shows the detection results (sample Nos. 1 to 14) of Test 1 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 11] FIG. 1 shows the detection results (sample Nos. 15 to 28) of Test 1 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 12] FIG. 1 shows the detection results (sample numbers 29 to 43) of test 1 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 13] FIG. 1 shows the detection results (sample Nos. 1 to 14) of Test 1 using the probes (SEQ ID NOs: 32 to 45) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 14] FIG. 1 shows the detection results (sample Nos. 15 to 28) of Test 1 using the probes (SEQ ID NOs: 32 to 45) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 15] FIG. 1 shows the detection results (sample numbers 29 to 43) of Test 1 using the probes (SEQ ID NOs: 32 to 45) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 16] FIG. 1 shows sample numbers and test strains of samples (Staphylococcus aureus, Escherichia coli, Shigella, Nagvibrio, and Salmonella) used in Test 2 for checking the presence or absence of false positives in the bacterial testing method according to an embodiment of the present invention. [Figure 17] FIG. 1 shows the detection results (sample numbers 44 to 59) of test 2 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 18] FIG. 1 shows the detection results (sample Nos. 44 to 59) of Test 2 using the probes (SEQ ID NOs: 32 to 45) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 19] FIG. 1 shows the test strains, DNA concentrations, and whether or not an internal control (IC) was added for samples (sample numbers 60 to 65) used in Test 3 for the bacteria testing method etc. according to an embodiment of the present invention. [Figure 20] FIG. 1 shows the test strains, DNA concentrations, and whether or not an internal control (IC) was added for samples (sample numbers 66 to 71) used in Test 3 for the bacteria testing method etc. according to an embodiment of the present invention. [Figure 21] FIG. 1 shows the detection results (sample numbers 60 to 71) of test 3 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 22] FIG. 1 shows the detection results (sample Nos. 60 to 71) of Test 3 using the probes (SEQ ID NOs: 32 to 45) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 23] This figure shows the base sequence (sequence number 46) of the IGS region of Listeria marti, which is the subject of the bacterial testing method of an embodiment of the present invention, and the base sequence (sequence number 47) of a probe for detecting Listeria marti, which were analyzed in the examples. [Figure 24] FIG. 1 shows the detection results (sample Nos. 1a to 14a) of Test 4 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Diagram 25] FIG. 1 shows the detection results (sample numbers 15a to 28a) of Test 4 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 26] FIG. 1 shows the detection results (sample Nos. 29a to 43a) of Test 4 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 27] FIG. 1 shows the detection results (sample Nos. 1a to 14a) of Test 4 using the probes (SEQ ID NOs: 32 to 45, 47) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 28] FIG. 1 shows the detection results (sample Nos. 15a to 28a) of Test 4 using the probes (SEQ ID NOs: 32 to 45, 47) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Figure 29] FIG. 1 shows the detection results (sample Nos. 29a to 43a) of Test 4 using the probes (SEQ ID NOs: 32 to 45, 47) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Diagram 30] FIG. 1 shows the detection results (sample Nos. 44a to 59a) of Test 5 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Diagram 31] FIG. 1 shows the detection results (sample Nos. 44a to 59a) of Test 5 using the probes (SEQ ID NOs: 32 to 45, 47) used in the bacteria inspection method etc. according to the embodiment of the present invention. [Diagram 32] FIG. 1 shows the detection results (sample Nos. 63a, 65a, 72a, 69a, 71a, and 73a) of Test 6 using the probes (SEQ ID NOs: 18 to 31) used in the bacteria inspection method according to an embodiment of the present invention. [Diagram 33] FIG. 1 shows the detection results (sample Nos. 63a, 65a, 72a, 69a, 71a, and 73a) of Test 6 using the probes (SEQ ID NOs: 32 to 45 and 47) used in the bacteria inspection method and the like according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, an embodiment of the bacteria inspection method, the bacteria inspection microarray, the bacteria inspection kit, the bacteria inspection probe set, and the bacteria inspection primer set 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.

[0018] The method for testing bacteria of the present embodiment is a method for testing the presence of bacteria in a sample, the target bacteria being selected from the Listeria genus bacteria described in the following (1) to (10): (1) Listeria floridensis, (2) Listeria weihenstephanensis, (3) Listeria cornellensis, (4) Listeria riparia, (5) Listeria grandensis, (6) Listeria booriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria fleischmannii, (10) Listeria marthii, This method is (a) preparing a reaction solution containing a primer set for amplifying the IGS (intergenic spacer) region of the rRNA gene; and (b) an amplification step of amplifying nucleic acid of the target bacterium contained in the sample using the reaction solution obtained in the preparation step.

[0019] Furthermore, the target bacteria preferably further include bacteria selected from the Listeria genus bacteria described in (11) to (17) below. (11) Listeria grayi (12) Listeria innocua (13) Listeria ivanovii (14) Listeria monocytogenes (15) Listeria rocourtiae (16) Listeria seeligeri (17) Listeria welshimeri

[0020] The genome sequences of the above 10 target bacteria (1) to (10) had not been reported in the DNA Data Bank of Japan (DDBJ) at the time of filing the present application. Therefore, in developing the bacterial testing method, bacterial testing microarray, bacterial testing kit, bacterial testing probe set, and bacterial testing primer set of the present embodiment, the IGS (intergenic spacer) region of these 10 bacterial species was first analyzed. The IGS region is a region located between the 16SrRNA and 23SrRNA of the rRNA gene, and we analyzed the base sequence of the region where we assumed that species-specific sequences exist.

