A method for identifying brucella melitensis based on specific sequences and SNP sites

Abstract

The application discloses a method for identifying sheep brucella based on specific sequences and SNP sites. The application provides application of the specific sequences and the SNP sites in identifying or assisting in identifying the sheep brucella. The application provides specific sequences of the sheep brucella, determines the bacterial species, is not generally identified into the brucella genus, and provides a reference for precise prevention and control. The application provides multiple selectable specific sequences, can realize single-bacterium multi-site coverage identification, and the result is more stable and accurate. The application provides multiple selectable specific sequences, meets identification of bacterial strain mutants, ensures that a suspected bacterial strain is not missed, and facilitates experts to perform bacterial species traceability analysis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for identifying Brucella ovis based on specific sequences and SNP sites. Background Technology

[0002] Brucella is a Gram-negative, aerobic, coccidial bacillus. It is non-capsulated, non-spore-forming, non-flagellated, and non-motile, and is the causative agent of brucellosis. Brucellosis is a zoonotic infectious disease that can cause abortions in livestock such as sheep, cattle, and pigs, and can also cause high fever in humans, making it one of the most serious infectious diseases affecting humans. The genus Brucella mainly comprises six species: *Brucella melitensis* (sheep), *Brucella abortus* (cattle), *Brucella suis* (swine), *Brucella anotomae* (sand rat), *Brucella canis* (canine), and *Brucella ovis* (sheep epididymis). Sheep brucellosis is the most widespread and damaging.

[0003] Brucellosis presents with very similar clinical symptoms, and mixed infections can be pathologically complex, often making it difficult to determine the causative pathogen. Traditional methods for Brucella classification primarily rely on bacteriology and serology, but these are time-consuming and cumbersome. In recent years, molecular biological diagnostics has advanced rapidly and accurately, becoming the most commonly used method for Brucella classification, although multiple primer pairs still need to be designed for detection. Currently, whole-genome sequencing of Brucella strains from different species has been completed, making it possible to screen for species-specific molecular markers across the entire genome. Screening for specific sequences and SNP sites at the whole-genome level provides specific DNA sequences for rapid molecular biological identification of Brucella, laying the foundation for biotyping. Summary of the Invention

[0004] To effectively solve the above-mentioned technical problems, the present invention provides a method for identifying Brucella ovis based on specific sequences and SNP sites.

[0005] In a first aspect, the present invention claims protection for the use of a particular substance in any of the following:

[0006] P1. Identification or auxiliary identification of Brucella mesenteriae;

[0007] P2. Prepare products for the identification or auxiliary identification of Brucella mesenteriae;

[0008] P3. Detection or auxiliary detection to determine whether the test strain is Brucella membranaceus;

[0009] P4. Prepare products for detecting or assisting in the detection of whether the test strain is Brucella membranaceus;

[0010] P5. To detect or assist in the detection of whether the target individual carries Brucella membranaceus;

[0011] P6. Prepare products for detecting or assisting in the detection of whether a target individual carries Brucella mesenteriae.

[0012] The specific substance is a substance capable of detecting all or part of the nucleotide polymorphisms in the 33 groups of SNP sites shown in (1)-(33) of the Brucella genome:

[0013] (1) SNP11 and SNP12: SNP11 is the 100th nucleotide of SEQ ID No.1 in the genome of Brucella, which is T or C; SNP12 is the 242nd nucleotide of SEQ ID No.1 in the genome of Brucella, which is A or G;

[0014] (2) SNP21, SNP22 and SNP23: SNP21 is the 88th nucleotide of SEQ ID No.2 in the genome of Brucella, and is T or C; SNP22 is the 114th nucleotide of SEQ ID No.2 in the genome of Brucella, and is G or T; SNP23 is the 185th nucleotide of SEQ ID No.2 in the genome of Brucella, and is T or C;

[0015] (3) SNP31 and SNP32: SNP31 is the 5th nucleotide of SEQ ID No.3 in the genome of Brucella, and is A or C; SNP32 is the 146th nucleotide of SEQ ID No.3 in the genome of Brucella, and is G or A;

[0016] (4) SNP41 and SNP42: SNP41 is the 26th nucleotide of SEQ ID No.4 in the genome of Brucella, which is C or G; SNP42 is the 108th nucleotide of SEQ ID No.4 in the genome of Brucella, which is T or C;

[0017] (5) SNP51 and SNP52: SNP51 is the 32nd nucleotide of SEQ ID No. 5 in the genome of Brucella, which is T or C; SNP52 is the 106th nucleotide of SEQ ID No. 5 in the genome of Brucella, which is G or C;

[0018] (6) SNP61 and SNP72: SNP61 is the 100th nucleotide of SEQ ID No. 6 in the genome of Brucella, and is A or G; SNP62 is the 135th nucleotide of SEQ ID No. 6 in the genome of Brucella, and is T or C;

[0019] (7) SNP71 and SNP72: SNP71 is the 88th nucleotide of SEQ ID No.7 in the genome of Brucella, which is T or C; SNP72 is the 139th nucleotide of SEQ ID No.7 in the genome of Brucella, which is C or T;

[0020] (8) SNP81, SNP82 and SNP83: SNP81 is the 100th nucleotide of SEQ ID No. 8 in the genome of Brucella, and is G or A; SNP82 is the 161st nucleotide of SEQ ID No. 8 in the genome of Brucella, and is A or G; SNP83 is the 194th nucleotide of SEQ ID No. 8 in the genome of Brucella, and is T or G;

[0021] (9) SNP91 and SNP92: SNP91 is the 100th nucleotide of SEQ ID No.9 in the genome of Brucella, and is A or G; SNP92 is the 220th nucleotide of SEQ ID No.9 in the genome of Brucella, and is T or C;

[0022] (10) SNP101 and SNP102: SNP101 is the 135th nucleotide of SEQ ID No.10 in the genome of Brucella, and is A or G; SNP102 is the 136th nucleotide of SEQ ID No.10 in the genome of Brucella, and is A or G;

[0023] (11) SNP111 and SNP112: SNP111 is the 100th nucleotide of SEQ ID No.11 in the genome of Brucella, and is A or G; SNP112 is the 154th nucleotide of SEQ ID No.11 in the genome of Brucella, and is T or C;

[0024] (12) SNP121 and SNP122: SNP121 is the 67th nucleotide of SEQ ID No. 12 in the genome of Brucella, which is T or C; SNP122 is the 250th nucleotide of SEQ ID No. 12 in the genome of Brucella, which is C or T;