[0021] Specifically, the IGS regions of the above 10 bacterial species were analyzed using the primer set shown in SEQ ID NOs: 1 and 2 in Figure 1. In the figure, in "F / R," F indicates the forward primer and R indicates the reverse primer.

[0022] The resulting nucleotide sequences are shown in Figures 2 and 3. Figure 2 shows the nucleotide sequences (SEQ ID NOs: 3 to 7, in order) in the IGS regions of Listeria floridensis, Listeria wehenstephanensis, Listeria cornelensis, Listeria riparia, and Listeria grandensis. Figure 3 shows the nucleotide sequences (SEQ ID NOs: 8 to 12, in order) in the IGS regions of Listeria boehriae, Listeria newyorkensis, Listeria aquatica, Listeria freshmannii, and Listeria marti.

[0023] Furthermore, for Listeria marti, in order to find a more effective probe, the IGS region was analyzed again to obtain the base sequence shown in FIG. 23 (SEQ ID NO: 46).

[0024] In addition, although not shown, the base sequences in the IGS regions of Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria locoutiae, Listeria siligeri, and Listeria welshimeri were obtained from DDBJ. The base sequences in the IGS regions of these 17 types of Listeria were then compared to determine the design locations for the probe set and primer set for testing bacteria of this embodiment.

[0025] Specifically, the primer set for amplifying the IGS (intergenic spacer) region of the rRNA gene contained in the reaction solution prepared in the above preparation step consists of a forward primer consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 16 in Figure 7, and a reverse primer consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 17.

[0026] In addition, the probe set selected from the IGS (intergenic spacer) region of the rRNA gene, which hybridizes with the nucleic acid amplified by the above amplification process, consists of at least one selected from the group consisting of probes consisting of oligonucleotides having the base sequences shown in SEQ ID NOs: 18 to 31 in Figure 8 and probes consisting of oligonucleotides having the base sequences shown in SEQ ID NOs: 32 to 43 in Figure 9. The probe for detecting the plasmid as an internal control used to determine whether DNA amplification by PCR was performed properly is shown in SEQ ID NO: 45 in FIG.

[0027] As for Listeria marti, a more effective probe was designed consisting of an oligonucleotide having the base sequence shown in SEQ ID NO:47 of FIG. In other words, it is also preferable that the probe set selected from the IGS (intergenic spacer) region of the rRNA gene, which hybridizes with the nucleic acid amplified by the above-mentioned amplification step, consists of at least one selected from the group consisting of probes consisting of oligonucleotides having the base sequences shown in SEQ ID NOs: 18 to 31 and probes consisting of oligonucleotides having the base sequences shown in SEQ ID NOs: 32 to 43 and 47.

[0028] The reaction solution contains genomic DNA extracted from cells of the Listeria genus. Genomic DNA can be extracted by a general method such as a kit or a DNA extraction device.

[0029] In the above amplification step, the target region 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.

[0030] 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.

[0031] 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.

[0032] In this embodiment, hybridization can be carried out as follows. That is, the amplification products obtained by the above amplification process are dropped onto the microarray for bacteria testing of this embodiment, and the presence or absence of the amplification products is confirmed by detecting the labels of the amplification products that have hybridized to the probes arranged thereon, thereby making it possible to identify Listeria bacteria present in the environment to be tested.

[0033] 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.

[0034] Furthermore, the microarray for testing bacteria of this embodiment is used to test for the presence of bacteria in a sample, and is characterized in that at least one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria genus bacteria described in (1) to (10) above is immobilized thereon. Furthermore, it is preferable that the microarray for bacterial testing of this embodiment further has at least one probe having a base sequence shown in SEQ ID NO: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria bacteria described in (11) to (17) above immobilized thereon. Furthermore, the microarray for bacteria testing of this embodiment may have all or part of the probes having the base sequences shown in SEQ ID NOs: 18 to 43 and 47 immobilized thereon.

[0035] Furthermore, the microarray for testing bacteria of this embodiment can be produced by using probes having the base sequences shown in SEQ ID NOs: 18 to 43 and 47, according to an existing general method. 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.

[0036] Furthermore, the bacteria testing kit of this embodiment is for testing the presence of bacteria in a sample, and is characterized by including a microarray on which at least one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43, and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria genus bacteria described in (1) to (10) above is immobilized, and a primer set consisting of primers having the base sequences shown in SEQ ID NOs: 16 and 17 for amplifying nucleic acid that hybridizes with the probe immobilized on the microarray. Furthermore, it is preferable that the bacteria testing kit of this embodiment further includes at least one probe having a base sequence as shown in SEQ ID NO: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria bacteria described in (11) to (17) above, immobilized thereon.

[0037] Furthermore, the probe set for testing bacteria of this embodiment is used to test for the presence of bacteria in a sample, and is characterized by comprising at least one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria described in (1) to (10) above. In addition, it is preferable that the probe set for testing bacteria of this embodiment further includes at least one of the probes having the base sequences shown in SEQ ID NOs: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria described in (11) to (17) above.

[0038] Furthermore, the primer set for testing bacteria of this embodiment is used to test for the presence of bacteria in a sample, and is characterized by comprising primers having the base sequences shown in SEQ ID NOs: 16 and 17 for amplifying nucleic acid that hybridizes with at least any one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria described in (1) to (10) above.

[0039] Furthermore, it is also preferable that the above-mentioned probe set includes at least one of the following probes (A) and (B), two or more of these, or all of these. (A) Each probe consisting of the base sequence shown in SEQ ID NOs: 18 to 43 and 47 (B) Each probe having an identity of 90% or more to the base sequence of each probe in (A) above.

[0040] The probe sets for testing bacteria according to this embodiment are all 21 to 36 bases long and can be synthesized by a general DNA synthesizer. The probes consisting of the base sequences shown in SEQ ID NOs: 18 to 43 and 47 used in the examples described later were all synthesized by a DNA synthesizer. The primer set for testing bacteria in this embodiment is also 19 to 22 bases long, and the primers consisting of the base sequences shown in SEQ ID NOs: 16 and 17 used in PCR in the examples were synthesized by a DNA synthesizer. The primers consisting of the base sequences shown in SEQ ID NOs: 1 and 2 used in PCR in the examples were also synthesized by a DNA synthesizer.

[0041] In this embodiment, the probes consisting of the base sequences shown in SEQ ID NOs: 18 to 43 and 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: 18 to 43 and 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: 18 to 43 and 47 can be used. Also, probes having base sequences complementary to these probes or probes consisting of the base sequences shown in SEQ ID NOs: 18 to 43 and 47 can be used.

[0042] The stringent conditions refer to conditions under which specific hybrids are formed and non-specific hybrids are not formed. For example, the conditions include conditions under which DNA having high homology (homology of 90% or more, preferably 95% or more) to DNA consisting of the nucleotide sequences shown in SEQ ID NOs: 18 to 43 and 47 hybridizes with DNA consisting of a nucleotide sequence complementary to each of the DNA consisting of the nucleotide sequences shown in SEQ ID NOs: 18 to 43 and 47. Usually, the conditions refer to a case where 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 the complete hybrid. Regarding the stringent conditions, the conditions described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), especially Section 11.45 "Conditions for Hybridization of Oligonucleotide Probes", can be used.

[0043] According to the bacterial testing method, the bacterial testing microarray, the bacterial testing kit, the bacterial testing probe set, and the bacterial testing primer set of this embodiment described above, it is possible to specifically and accurately identify all 17 types of Listeria, including the above-mentioned Listeria strains (1) to (10), which could not be tested for until now. EXAMPLES

[0044] Below, we will specifically explain the experiments conducted to develop the bacterial testing method, bacterial testing microarray, bacterial testing kit, bacterial testing probe set, and bacterial testing primer set according to the embodiments of the present invention, as well as the tests conducted to confirm the effects of these.

[0045] [Experiment 1] The genome sequences of the Listeria bacteria described in (1) to (10) above, which are the targets of the bacteria testing method and the like of this embodiment, had not been reported to the DNA Data Bank of Japan (DDBJ) at the time of filing this application. For this purpose, we first analyzed the IGS (intergenic spacer) regions of these 10 bacterial species.

[0046] The following target bacterial strains were used, all obtained from DSM (Deutsche Sammlung von Mikroorganismen). (1) Listeria floridensis (DSM 26687) (2) Listeria wehenstephanensis (DSM 24698) (3) Listeria cornelensis (DSM 26689) (4) Listeria riparia (DSM 26685) (5) Listeria grandensis (DSM 26688) (6) Listeria boehriae (DSM 28860) (7) Listeria newyorkensis (DSM 28861) (8) Listeria aquatica (DSM 26686) (9) Listeria freshmanni (DSM 25003) (10) Listeria Marti (DSM 23813)

[0047] These Listeria strains were individually cultured using Brain Heart Infusion (Difco) at 37°C for 1 to 2 days. Next, genomic DNA of Listeria was extracted according to the instructions of the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (RBC Bioscience).

[0048] The PCR reaction solution used contained a primer set consisting of a forward primer with the nucleotide sequence shown in SEQ ID NO: 1 for amplifying the IGS region of Listeria DNA and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 2. These primers were synthesized by Sigma-Aldrich Japan LLC.

[0049] As a PCR reaction solution, 20 μl of the following composition was prepared for each strain. 1. PCR buffer (QIAGEN) 2.0 μl 2.dNTP mix (Cat No.BR0600503, manufactured by biotechrabbit) 0.4μl 3. 10 μM forward primer (SEQ ID NO: 1) 0.2 μl 4. 10 μM reverse primer (SEQ ID NO: 2) 0.2 μl 5. HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μl 6.Template DNA (10ng / μl) 2.0μl 7. Sterile water (add water until the total volume is 20.0 μl)

[0050] Using each of the above PCR reaction solutions, DNA was amplified under the following conditions using a nucleic acid amplifier (Master cycler ep, Eppendorf). (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

[0051] Next, the PCR amplification products were electrophoresed using an electrophoresis device (MupidexU, Mupid Co., Ltd.) A gel imaging device (SCOPE WD, Optima Co., Ltd.) was used as a detector for the amplification products. Next, the amplified products were excised and purified using an excision / DNA purification kit, HiYield Gel / PCR DNA Fragments Extraction Kit (RBC Bioscience).

[0052] Furthermore, the purified DNA was sequenced using a DNA sequencing reaction apparatus (Big Dye® X terminator, Thermo Fisher Scientific). After sequencing, the DNA was purified and electrophoresed and analyzed using a DNA sequencer (3130xl Genetic Analyzer, 3730xl DNA Analyzer, Thermo Fisher Scientific). These sequences, electrophoresis, and analyses were performed by FASMAC Corporation. The resulting base sequences are shown in Figures 2, 3, and 23.