[0025] (13) SNP131 and SNP132: SNP131 is the 100th nucleotide of SEQ ID No.13 in the genome of Brucella, and is C or T; SNP132 is the 136th nucleotide of SEQ ID No.13 in the genome of Brucella, and is A or G;

[0026] (14) SNP141, SNP142 and SNP143: SNP141 is the 31st nucleotide of SEQ ID No. 14 in the genome of Brucella, and is G or A; SNP142 is the 105th nucleotide of SEQ ID No. 14 in the genome of Brucella, and is A or G; SNP143 is the 109th nucleotide of SEQ ID No. 14 in the genome of Brucella, and is T or C;

[0027] (15) SNP151 and SNP152: SNP151 is the 100th nucleotide of SEQ ID No.15 in the genome of Brucella, and is G or C; SNP152 is the 168th nucleotide of SEQ ID No.15 in the genome of Brucella, and is T or C;

[0028] (16) SNP161 and SNP162: SNP161 is the 100th nucleotide of SEQ ID No.16 in the genome of Brucella, and is A or G; SNP162 is the 114th nucleotide of SEQ ID No.16 in the genome of Brucella, and is T or C;

[0029] (17) SNP171 and SNP172: SNP171 is the 8th nucleotide of SEQ ID No. 17 in the genome of Brucella, and is T or A; SNP172 is the 87th nucleotide of SEQ ID No. 17 in the genome of Brucella, and is A or G;

[0030] (18) SNP181 and SNP182: SNP181 is the 100th nucleotide of SEQ ID No. 18 in the genome of Brucella, and is A or G; SNP182 is the 188th nucleotide of SEQ ID No. 18 in the genome of Brucella, and is T or G;

[0031] (19) SNP191 and SNP192: SNP191 is the 100th nucleotide of SEQ ID No.19 in the genome of Brucella, and is A or G; SNP192 is the 169th nucleotide of SEQ ID No.19 in the genome of Brucella, and is T or C;

[0032] (20) SNP201 and SNP202: SNP201 is the 62nd nucleotide of SEQ ID No. 20 in the genome of Brucella, and is A or C; SNP202 is the 89th nucleotide of SEQ ID No. 20 in the genome of Brucella, and is G or A;

[0033] (21) SNP211 and SNP212: SNP211 is the 100th nucleotide of SEQ ID No. 21 in the genome of Brucella, which is T or C; SNP212 is the 224th nucleotide of SEQ ID No. 21 in the genome of Brucella, which is A or G;

[0034] (22) SNP221 and SNP222: SNP221 is the 100th nucleotide of SEQ ID No. 22 in the genome of Brucella, and is A or C; SNP222 is the 253rd nucleotide of SEQ ID No. 22 in the genome of Brucella, and is C or A;

[0035] (23) SNP231 and SNP232: SNP231 is the 100th nucleotide of SEQ ID No. 23 in the genome of Brucella, and is G or C; SNP232 is the 120th nucleotide of SEQ ID No. 23 in the genome of Brucella, and is T or C;

[0036] (24) SNP241 and SNP242: SNP241 is the 100th nucleotide of SEQ ID No. 24 in the genome of Brucella, which is T or G; SNP242 is the 101st nucleotide of SEQ ID No. 24 in the genome of Brucella, which is C or T;

[0037] (25) SNP251 and SNP252: SNP251 is the 100th nucleotide of SEQ ID No. 25 in the genome of Brucella, and is C or A; SNP252 is the 197th nucleotide of SEQ ID No. 25 in the genome of Brucella, and is G or C;

[0038] (26) SNP261 and SNP262: SNP261 is the 100th nucleotide of SEQ ID No. 26 in the genome of Brucella, which is T or C; SNP262 is the 241st nucleotide of SEQ ID No. 26 in the genome of Brucella, which is C or T;

[0039] (27) SNP271 and SNP272: SNP271 is the 100th nucleotide of SEQ ID No. 27 in the genome of Brucella, and is C or A; SNP272 is the 229th nucleotide of SEQ ID No. 27 in the genome of Brucella, and is T or G;

[0040] (28) SNP281 and SNP282: SNP281 is the 61st nucleotide of SEQ ID No. 28 in the genome of Brucella, and is C or T; SNP282 is the 97th nucleotide of SEQ ID No. 28 in the genome of Brucella, and is G or A;

[0041] (29) SNP291 and SNP292: SNP291 is the 12th nucleotide of SEQ ID No. 29 in the genome of Brucella, and is G or A; SNP292 is the 119th nucleotide of SEQ ID No. 29 in the genome of Brucella, and is A or G;

[0042] (30) SNP301 and SNP302: SNP301 is the 100th nucleotide of SEQ ID No. 30 in the genome of Brucella, and is C or T; SNP302 is the 270th nucleotide of SEQ ID No. 30 in the genome of Brucella, and is A or G;

[0043] (31) SNP311, SNP312 and SNP313: SNP311 is the 42nd nucleotide of SEQ ID No. 31 in the genome of Brucella, and is A or G; SNP312 is the 83rd nucleotide of SEQ ID No. 31 in the genome of Brucella, and is A or G; SNP313 is the 84th nucleotide of SEQ ID No. 31 in the genome of Brucella, and is A or G;

[0044] (32) SNP321 and SNP322: SNP321 is the 28th nucleotide of SEQ ID No.32 in the genome of Brucella, and is A or T; SNP322 is the 171st nucleotide of SEQ ID No.32 in the genome of Brucella, and is A or G;

[0045] (33) SNP331 and SNP332: SNP331 is the 40th nucleotide of SEQ ID No.33 in the genome of Brucella, and is A or G; SNP332 is the 178th nucleotide of SEQ ID No.33 in the genome of Brucella, and is T or C.

[0046] Furthermore, the specific substance is a probe set, probe, reagent, or kit for identifying or assisting in the identification of Brucella ovis; the reagent or kit contains the probe set or the probe.