[0053] FIG. 2 shows the base sequences (SEQ ID NOs: 3 to 7, in order) in the IGS regions of (1) Listeria floridensis, (2) Listeria wehenstephanensis, (3) Listeria cornelensis, (4) Listeria riparia, and (5) Listeria grandensis. FIG. 3 also shows the base sequences (SEQ ID NOs: 8 to 12, in order) in the IGS regions of (6) Listeria boehriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria freshmannii, and (10) Listeria marti. Furthermore, Figure 23 shows (10) the base sequence (sequence number 46) in the IGS region of Listeria marti. Based on these base sequences and the like, a probe set for testing bacteria and a primer set for testing bacteria of this embodiment were designed.

[0054] [Test 1] Tests were carried out to confirm the effects of the bacteria inspection method, the bacteria inspection microarray, the bacteria inspection kit, the bacteria inspection probe set, and the bacteria inspection primer set of the present embodiment. The probe set for testing bacteria in this embodiment was designed by comparing the IGS regions of all 17 types of Listeria and identifying highly specific regions. In this test, the specificity (inclusiveness) of these probe sets was verified.

[0055] The following target bacterial strains were used, as shown in Figures 4 and 5. These were obtained from the following institutions. ATCC: American Type Culture Collection NCTC: National Collection of Type Cultures DSM: Deutsche Sammlung von Mikroorganismen GTC: Gifu University RIMD: Osaka University Research Institute for Microbial Diseases

[0056] Sample numbers 1 to 6: Listeria grayi, ATCC 25401, ATCC 700545, ATCC 19120, ATCC 25400, ATCC 25403, ATCC 19120 Sample No. 7~8: Listeria innocua, ATCC 33090, NCTC 11288 Sample numbers 9 to 10: Listeria ivanovii subsp. ivanovii ATCC 19119, Listeria ivanovii subsp. londoniensis ATCC BAA-139 Sample numbers 11 to 22: Listeria monocytogenes, ATCC 7644, SLR 2249, ATCC 19112, ATCC 19111, ATCC 15313, ATCC 19115, ATCC 13932, NCTC 10890, ATCC BAA-751, ATCC 19114, ATCC 19118, NCTC 11994 Sample No. 23: Listeria rocourtiae DSM 22097 Sample numbers 24 to 27: Listeria seeligeri, ATCC 51334, ATCC 35967, ATCC 51335, ATCC 35967 (sample numbers 25 and 27 are the same strain) Sample numbers 28 to 31: Listeria welshimeri, ATCC 43549, ATCC 49591, DSM 15452, ATCC 35897

[0057] Sample No. 32: Listeria floridensis DSM 26687 Sample numbers 33 to 34: Listeria weihenstephanensis, DSM 24698 and DSM 24699, respectively Sample No. 35: Listeria cornellensis DSM 26689 Sample No. 36: Listeria riparia DSM 26685 Sample No. 37: Listeria grandensis DSM 26688 Sample No. 38: Listeria booriae DSM 28860 Sample No. 39: Listeria newyorkensis DSM 28861 Sample No. 40: Listeria aquatica DSM 26686 Sample No. 41: Listeria fleischmannii subsp. fleischmannii DSM 25003 Sample No. 42: Listeria marthii DSM 23813 Sample No. 43: Negative control (sterile water was added instead of template)

[0058] These Listeria strains were individually cultured using Brain Heart Infusion (Difco) at 37°C for 1 to 2 days. Next, genomic DNA of Listeria was extracted according to the instructions of the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (RBC Bioscience).

[0059] The PCR reaction solution used consistently contained a primer set consisting of a forward primer having the base sequence shown in SEQ ID NO: 16 and a reverse primer having the base sequence shown in SEQ ID NO: 17 for amplifying the IGS region of Listeria DNA. In addition, to determine whether the DNA was amplified properly by the PCR reaction, an internal control was used. Plasmid DNA was used as the internal control, and a target DNA having the base sequence shown in SEQ ID NO: 15 was amplified using a primer set consisting of a forward primer having the base sequence shown in SEQ ID NO: 13 and a reverse primer having the base sequence shown in SEQ ID NO: 14. These primers were synthesized by Sigma-Aldrich Japan LLC. The target DNA of the plasmid was synthesized by FASMAC Co., Ltd.

[0060] As a PCR reaction solution, 20 μl of the following composition was prepared for each strain. 1. PCR buffer (QIAGEN) 2.0 μl 2.dNTP mix (Cat No.BR0600503, manufactured by biotechrabbit) 0.4μl 3. 10 μM forward primer (SEQ ID NO: 16) 1.0 μl 4. 10 μM reverse primer (SEQ ID NO: 17) 0.1 μl 5. 10 μM IC forward primer (SEQ ID NO: 13) 0.5 μl 6.1 μM IC reverse primer (SEQ ID NO: 14) 1.0 μl 7. 100 pg / μl plasmid solution (SEQ ID NO: 15) 1.0 μl 8. HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μl 9. Template DNA (10 ng / PCR reaction solution) 2.0 μl 10. Sterile water (add water until the total volume is 20.0 μl)

[0061] Using each of the above PCR reaction solutions, DNA was amplified under the following conditions using a nucleic acid amplifier (Master cycler ep, Eppendorf). (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

[0062] A DNA chip (manufactured by Toyo Seikan Group Holdings Co., Ltd.) was used as the microarray for testing Listeria bacteria, and the probes of SEQ ID NOs: 18 to 45 shown in Figures 8 and 9 were arranged on it. Each probe was synthesized by Sigma-Aldrich Japan LLC. Each probe was immobilized on the substrate using a DNA spotter.