[0047] In a specific embodiment of the present invention, the probe group consists of probes 1 to 33, wherein the probe is any one of probes 1 to 33:

[0048] Probe 1: Single-stranded DNA as shown in SEQ ID No. 1, with nucleotide T at position 100 and nucleotide A at position 242;

[0049] Probe 2: Single-stranded DNA as shown in SEQ ID No. 2, with nucleotide T at position 88, G at position 114, and T at position 185;

[0050] Probe 3: Single-stranded DNA as shown in SEQ ID No. 3, with nucleotide A at position 5 and nucleotide G at position 146;

[0051] Probe 4: Single-stranded DNA as shown in SEQ ID No. 4, with nucleotide C at position 26 and T at position 108;

[0052] Probe 5: Single-stranded DNA as shown in SEQ ID No. 5, with nucleotide T at position 32 and G at position 106;

[0053] Probe 6: Single-stranded DNA as shown in SEQ ID No. 6, with nucleotide A at position 100 and nucleotide T at position 135;

[0054] Probe 7: Single-stranded DNA as shown in SEQ ID No. 7, with nucleotide T at position 88 and C at position 139;

[0055] Probe 8: Single-stranded DNA as shown in SEQ ID No. 8, with nucleotide 100 being G, nucleotide 161 being A, and nucleotide 194 being T;

[0056] Probe 9: Single-stranded DNA as shown in SEQ ID No. 9, with nucleotide A at position 100 and nucleotide T at position 220;

[0057] Probe 10: Single-stranded DNA as shown in SEQ ID No. 10, with nucleotide A at position 135 and nucleotide A at position 136;

[0058] Probe 11: Single-stranded DNA as shown in SEQ ID No. 11, with nucleotide A at position 100 and nucleotide T at position 154;

[0059] Probe 12: Single-stranded DNA as shown in SEQ ID No. 12, with nucleotide T at position 67 and C at position 250;

[0060] Probe 13: Single-stranded DNA as shown in SEQ ID No. 13, with nucleotide C at position 100 and nucleotide A at position 136;

[0061] Probe 14: Single-stranded DNA as shown in SEQ ID No. 14, with nucleotide G at position 31, A at position 105, and T at position 109;

[0062] Probe 15: Single-stranded DNA as shown in SEQ ID No. 15, with nucleotide 100 being G and nucleotide 168 being T;

[0063] Probe 16: Single-stranded DNA as shown in SEQ ID No. 16, with nucleotide A at position 100 and nucleotide T at position 114;

[0064] Probe 17: Single-stranded DNA as shown in SEQ ID No. 17, with nucleotide T at position 8 and nucleotide A at position 87;

[0065] Probe 18: Single-stranded DNA as shown in SEQ ID No. 18, with nucleotide A at position 100 and nucleotide T at position 188;

[0066] Probe 19: Single-stranded DNA as shown in SEQ ID No. 19, with nucleotide A at position 100 and nucleotide T at position 169;

[0067] Probe 20: Single-stranded DNA as shown in SEQ ID No. 20, with nucleotide A at position 62 and nucleotide G at position 89;

[0068] Probe 21: Single-stranded DNA as shown in SEQ ID No. 21, with nucleotide 100 being T and nucleotide 224 being A;

[0069] Probe 22: Single-stranded DNA as shown in SEQ ID No. 22, with nucleotide A at position 100 and nucleotide C at position 253;

[0070] Probe 23: Single-stranded DNA as shown in SEQ ID No. 23, with nucleotide 100 being G and nucleotide 120 being T;

[0071] Probe 24: Single-stranded DNA as shown in SEQ ID No. 24, with nucleotide 100 being T and nucleotide 101 being C;

[0072] Probe 25: Single-stranded DNA as shown in SEQ ID No. 25, with nucleotide C at position 100 and G at position 197;

[0073] Probe 26: Single-stranded DNA as shown in SEQ ID No. 26, with nucleotide 100 being T and nucleotide 241 being C;

[0074] Probe 27: Single-stranded DNA as shown in SEQ ID No. 27, with nucleotide C at position 100 and T at position 229;

[0075] Probe 28: Single-stranded DNA as shown in SEQ ID No. 28, with nucleotide C at position 61 and G at position 97;

[0076] Probe 29: Single-stranded DNA as shown in SEQ ID No. 29, with nucleotide G at position 12 and nucleotide A at position 119;

[0077] Probe 30: Single-stranded DNA as shown in SEQ ID No. 30, with nucleotide C at position 100 and nucleotide A at position 270;

[0078] Probe 31: Single-stranded DNA as shown in SEQ ID No. 31, with nucleotide A at position 42, nucleotide A at position 83, and nucleotide A at position 84;

[0079] Probe 32: Single-stranded DNA as shown in SEQ ID No. 32, with nucleotide A at position 28 and nucleotide A at position 171;

[0080] Probe 33: Single-stranded DNA as shown in SEQ ID No. 33, with nucleotide A at position 40 and nucleotide T at position 178.

[0081] Secondly, the present invention claims a method for detecting or assisting in the detection of whether a test strain is Brucella membranaceus.

[0082] The method for detecting or assisting in the detection of whether a test strain is Brucella membranaceus, as claimed in this invention, may include the following steps:

[0083] (A1) Perform whole-genome sequencing on the test strain;

[0084] (A2) Align the sequencing results with the sequence of each probe in the probe set described above;

[0085] (A3) Based on the sequence alignment results of (A2), determine whether the test strain is Brucella meliottii as follows: If any probe sequence from the probe set is present in the sequencing results (if any one is detected, it can be identified as Brucella meliottii; multiple probes can ensure accurate and rapid identification of Brucella meliottii even in cases of poor sequencing quality or mutations at some sites (artificial or natural), and the more matching probes, the higher the accuracy of identifying Brucella meliottii), then the test strain is or is a candidate for Brucella meliottii; if no probe sequence from the probe set is present in the sequencing results, then the test strain is not or is not a candidate for Brucella meliottii.

[0086] Thirdly, the present invention claims a method for detecting or assisting in the detection of whether a target individual carries Brucella mesenteriae.

[0087] The method for detecting or assisting in the detection of whether a target individual carries Brucella mesenteriae, as claimed in this invention, may include the following steps:

[0088] (B1) The bacterial whole genome was extracted from the test sample of the target individual and then sequenced;

[0089] (B2) Perform sequence alignment of the sequencing results with each probe in the probe set described above;

[0090] (B3) Based on the sequence alignment results of (B2), determine whether the target individual carries Brucella meliottii as follows: If any probe sequence from the probe set is present in the sequencing results (if any one of them is detected, it can be identified as Brucella meliottii; multiple probes can ensure accurate and rapid identification of Brucella meliottii even in cases of poor sequencing quality or mutations at some sites (artificial or natural), and the more matching probes, the higher the accuracy of determining that the individual carries Brucella meliottii), then the target individual carries or is a candidate for carrying Brucella meliottii; if no probe sequence from the probe set is present in the sequencing results, then the target individual does not carry or is a candidate for not carrying Brucella meliottii.

[0091] In all the above aspects, the strain to be tested can be any strain, including but not limited to Brucella.

[0092] Fourthly, the present invention claims a method for detecting or assisting in the detection of whether the Brucella to be tested is Brucella melitensis.

[0093] The method for detecting or assisting in the detection of whether a Brucella aspirate is Brucella mesenteriae, as claimed in this invention, may include the following steps:

[0094] (C1) Detect all or part of the nucleotide polymorphisms in the 33 SNP sites mentioned above in the genome of the Brucella bacteria to be tested:

[0095] (C2) Based on the results obtained in (C1), determine whether the Brucella to be tested is Brucella melitensis as follows: If at least one of the 33 sets of SNP sites' nucleotide polymorphisms matches the corresponding Brucella melitensis criteria (if any one set of SNP sites is detected, it can be identified as Brucella melitensis; multiple sets of SNP sites can ensure accurate and rapid identification of Brucella melitensis even with poor sequencing quality or variations at some sites (artificial or natural); the more matching sites, the higher the accuracy of identifying Brucella melitensis), then the Brucella to be tested is Brucella melitensis; if none of the 33 sets of SNP sites' nucleotide polymorphisms matches the corresponding Brucella melitensis criteria, then the Brucella to be tested is not Brucella melitensis.

[0096] The criteria for determining Brucella adenosine mononucleosis in sheep are as follows:

[0097] (a1) The criteria for determining the Brucella ovis to be tested for SNP11 and SNP12 are: the nucleotide at the position of SNP11 is T and the nucleotide at the position of SNP12 is A;

[0098] (a2) The criteria for determining the Brucella aureus to be tested for SNP21, SNP22 and SNP23 are as follows: the nucleotide at the position of SNP21 is T, the nucleotide at the position of SNP22 is G and the nucleotide at the position of SNP23 is T.

[0099] (a3) The criteria for determining the Brucella aureus to be tested for SNP31 and SNP32 are as follows: the nucleotide at the position of SNP31 is A and the nucleotide at the position of SNP32 is G;

[0100] (a4) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP41 and SNP42 are as follows: the nucleotide at the position of SNP41 is C and the nucleotide at the position of SNP42 is T.

[0101] (a5) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP51 and SNP52 are as follows: the nucleotide at the position of SNP51 is T and the nucleotide at the position of SNP52 is G.

[0102] (a6) The criteria for determining the Brucella aureus to be tested for SNP61 and SNP62 are as follows: the nucleotide at the position of SNP61 is A and the nucleotide at the position of SNP62 is T.

[0103] (a7) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP71 and SNP72 are as follows: the nucleotide at the position of SNP71 is T and the nucleotide at the position of SNP72 is C.

[0104] (a8) The criteria for determining the Brucella aureus to be tested for SNP81, SNP82 and SNP83 are as follows: the nucleotide at the position of SNP81 is G, the nucleotide at the position of SNP82 is A and the nucleotide at the position of SNP83 is T.

[0105] (a9) The criteria for determining the Brucella aureus to be tested for SNP91 and SNP92 are as follows: the nucleotide at the position of SNP91 is A and the nucleotide at the position of SNP92 is T.

[0106] (a10) For SNP101 and SNP102, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP101 is A, and the nucleotide at the position of SNP102 is A;

[0107] (a11) For SNP111 and SNP112, the criteria for determining the sheep Brucella to be tested are: the nucleotide at the position of SNP111 is A, and the nucleotide at the position of SNP112 is T;

[0108] (a12) For SNP121 and SNP122, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP121 is T, and the nucleotide at the position of SNP122 is C;

[0109] (a13) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP131 and SNP132 are as follows: the nucleotide at the position of SNP131 is C and the nucleotide at the position of SNP132 is A.

[0110] (a14) For SNP141, SNP142 and SNP143, the criteria for determining the sheep Brucella to be tested are: the nucleotide at the position of SNP141 is G, the nucleotide at the position of SNP142 is A and the nucleotide at the position of SNP143 is T.

[0111] (a15) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP151 and SNP152 are as follows: the nucleotide at the position of SNP151 is G and the nucleotide at the position of SNP152 is T.

[0112] (a16) For SNP161 and SNP162, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP161 is A, and the nucleotide at the position of SNP162 is T;

[0113] (a17) For SNP171 and SNP172, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP171 is T, and the nucleotide at the position of SNP172 is A;

[0114] (a18) For SNP181 and SNP182, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP181 is A, and the nucleotide at the position of SNP182 is T;

[0115] (a19) For SNP191 and SNP192, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP191 is A, and the nucleotide at the position of SNP192 is T;

[0116] (a20) For SNP201 and SNP202, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP201 is A, and the nucleotide at the position of SNP202 is G;

[0117] (a21) For SNP211 and SNP212, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP211 is T, and the nucleotide at the position of SNP212 is A;

[0118] (a22) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP221 and SNP222 are as follows: the nucleotide at the position of SNP221 is A and the nucleotide at the position of SNP222 is C.

[0119] (a23) For SNP231 and SNP232, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP231 is G, and the nucleotide at the position of SNP232 is T;

[0120] (a24) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP241 and SNP242 are as follows: the nucleotide at the position of SNP241 is T and the nucleotide at the position of SNP242 is C.

[0121] (a25) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP251 and SNP252 are as follows: the nucleotide at the position of SNP251 is C and the nucleotide at the position of SNP252 is G.

[0122] (a26) For SNP261 and SNP262, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP261 is T, and the nucleotide at the position of SNP262 is C;

[0123] (a27) For SNP271 and SNP272, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP271 is C, and the nucleotide at the position of SNP272 is T;

[0124] (a28) For SNP281 and SNP282, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP281 is C, and the nucleotide at the position of SNP282 is G;

[0125] (a29) The criteria for determining the *Brucella aegyptiacus* to be tested for SNP291 and SNP292 are as follows: the nucleotide at the position of SNP291 is G and the nucleotide at the position of SNP292 is A.

[0126] (a30) For SNP301 and SNP302, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP301 is C, and the nucleotide at the position of SNP302 is A;

[0127] (a31) For SNP311, SNP312 and SNP313, the criteria for determining the Brucella aureus to be tested in sheep are: the nucleotide at the position of SNP311 is A, the nucleotide at the position of SNP312 is A and the nucleotide at the position of SNP313 is A.

[0128] (a32) The criteria for determining the Brucella aureus to be tested for SNP321 and SNP322 are as follows: the nucleotide at the position of SNP321 is A and the nucleotide at the position of SNP322 is A.

[0129] (a33) The criteria for determining the *Brucella mollissima* to be tested in sheep for SNP331 and SNP332 are: the nucleotide at the position of SNP331 is A and the nucleotide at the position of SNP332 is T.

[0130] Fifthly, the present invention claims protection for probe sets used for the identification or auxiliary identification of Brucella mesenteriae.