[0063] SEQ ID NO: 18 shows the base sequence of a probe for detecting the genus Listeria. SEQ ID NO: 19 shows the base sequence of a probe for detecting Listeria greyi. SEQ ID NOs: 20 to 21 show the base sequence of a probe for detecting Listeria innocua. SEQ ID NOs: 22 to 23 show the base sequence of a probe for detecting Listeria ivanovii. SEQ ID NOs: 24 to 25 show the base sequence of a probe for detecting Listeria monocytogenes. SEQ ID NOs: 26 to 28 show the base sequence of a probe for detecting Listeria locoutiae. SEQ ID NO: 29 shows the base sequence of a probe for detecting Listeria siligeri. SEQ ID NOs: 30 to 31 show the base sequence of a probe for detecting Listeria welshimeri.

[0064] SEQ ID NO: 32-33 show the base sequence of a probe for detecting Listeria floridensis. SEQ ID NO: 34 shows the base sequence of a probe for detecting Listeria wehenstephanensis. SEQ ID NO: 35 shows the base sequence of a probe for detecting Listeria cornelensis. SEQ ID NO: 36 shows the base sequence of a probe for detecting Listeria riparia. SEQ ID NO: 37 shows the base sequence of a probe for detecting Listeria grandensis. SEQ ID NO: 38-39 show the base sequence of a probe for detecting Listeria booriae. SEQ ID NO: 40-41 show the base sequence of a probe for detecting Listeria newyorkensis. SEQ ID NO: 42 shows the base sequence of a probe for detecting Listeria aquatica. SEQ ID NO: 43 shows the base sequence of a probe for detecting Listeria freshmannii. SEQ ID NO: 44 shows the base sequence of a probe for detecting Listeria marti. SEQ ID NO: 45 shows the base sequence of a probe for detecting a plasmid.

[0065] The reaction solution for hybridization was prepared as follows. 2 μL of hybridization buffer and 4 μL of the amplified 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 (Dako) under hybridization conditions of 45° C. / 1 hour.

[0066] 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. The results are shown in Figures 10 to 15. In these figures, when the S / N ratio value of any of the probe sets corresponding to the Listeria bacteria to be detected was positive, it was determined that the Listeria bacteria had been detected. In addition, in these figures, positive S / N ratio values ​​are indicated by a thick frame.

[0067] As shown in these figures, the probe shown in SEQ ID NO: 18 is positive for all 17 types of Listeria. It is also clear that the probes shown in SEQ ID NOs: 19 to 43 are able to detect the respective target bacteria of the genus Listeria. Thus, it was revealed that the probe set for testing bacteria of this embodiment exhibits excellent specificity (inclusiveness) except for SEQ ID NO: 44 for detecting Listeria marti. Therefore, for Listeria marti, a new probe was developed as shown in SEQ ID NO: 47. Then, in order to confirm the specificity (comprehensiveness) of this embodiment including this probe, Test 4 described below was carried out.

[0068] [Test 2] In this test, the specificity (exclusivity) of the probe set for testing bacteria of this embodiment was verified. As the target bacterial strains, the following were used, as shown in FIG.

[0069] Sample No. 44: Staphylococcus aureus GTC DY 0073 Sample No. 45: Escherichia coli RIMD 0509516 Sample No. 46: Shigella dysenteriae GTC 00100 Sample No. 47: Vibrio mimicus RIMD 2218001

[0070] Sample numbers 48-58: Salmonella in order Salmonella enterica subsp salamae GTC 1731, Salmonella enterica subsp enterica serovar Arizonae GTC 1732, Salmonella enterica subsp enterica serovar Cerro GTC 2554, Salmonella enterica subsp enterica serovar Landau GTC 2614, Salmonella enterica subsp enterica serovar Arizonae GTC 3820, Salmonella enterica subsp enterica serovar Enteritidis GTC 3838, Salmonella enterica subsp enterica serovar Aberdeen GTC 12694, Salmonella enterica subsp enterica serovar Kentucky GTC 12786, Salmonella enterica subsp enterica serovar Essen GTC 12814, Salmonella enterica subsp enterica va Newport GTC 12819, Salmonella enterica subsp enterica serovar Niarembe GTC 12820 Sample No. 59: Negative control (sterile water was added instead of template)

[0071] These bacteria were cultured individually for each strain. The culture was carried out at 37°C for 1 to 2 days using Brain Heart Infusion (Difco) or 1.0% sodium chloride-added nutrient bouillon medium (Nissui Pharmaceutical). Next, the genomic DNA of these bacteria was extracted according to the instructions for the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (RBC Bioscience).

[0072] As in Test 1, the PCR reaction solution used contained a primer set consisting of a forward primer having the base sequence shown in SEQ ID NO: 16 and a reverse primer having the base sequence shown in SEQ ID NO: 17 for amplifying the IGS region of Listeria DNA. In addition, to determine whether the DNA was amplified properly by the PCR reaction, an internal control was used. Plasmid DNA was used as the internal control, and a target DNA having the base sequence shown in SEQ ID NO: 15 was amplified using a primer set consisting of a forward primer having the base sequence shown in SEQ ID NO: 13 and a reverse primer having the base sequence shown in SEQ ID NO: 14. These primers were synthesized by Sigma-Aldrich Japan LLC. The target DNA of the plasmid was synthesized by FASMAC Co., Ltd.