[0131] The probe set claimed in this invention for identifying or assisting in the identification of Brucella ovis is the probe set described above.

[0132] Sixthly, the present invention claims protection for probes used to identify or assist in the identification of Brucella mesenteriae.

[0133] The probes claimed in this invention for identifying or assisting in the identification of Brucella ovis are any of the probes in the probe group described above.

[0134] Seventhly, the present invention claims protection for reagents or kits used for identifying or assisting in the identification of Brucella ovis.

[0135] The present invention claims a reagent or kit for identifying or assisting in the identification of Brucella ovis, containing the probe set or probes described above.

[0136] All of the above applications are non-disease diagnostic applications; all of the methods are non-disease diagnostic methods.

[0137] The beneficial effects of this invention are:

[0138] This invention provides specific sequences for direct identification of *Brucella mellitus*, pinpointing the species rather than simply identifying it as a genus *Brucella*, thus providing a reference for precise disease control. This invention offers multiple selectable specific sequences, enabling multi-site coverage identification of single bacteria, resulting in more stable and accurate results. This invention also provides multiple selectable specific sequences to meet the identification needs of strain mutants, ensuring that no suspected strains are missed, and facilitating expert analysis for strain tracing. Attached Figure Description

[0139] Figure 1 This is a strategy for extracting specific tags.

[0140] Figure 2 The results of the Bmelitensis_Probe01 probe in the NCBI library using Blast testing.

[0141] Figure 3 Here is an example of the specific Blast results for Bmelitensis_Probe01 in a single strain.

[0142] Figure 4Examples of the practicality and specificity of specific probes.

[0143] Figure 5 This allows for the practical and specific application of specific probes using literature data. Detailed Implementation

[0144] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0145] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0146] The Brucella melitensis genome sequence described in this article is the chromosomal sequence of Brucella melitensis bv.1str.16M (GenBank: GCF_000007125.1, AE008917, AE008918), and the nucleic acid sequence positions are also calculated based on Brucella melitensis bv.1str.16M (GenBank: GCF_000007125.1, AE008917, AE008918).

[0147] Example 1: Establishment and Application of a Method for Identifying Brucella ovis Based on Specific Sequences and SNP Sites I. Fabrication Process of the Brucella ovis Electron Probe of this Invention

[0148] 1. Downloaded the genome sequences of Brucella from the NCBI Genome Database, totaling 800 strains of 12 species (Table 1), and numbered these sequences.

[0149] Table 1. Information on Brucella species and their numbers

[0150]

[0151] 2. To ensure the reliability of the results, the quality of the downloaded genome sequences was analyzed using CheckM software, along with whole-genome SNP analysis and whole-genome core gene clustering analysis. Strains with obvious classification errors were removed, leaving 789 strains from 11 species for analysis (Table 2).

[0152] Table 2. Information on the remaining strains used in the analysis

[0153]

[0154] 3. Using the chromosome sequence of Brucella melitensis bv.1str.16M (GenBank: GCF_000007125.1, AE008917, AE008918) as the reference sequence, sequence tags were extracted according to window=100bp and step=1bp, resulting in 3,294,832 100bp sequence tags. Figure 1 As shown.

[0155] 4. Using a local BLAST (BLAST-2.7.1+), a database was established with the genome sequences of other Brucella species (436 strains) besides *Brucella melanogaster* (353 strains) as the target sequences, and 3,294,832 sequence tags as the query sequences. A Python program was used to remove all the retrieved sequences (Identical = 100%, alignment length = 100 bp). This step identified 2,954,355 sequence tags in the genome sequences of the other Brucella species (436 strains), and after removing these tags, 340,477 sequence tags remained.

[0156] 5. Locate sequence tags present in all 353 strains of Brucella mesenteriae.

[0157] A Python program was written to query the genome sequences of all 353 strains of Brucella melanogaster using the 340,477 sequence tags obtained in "Step 4". Sequence tags that were not present in the genome of any strain were removed. As a result, we obtained 73,142 sequence tags that were present in the genomes of all 353 strains of Brucella melanogaster.

[0158] 6. Based on the overlap between fragments, 73,142 sequence tags were fused to obtain 1,282 specific genomic fragments, which ranged in length from 100bp to 369bp.

[0159] 7. Using these 1282 fragments, perform a local BLAST query on all Brucella genomes. Based on the "subject start" and "subject end" information in the BLAST results, extract the information of each fragment in each genome. Construct a FASTA file from the fragments that match the genomes of all strains for each of these 1282 fragments, resulting in a total of 1282 FASTA files.

[0160] 8. Use megaX software to compare the 1282 FASTA format files obtained in step 7 to find the specific SNP site that can distinguish Brucella melitensis: this site is the same base in all Brucella melitensis strains, but is a different base in all No-B. melitensis strains.

[0161] 9. A total of 1084 sequences contained one (or more) SNP sites. For ease of use, all sequences containing two or more SNP sites were selected as specific identification sequences for B. melitensis. A total of 33 specific identification sequences (Table 3), i.e., 33 probes, are as follows:

[0162] Probe 1: Single-stranded DNA as shown in SEQ ID No. 1, with nucleotide T at position 100 and nucleotide A at position 242;

[0163] Probe 2: Single-stranded DNA as shown in SEQ ID No. 2, with nucleotide T at position 88, G at position 114, and T at position 185;

[0164] Probe 3: Single-stranded DNA as shown in SEQ ID No. 3, with nucleotide A at position 5 and nucleotide G at position 146;

[0165] Probe 4: Single-stranded DNA as shown in SEQ ID No. 4, with nucleotide C at position 26 and T at position 108;

[0166] Probe 5: Single-stranded DNA as shown in SEQ ID No. 5, with nucleotide T at position 32 and G at position 106;

[0167] Probe 6: Single-stranded DNA as shown in SEQ ID No. 6, with nucleotide A at position 100 and nucleotide T at position 135;

[0168] Probe 7: Single-stranded DNA as shown in SEQ ID No. 7, with nucleotide T at position 88 and C at position 139;

[0169] Probe 8: Single-stranded DNA as shown in SEQ ID No. 8, with nucleotide 100 being G, nucleotide 161 being A, and nucleotide 194 being T;

[0170] Probe 9: Single-stranded DNA as shown in SEQ ID No. 9, with nucleotide A at position 100 and nucleotide T at position 220;

[0171] Probe 10: Single-stranded DNA as shown in SEQ ID No. 10, with nucleotide A at position 135 and nucleotide A at position 136;

[0172] Probe 11: Single-stranded DNA as shown in SEQ ID No. 11, with nucleotide A at position 100 and nucleotide T at position 154;

[0173] Probe 12: Single-stranded DNA as shown in SEQ ID No. 12, with nucleotide T at position 67 and C at position 250;

[0174] Probe 13: Single-stranded DNA as shown in SEQ ID No. 13, with nucleotide C at position 100 and nucleotide A at position 136;

[0175] Probe 14: Single-stranded DNA as shown in SEQ ID No. 14, with nucleotide G at position 31, A at position 105, and T at position 109;

[0176] Probe 15: Single-stranded DNA as shown in SEQ ID No. 15, with nucleotide 100 being G and nucleotide 168 being T;

[0177] Probe 16: Single-stranded DNA as shown in SEQ ID No. 16, with nucleotide A at position 100 and nucleotide T at position 114;

[0178] Probe 17: Single-stranded DNA as shown in SEQ ID No. 17, with nucleotide T at position 8 and nucleotide A at position 87;

[0179] Probe 18: Single-stranded DNA as shown in SEQ ID No. 18, with nucleotide A at position 100 and nucleotide T at position 188;

[0180] Probe 19: Single-stranded DNA as shown in SEQ ID No. 19, with nucleotide A at position 100 and nucleotide T at position 169;

[0181] Probe 20: Single-stranded DNA as shown in SEQ ID No. 20, with nucleotide A at position 62 and nucleotide G at position 89;

[0182] Probe 21: Single-stranded DNA as shown in SEQ ID No. 21, with nucleotide 100 being T and nucleotide 224 being A;

[0183] Probe 22: Single-stranded DNA as shown in SEQ ID No. 22, with nucleotide A at position 100 and nucleotide C at position 253;

[0184] Probe 23: Single-stranded DNA as shown in SEQ ID No. 23, with nucleotide 100 being G and nucleotide 120 being T;

[0185] Probe 24: Single-stranded DNA as shown in SEQ ID No. 24, with nucleotide 100 being T and nucleotide 101 being C;

[0186] Probe 25: Single-stranded DNA as shown in SEQ ID No. 25, with nucleotide C at position 100 and G at position 197;

[0187] Probe 26: Single-stranded DNA as shown in SEQ ID No. 26, with nucleotide 100 being T and nucleotide 241 being C;

[0188] Probe 27: Single-stranded DNA as shown in SEQ ID No. 27, with nucleotide C at position 100 and T at position 229;

[0189] Probe 28: Single-stranded DNA as shown in SEQ ID No. 28, with nucleotide C at position 61 and G at position 97;

[0190] Probe 29: Single-stranded DNA as shown in SEQ ID No. 29, with nucleotide G at position 12 and nucleotide A at position 119;

[0191] Probe 30: Single-stranded DNA as shown in SEQ ID No. 30, with nucleotide C at position 100 and nucleotide A at position 270;

[0192] Probe 31: Single-stranded DNA as shown in SEQ ID No. 31, with nucleotide A at position 42, nucleotide A at position 83, and nucleotide A at position 84;

[0193] Probe 32: Single-stranded DNA as shown in SEQ ID No. 32, with nucleotide A at position 28 and nucleotide A at position 171;

[0194] Probe 33: Single-stranded DNA as shown in SEQ ID No. 33, with nucleotide A at position 40 and nucleotide T at position 178.

[0195] 10. To test the specificity of these 33 electron probes in a larger database, an online BLAST query was performed on these 33 electron probes (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that all sequences with Query Coverage = 100% and percent identity = 100% were B. melitensis genome sequences, indicating that these 33 sequences have very good specificity.

[0196] 11. Taking Bmelitensis_Probe01 as an example, the test results are as follows: Figure 2 The results showed that these electronic probes could only achieve 100% Query Cover and Per.Ident (Percentage of Identity) values ​​in B.melitensis. See the specific example below. Figure 3 The sequences were completely identical. Since NCBI Blast only displays 100 records, strains that were not completely identical were not shown.

[0197] Table 3. Specific electron probe sequences and SNP sites of the present invention

[0198]

[0199]

[0200]

[0201]

[0202] Note: In the table, "Sequence ( w In the column “ith SNP)”, the nucleotides in square brackets are SNP sites. The preceding nucleotides are polymorphisms in Brucella ovis, and the following nucleotides are polymorphisms in other Brucella species.

[0203] II. Application Examples

[0204] The inventors conducted in-depth research on the whole genome sequences of *Brucella mellitus* and non-*Brucella mellitus* strains in the NCBI database. Using a local BLAST database, they developed a Python program to perform extensive screening and comparison, identifying conserved DNA regions in the *Brucella mellitus* genome sequence. Specific probe sequences were then selected and used to detect sequencing results (including second- or third-generation sequencing) of unknown samples. Since up to 33 specific probes were used, a positive result was determined as long as any one probe was detected. The more probes detected, the higher the reliability, ensuring that even genome sequences with poor sequencing quality and low coverage can be queried using these electron probes for rapid, simple, and efficient identification of whether a sample is *Brucella mellitus*.

[0205] The method of this invention provides a simple and rapid procedure for identifying whether a target individual is infected with Brucella membranaceus:

[0206] (1) Collect samples and extract their genomes. Bacterial whole genome extraction was performed according to the steps of the Bacterial Genomic DNA Extraction kit.

[0207] (2) The genome sample was sequenced using a sequencer (either second-generation or third-generation);

[0208] (3) Use bioinformatics software to compare and analyze the sequencing results with the probe sequences;

[0209] (4) If the sequencing result matches 100% with any of the probes, the result is positive and the sample is Brucella melitensis. The more probes detected, the more reliable the result. Multiple electron probes can avoid incorrect identification due to poor sequencing quality or artificially modified bacteria. If none of the probes are detected, the result is negative.

[0210] Example 1:

[0211] (5) Genome Acquisition of Example Test Strains: We downloaded 1240 Brucella genomes from the website, of which 758 strains overlapped with the 800 strains used previously, and 482 strains were newly added to the genome. The sequencing quality of these 482 strains was also tested using CheckM, and whole-genome SNP analysis was performed. Strains with obvious classification errors (8 strains) were removed, leaving 474 strains. Then, strains classified only to the genus level (10 strains) were removed, leaving 464 usable strains. Information is shown in Table 4 below.

[0212] Table 4. Information on strains used in the example tests

[0213]

[0214]

[0215] Download website: https: / / www.bv-brc.org / view / Taxonomy / 234#view_tab=genomes

[0216] (6) Using the 33 probes we obtained (Table 3), we identified and analyzed these 464 bacterial strains (Table 4) and 10271 bacterial strains of the genera (species) Bacillus, Acinetobacter, Yersinia, etc., downloaded from the internet (Table 5). The results showed that all 94 Brucella melitensis_add strains contained all 33 specific tags, while no probes were found in the other strains. Figure 4 This application example further demonstrates the specificity and practicality of these probes.