[0073] As a PCR reaction solution, 20 μl of the following composition was prepared for each strain. 1. PCR buffer (QIAGEN) 2.0 μl 2.dNTP mix (Cat No.BR0600503, manufactured by biotechrabbit) 0.4μl 3. 10 μM forward primer (SEQ ID NO: 16) 1.0 μl 4. 10 μM reverse primer (SEQ ID NO: 17) 0.1 μl 5. 10 μM IC forward primer (SEQ ID NO: 13) 0.5 μl 6.1 μM IC reverse primer (SEQ ID NO: 14) 1.0 μl 7. 100 pg / μl plasmid solution (SEQ ID NO: 15) 1.0 μl 8. HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μl 9. Template DNA (10 ng / PCR reaction solution) 2.0 μl 10. 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, Eppendorf). (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] As in Test 1, a DNA chip (manufactured by Toyo Seikan Group Holdings Co., Ltd.) was used as the microarray for testing, and the probes of SEQ ID NOs: 18 to 45 shown in Figures 8 and 9 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.

[0076] The reaction solution for hybridization was prepared in the same manner as in Test 1, as follows. 2 μL of hybridization buffer and 4 μL of the amplified 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 (Dako) under hybridization conditions of 45° C. / 1 hour.

[0077] 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. The results are shown in Figures 17 to 18. In these figures, positive S / N ratio values ​​are indicated by bold frames.

[0078] As shown in these figures, the internal control probe shown in SEQ ID NO:45 was positive for all samples shown in sample numbers 44 to 59. On the other hand, the other probes showed negative results for all bacteria in sample numbers 44 to 58, and no false positives were observed. Thus, it has become clear that the probe set for testing bacteria of this embodiment exhibits excellent specificity (exclusivity).

[0079] [Test 3] In this test, a sample containing a mixture of multiple Listeria species was used to verify the specificity (simultaneous detection) of the probe set for testing bacteria of this embodiment.

[0080] As the target bacterial strains, the following combinations were used for each sample number, as shown in Figures 19 and 20. Sample numbers 60 to 65 were used as internal controls to which no plasmid was added, and sample numbers 66 to 71 were used to which an internal control was added.

[0081] Listeria grayi ATCC 19120 Listeria innocua NCTC 11288 Listeria ivanovii subsp. ivanovii ATCC 19119 Listeria monocytogenes NCTC 11994 Listeria rocourtiae DSM 22097 Listeria seeligeri ATCC 35967 Listeria welshimeri ATCC 35897

[0082] Listeria floridensis DSM 26687 Listeria weihenstephanensis DSM 24698 Listeria cornellensis DSM 26689 Listeria riparia DSM 26685 Listeria grandensis DSM 26688 Listeria booriae DSM 28860 Listeria newyorkensis DSM 28861 Listeria aquatica DSM 26686 Listeria fleischmannii subsp. fleischmannii DSM 25003 Listeria marthii DSM 23813

[0083] Sample No. 60, 61: Listeria boreale, Listeria newyorkensis, Listeria freshmannii Sample No. 62: Listeria floridensis, Listeria wehenstephanensis, Listeria cornelensis, Listeria riparia, Listeria grandensis, Listeria boeoriae, Listeria newyorkensis, Listeria aquatica, Listeria freshmannii Sample No. 63: Sample No. 62 with the addition of Listeria Marti Sample No. 64: All 17 types of Listeria spp. except Listeria marti Sample No. 65: All 17 types of Listeria spp. Samples 66-71 contain the same strains as samples 60-65, respectively, and contain an internal control.

[0084] These Listeria strains were individually cultured using Brain Heart Infusion (Difco) at 37°C for 1 to 2 days. Next, genomic DNA of Listeria was extracted according to the instructions of the DNA extraction kit, RBC Genomic DNA Extraction Kit Mini (RBC Bioscience).

[0085] As in Test 1, the PCR reaction solution used contained a primer set consisting of a forward primer having the base sequence shown in SEQ ID NO: 16 and a reverse primer having the base sequence shown in SEQ ID NO: 17 for amplifying the IGS region of Listeria DNA. In addition, in order to determine whether or not the DNA was properly amplified by the PCR reaction, an internal control was used in sample numbers 66 to 71. Plasmid DNA was used as the internal control, and a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 13 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 14 was used to amplify the target DNA consisting of the base sequence shown in SEQ ID NO: 15. These primers were synthesized by Sigma-Aldrich Japan LLC. The target DNA of the plasmid was synthesized by FASMAC Co., Ltd.

[0086] As the PCR reaction solution, 20 μl of the following composition was prepared for each sample number. 1. PCR buffer (QIAGEN) 2.0 μl 2.dNTP mix (Cat No.BR0600503, manufactured by biotechrabbit) 0.4μl 3. 10 μM forward primer (SEQ ID NO: 16) 1.0 μl 4. 10 μM reverse primer (SEQ ID NO: 17) 0.1 μl 5. 10 μM IC forward primer (SEQ ID NO: 13) 0.5 μl 6.1 μM IC reverse primer (SEQ ID NO: 14) 1.0 μl 7. 100 pg / μl plasmid solution (SEQ ID NO: 15) 1.0 μl 8. HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 μl 9. Template DNA (10 ng / PCR reaction solution) 2.0 μl 10. Sterile water (add water until the total volume is 20.0 μl) For sample numbers 60 and 66, the template DNA was 10 ng of each strain per PCR reaction solution, and for sample numbers 61 to 65 and 67 to 71, the template DNA was 1 ng of each strain per PCR reaction solution.