[0217] Table 5. Summary of strains used in the case studies

[0218]

[0219] Example 2:

[0220] (8) Some strains were obtained from 5 articles, and the specific strain information is shown in Table 6. After simple sequence splicing of these SRA files, the above 33 electronic probes were used for comparison. The results showed that all 89 strains of Brucellamelitensis in the literature [1] contained the above 33 specific probes, and all 21 strains of Brucellamelitensis in the literature [2] contained the above 33 specific probes (such as... Figure 5 The upper half of the DNA strand in the sample). No other Brucella strain contains any of the 33 specific probes (e.g., the upper half of the sample). Figure 5 (The lower half of the text). As shown in Tables 7 and 8, the results are completely consistent with those in the literature, demonstrating speed and accuracy. This application example further proves the specificity and practicality of these probes.

[0221] Table 6. Specific strain information from the literature

[0222]

[0223]

[0224] Table 7. Information and identification results of 89 strains in reference [1]

[0225]

[0226]

[0227] Table 8. Information and identification results of 29 strains in reference [2]

[0228] SRA Number BioProject Number Number in the Literature Identification Result of the Literature Identification Result of the Present Invention SRR12368024 PRJNA650270 18RB17241 Brucella melitensis Brucella melitensis SRR12368025 PRJNA650270 18RB17240 Brucella melitensis Brucella melitensis SRR12368026 PRJNA650270 18RB17238 Brucella melitensis Brucella melitensis SRR12368027 PRJNA650270 18RB17236 Brucella melitensis Brucella melitensis SRR12368028 PRJNA650270 18RB17235 Brucella melitensis Brucella melitensis SRR12368030 [[ID=四十二]]PRJNA650270 18RB17230 Brucella melitensis Brucella melitensis SRR12368031 PRJNA650270 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Brucella melitensis Brucella melitensis SRR12368043 PRJNA650270 18RB17249 Brucella melitensis Brucella melitensis SRR12368044 PRJNA650270 18RB17248 Brucella melitensis Brucella melitensis SRR12368045 PRJNA650270 18RB17247 Brucella melitensis Brucella melitensis SRR12368046 PRJNA650270 18RB17246 Brucella melitensis Brucella melitensis SRR12368048 PRJNA650270 18RB17244 Brucella melitensis Brucella melitensis SRR12368051 PRJNA650270 18RB17228 Brucella melitensis Brucella melitensis SRR12368052 PRJNA650270 18RB17227 Brucella melitensis Brucella melitensis SRR12368029 PRJNA650270 18RB17233 Brucella abortion Non-Brucella melitensis SRR12368032 PRJNA650270 18RB17259 Brucella abortion Non-Brucella melitensis SRR12368034 PRJNA650270 18RB17257 Brucella abortion Non-Brucella melitensis SRR12368035 PRJNA650270 18RB17256 Brucella abortion Non-Brucella melitensis SRR12368036 PRJNA650270 18RB17255 Brucella abortion Non-Brucella melitensis SRR12368047 PRJNA650270 18RB17245 Brucella abortion Non-Brucella melitensis SRR12368049 PRJNA650270 18RB17243 Brucella abortion Non-Brucella melitensis SRR12368050 PRJNA650270 18RB17242 Brucella abortion Non-Brucella melitensis