[0087] Using each of the above PCR reaction solutions, DNA was amplified under the following conditions using a nucleic acid amplifier (Master cycler ep, Eppendorf). (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

[0088] As in Test 1, a DNA chip (manufactured by Toyo Seikan Group Holdings Co., Ltd.) was used as the microarray for testing Listeria, and the probes of SEQ ID NOs: 18 to 45 shown in Figures 8 and 9 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.

[0089] The reaction solution for hybridization was prepared in the same manner as in Test 1, as follows. 2 μL of hybridization buffer and 4 μL of the amplified 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 (Dako) under hybridization conditions of 45° C. / 1 hour.

[0090] 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. The results are shown in Figures 21 to 22. In these figures, when the S / N ratio value of any of the probe sets corresponding to the Listeria bacteria to be detected was positive, it was determined that the Listeria bacteria had been detected. In addition, in these figures, positive S / N ratio values ​​are indicated by a thick frame.

[0091] As shown in these figures, in sample numbers 60 to 62, 64, 66 to 68, and 70, all Listeria genus bacteria were detected simultaneously. In samples 63, 65, 69, and 71, Listeria marti could not be detected simultaneously, but all other Listeria species were detected simultaneously, demonstrating that the bacterial testing probe set of this embodiment generally exhibits excellent specificity even in simultaneous detection.

[0092] [Test 4] Tests to confirm the specificity (inclusiveness) of the bacterial testing method, bacterial testing microarray, bacterial testing kit, bacterial testing probe set, and bacterial testing primer set of this embodiment, including the new probe shown in sequence number 47 for detecting Listeria marti, were conducted in the same manner as in Test 1.

[0093] That is, the same target bacterial strains as those in Test 1 were used, and samples identical to Sample Nos. 1 to 43 in Test 1 were prepared as Sample Nos. 1a to 43a, respectively, and cultured in the same manner as in Test 1. Then, PCR was performed in the same manner as in Test 1 using genomic DNA extracted from the obtained Listeria bacteria.

[0094] As a microarray for testing Listeria, a DNA chip was used on which, in addition to the probe of Test 1, the probe of SEQ ID NO: 47 for detecting Listeria marti was arranged. Hybridization was then carried out and the fluorescence intensity of each probe was measured, the results of which are shown in Figures 24 to 29.

[0095] As shown in these figures, the probe shown in SEQ ID NO: 18 is positive for all 17 types of Listeria. It is also clear that the probes shown in SEQ ID NOs: 19 to 43 and 47 are able to detect the respective target bacteria of the genus Listeria. Thus, it was revealed that the probe set for testing bacteria of this embodiment exhibits excellent specificity (inclusiveness).

[0096] [Test 5] Tests to verify the specificity (exclusivity) of the bacterial testing method, bacterial testing microarray, bacterial testing kit, bacterial testing probe set, and bacterial testing primer set of this embodiment, including the new probe shown in sequence number 47 for detecting Listeria marti, were conducted in the same manner as in Test 2.

[0097] That is, the same target bacterial strains as those in Test 2 were used, and samples identical to sample numbers 44 to 59 in Test 2 were prepared as sample numbers 44a to 59a, respectively, and cultured in the same manner as in Test 2. Then, PCR was performed in the same manner as in Test 2 using genomic DNA extracted from the obtained Listeria bacteria.

[0098] As a microarray for testing Listeria, a DNA chip was used on which, in addition to the probe of Test 2, the probe of SEQ ID NO: 47 for detecting Listeria marti was arranged. Hybridization was then carried out and the fluorescence intensity of each probe was measured, the results of which are shown in Figures 30 and 31.

[0099] As shown in these figures, the internal control probe shown in SEQ ID NO: 45 was positive for all samples shown in sample numbers 44a to 59a. On the other hand, the other probes showed negative results for all bacteria in sample numbers 44a to 58a, and no false positives were observed. Thus, it has become clear that the probe set for testing bacteria of this embodiment exhibits excellent specificity (exclusivity).

[0100] [Test 6] Tests to verify the specificity (simultaneous detection) of the bacterial testing method, bacterial testing microarray, bacterial testing kit, bacterial testing probe set, and bacterial testing primer set of this embodiment, including the new probe shown in sequence number 47 for detecting Listeria marti, were conducted in the same manner as in Test 3.

[0101] That is, using the same target bacterial strain as in Test 3, samples identical to Sample Nos. 63, 65, 69, and 71 in Test 3 were prepared as Sample Nos. 63a, 65a, 69a, and 71a, respectively, and cultured in the same manner as in Test 3. Sample Nos. 72a and 73a were also prepared as negative controls (sterile water was added instead of template). Sample No. 72a was an internal control to which no plasmid was added, and Sample No. 73a was an internal control to which an internal control was added. Then, PCR was performed in the same manner as in Test 3 using genomic DNA extracted from the obtained Listeria bacteria.

[0102] As a microarray for testing Listeria, a DNA chip was used on which, in addition to the probe of Test 3, the probe of SEQ ID NO: 47 for detecting Listeria marti was arranged. Hybridization was then carried out and the fluorescence intensity of each probe was measured, the results of which are shown in Figures 32 and 33.

[0103] As shown in these figures, in samples 63a, 65a, 69a, and 71a, all Listeria species were simultaneously detected except for the probe of SEQ ID NO: 44 for detecting Listeria marti. In addition, Listeria marti was simultaneously detected by the probe of SEQ ID NO: 47. Thus, it has become clear that the probe set for testing bacteria according to this embodiment exhibits excellent specificity even in simultaneous detection.

[0104] 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 probes arranged on the microarray for testing bacteria of this embodiment are 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 Listeria genus 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]

[0105] INDUSTRIAL APPLICABILITY The present invention can be suitably used for specific and multiple detection of Listeria bacteria in environmental inspection, food inspection, and the like.

Claims

1. A method for testing the presence of bacteria in a sample, the target bacteria being selected from the Listeria genus bacteria described in (1) to (10) below, (1) Listeria floridensis, (2) Listeria weihenstephanensis, (3) Listeria cornellensis, (4) Listeria riparia, (5) Listeria grandensis, (6) Listeria booriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria fleischmannii, (10) Listeria marthii, The method comprises: (a) preparing a reaction solution containing a primer set for amplifying an IGS (intergenic spacer) region of an rRNA gene; and (b) an amplification step of amplifying the nucleic acid of the target bacterium contained in the sample using the reaction solution obtained in the preparation step, The presence of the Listeria bacteria described in (1) to (10) is simultaneously examined by hybridization between all of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria bacteria described in (1) to (10) and the nucleic acid amplified by the amplification step. A method for testing bacteria.

2. The method for testing bacteria according to claim 1, characterized in that the target bacteria further include bacteria selected from the Listeria genus bacteria described in (11) to (17) below, and the probe further includes at least one of the probes having the base sequences shown in SEQ ID NOs: 19 to 31. (11) Listeria grayi (12) Listeria innocua (13) Listeria ivanovii (14) Listeria monocytogenes (15) Listeria rocourtiae (16) Listeria seeligeri (17) Listeria welshimeri

3. 3. The method for detecting bacteria according to claim 1, wherein the primer set contained in the reaction solution consists of primers having the base sequences shown in SEQ ID NOs: 16 and 17.

4. The method for testing bacteria according to any one of claims 1 to 3, characterized in that the presence of the target bacteria is tested by hybridization between at least one of probes having a base sequence shown in SEQ ID NOs: 18 to 43, and 47 selected from the IGS (intergenic spacer) region of the rRNA gene and the nucleic acid amplified in the amplification step.

5. A microarray for testing bacteria for testing the presence of bacteria in a sample, comprising probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria as described in (1) to (10) below, immobilized thereon. A microarray for testing bacteria. (1) Listeria floridensis (2) Listeria weihenstephanensis (3) Listeria cornellensis (4) Listeria riparia (5) Listeria grandensis (6) Listeria booriae (7) Listeria newyorkensis (8) Listeria aquatica (9) Listeria fleischmannii (10) Listeria marthii

6. The microarray for testing bacteria according to claim 5, further comprising at least one probe having a base sequence as shown in SEQ ID NO: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of the Listeria bacterium described in (11) to (17) below, immobilized thereon. (11) Listeria grayi (12) Listeria innocua (13) Listeria ivanovii (14) Listeria monocytogenes (15) Listeria rocourtiae (16) Listeria seeligeri (17) Listeria welshimeri

7. A bacteria testing kit for testing the presence of bacteria in a sample, comprising: a microarray on which all of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria described in (1) to (10) below are immobilized; A primer set consisting of primers having the base sequences shown in SEQ ID NOs: 16 and 17 for amplifying a nucleic acid that hybridizes with the probe. A bacteria testing kit comprising: (1) Listeria floridensis (2) Listeria weihenstephanensis (3) Listeria cornellensis (4) Listeria riparia (5) Listeria grandensis (6) Listeria booriae (7) Listeria newyorkensis (8) Listeria aquatica (9) Listeria fleischmannii (10) Listeria marthii

8. The bacteria testing kit according to claim 7, further comprising the microarray on which at least one of probes having a base sequence shown in SEQ ID NO: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria described in (11) to (17) below is immobilized. (11) Listeria grayi (12) Listeria innocua (13) Listeria ivanovii (14) Listeria monocytogenes (15) Listeria rocourtiae (16) Listeria seeligeri (17) Listeria welshimeri

9. A probe set for testing the presence of bacteria in a sample, comprising all of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria as described in (1) to (10) below. A probe set for testing bacteria, comprising: (1) Listeria floridensis (2) Listeria weihenstephanensis (3) Listeria cornellensis (4) Listeria riparia (5) Listeria grandensis (6) Listeria booriae (7) Listeria newyorkensis (8) Listeria aquatica (9) Listeria fleischmannii (10) Listeria marthii

10. The probe set for testing bacteria according to claim 9, further comprising at least one of probes having a base sequence shown in SEQ ID NO: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria described in (11) to (17) below. (11) Listeria grayi (12) Listeria innocua (13) Listeria ivanovii (14) Listeria monocytogenes (15) Listeria rocourtiae (16) Listeria seeligeri (17) Listeria welshimeri

11. A primer set for testing the presence of bacteria in a sample, comprising primers having the base sequences shown in SEQ ID NOs: 16 and 17 for use in a method for testing bacteria using all of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria bacteria as described in (1) to (10) below. A primer set for testing bacteria, characterized in that (1) Listeria floridensis (2) Listeria weihenstephanensis (3) Listeria cornellensis (4) Listeria riparia (5) Listeria grandensis (6) Listeria booriae (7) Listeria newyorkensis (8) Listeria aquatica (9) Listeria fleischmannii (10) Listeria marthii

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