[0229] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of a specific substance in any of the following: P1. Prepare products for the identification or auxiliary identification of Brucella mesenteriae; P2. Prepare products for detecting or assisting in the detection of whether the test strain is Brucella membranaceus; P3. Prepare products for detecting or assisting in the detection of whether a target individual carries Brucella membranaceus; The specific substance is one that can be used to detect all nucleotide polymorphisms in the 33 groups of SNP sites shown in (1)-(33) of the Brucella genome: (1) SNP11 and SNP12: SNP11 is the 100th nucleotide of SEQ ID No.1 in the genome of Brucella, which is T or C; SNP12 is the 242nd nucleotide of SEQ ID No.1 in the genome of Brucella, which is A or G; (2) SNP21, SNP22 and SNP23: SNP21 is the 88th nucleotide of SEQ ID No.2 in the genome of Brucella, which is T or C; SNP22 is the 114th nucleotide of SEQ ID No.2 in the genome of Brucella, which is G or T; SNP23 is the 185th nucleotide of SEQ ID No.2 in the genome of Brucella, which is T or C; (3) SNP31 and SNP32: SNP31 is the 5th nucleotide of SEQ ID No.3 in the genome of Brucella, and is A or C; SNP32 is the 146th nucleotide of SEQ ID No.3 in the genome of Brucella, and is G or A; (4) SNP41 and SNP42: SNP41 is the 26th nucleotide of SEQ ID No.4 in the genome of Brucella, which is C or G; SNP42 is the 108th nucleotide of SEQ ID No.4 in the genome of Brucella, which is T or C; (5) SNP51 and SNP52: SNP51 is the 32nd nucleotide of SEQ ID No. 5 in the genome of Brucella, which is T or C; SNP52 is the 106th nucleotide of SEQ ID No. 5 in the genome of Brucella, which is G or C; (6) SNP61 and SNP62: SNP61 is the 100th nucleotide of SEQ ID No. 6 in the genome of Brucella, and is A or G; SNP62 is the 135th nucleotide of SEQ ID No. 6 in the genome of Brucella, and is T or C; (7) SNP71 and SNP72: SNP71 is the 88th nucleotide of SEQ ID No.7 in the genome of Brucella, which is T or C; SNP72 is the 139th nucleotide of SEQ ID No.7 in the genome of Brucella, which is C or T; (8) SNP81, SNP82 and SNP83: SNP81 is the 100th nucleotide of SEQ ID No. 8 in the genome of Brucella, and is G or A; SNP82 is the 161st nucleotide of SEQ ID No. 8 in the genome of Brucella, and is A or G; SNP83 is the 194th nucleotide of SEQ ID No. 8 in the genome of Brucella, and is T or G; (9) SNP91 and SNP92: SNP91 is the 100th nucleotide of SEQ ID No.9 in the genome of Brucella, and is A or G; SNP92 is the 220th nucleotide of SEQ ID No.9 in the genome of Brucella, and is T or C; (10) SNP101 and SNP102: SNP101 is the 135th nucleotide of SEQ ID No.10 in the genome of Brucella, and is A or G; SNP102 is the 136th nucleotide of SEQ ID No.10 in the genome of Brucella, and is A or G; (11) SNP111 and SNP112: SNP111 is the 100th nucleotide of SEQ ID No.11 in the genome of Brucella, and is A or G; SNP112 is the 154th nucleotide of SEQ ID No.11 in the genome of Brucella, and is T or C; (12) SNP121 and SNP122: SNP121 is the 67th nucleotide of SEQ ID No.12 in the genome of Brucella, which is T or C; SNP122 is the 250th nucleotide of SEQ ID No.12 in the genome of Brucella, which is C or T; (13) SNP131 and SNP132: SNP131 is the 100th nucleotide of SEQ ID No.13 in the genome of Brucella, and is C or T; SNP132 is the 136th nucleotide of SEQ ID No.13 in the genome of Brucella, and is A or G; (14) SNP141, SNP142 and SNP143: SNP141 is the 31st nucleotide of SEQ ID No. 14 in the genome of Brucella, and is G or A; SNP142 is the 105th nucleotide of SEQ ID No. 14 in the genome of Brucella, and is A or G; SNP143 is the 109th nucleotide of SEQ ID No. 14 in the genome of Brucella, and is T or C; (15) SNP151 and SNP152: SNP151 is the 100th nucleotide of SEQ ID No.15 in the genome of Brucella, and is G or C; SNP152 is the 168th nucleotide of SEQ ID No.15 in the genome of Brucella, and is T or C; (16) SNP161 and SNP162: SNP161 is the 100th nucleotide of SEQ ID No.16 in the genome of Brucella, and is A or G; SNP162 is the 114th nucleotide of SEQ ID No.16 in the genome of Brucella, and is T or C; (17) SNP171 and SNP172: SNP171 is the 8th nucleotide of SEQ ID No.17 in the genome of Brucella, which is T or A; SNP172 is the 87th nucleotide of SEQ ID No.17 in the genome of Brucella, which is A or G; (18) SNP181 and SNP182: SNP181 is the 100th nucleotide of SEQ ID No.18 in the genome of Brucella, and is A or G; SNP182 is the 188th nucleotide of SEQ ID No.18 in the genome of Brucella, and is T or G; (19) SNP191 and SNP192: SNP191 is the 100th nucleotide of SEQ ID No.19 in the genome of Brucella, and is A or G; SNP192 is the 169th nucleotide of SEQ ID No.19 in the genome of Brucella, and is T or C; (20) SNP201 and SNP202: SNP201 is the 62nd nucleotide of SEQ ID No. 20 in the genome of Brucella, and is A or C; SNP202 is the 89th nucleotide of SEQ ID No. 20 in the genome of Brucella, and is G or A; (21) SNP211 and SNP212: SNP211 is the 100th nucleotide of SEQ ID No.21 in the genome of Brucella, which is T or C; SNP212 is the 224th nucleotide of SEQ ID No.21 in the genome of Brucella, which is A or G; (22) SNP221 and SNP222: SNP221 is the 100th nucleotide of SEQ ID No. 22 in the genome of Brucella, and is A or C; SNP222 is the 253rd nucleotide of SEQ ID No. 22 in the genome of Brucella, and is C or A; (23) SNP231 and SNP232: SNP231 is the 100th nucleotide of SEQ ID No. 23 in the genome of Brucella, and is G or C; SNP232 is the 120th nucleotide of SEQ ID No. 23 in the genome of Brucella, and is T or C; (24) SNP241 and SNP242: SNP241 is the 100th nucleotide of SEQ ID No.24 in the genome of Brucella, which is T or G; SNP242 is the 101st nucleotide of SEQ ID No.24 in the genome of Brucella, which is C or T; (25) SNP251 and SNP252: SNP251 is the 100th nucleotide of SEQ ID No. 25 in the genome of Brucella, and is C or A; SNP252 is the 197th nucleotide of SEQ ID No. 25 in the genome of Brucella, and is G or C; (26) SNP261 and SNP262: SNP261 is the 100th nucleotide of SEQ ID No.26 in the genome of Brucella, which is T or C; SNP262 is the 241st nucleotide of SEQ ID No.26 in the genome of Brucella, which is C or T; (27) SNP271 and SNP272: SNP271 is the 100th nucleotide of SEQ ID No. 27 in the genome of Brucella, and is C or A; SNP272 is the 229th nucleotide of SEQ ID No. 27 in the genome of Brucella, and is T or G; (28) SNP281 and SNP282: SNP281 is the 61st nucleotide of SEQ ID No. 28 in the genome of Brucella, and is C or T; SNP282 is the 97th nucleotide of SEQ ID No. 28 in the genome of Brucella, and is G or A; (29) SNP291 and SNP292: SNP291 is the 12th nucleotide of SEQ ID No. 29 in the genome of Brucella, and is G or A; SNP292 is the 119th nucleotide of SEQ ID No. 29 in the genome of Brucella, and is A or G; (30) SNP301 and SNP302: SNP301 is the 100th nucleotide of SEQ ID No. 30 in the genome of Brucella, and is C or T; SNP302 is the 270th nucleotide of SEQ ID No. 30 in the genome of Brucella, and is A or G; (31) SNP311, SNP312 and SNP313: SNP311 is the 42nd nucleotide of SEQ ID No. 31 in the genome of Brucella, and is A or G; SNP312 is the 83rd nucleotide of SEQ ID No. 31 in the genome of Brucella, and is A or G; SNP313 is the 84th nucleotide of SEQ ID No. 31 in the genome of Brucella, and is A or G; (32) SNP321 and SNP322: SNP321 is the 28th nucleotide of SEQ ID No.32 in the genome of Brucella, and is A or T; SNP322 is the 171st nucleotide of SEQ ID No.32 in the genome of Brucella, and is A or G; (33) SNP331 and SNP332: SNP331 is the 40th nucleotide of SEQ ID No.33 in the genome of Brucella, and is A or G; SNP332 is the 178th nucleotide of SEQ ID No.33 in the genome of Brucella, and is T or C.

2. The application according to claim 1, characterized in that: The specific substance is a probe set used to identify or assist in the identification of Brucella ovis.

3. The application according to claim 2, characterized in that: The probe group consists of probes 1 to 33 as follows: Probe 1: Single-stranded DNA as shown in SEQ ID No. 1, with nucleotide T at position 100 and nucleotide A at position 242; Probe 2: Single-stranded DNA as shown in SEQ ID No. 2, with nucleotide T at position 88, G at position 114, and T at position 185; Probe 3: Single-stranded DNA as shown in SEQ ID No. 3, with nucleotide A at position 5 and nucleotide G at position 146; Probe 4: Single-stranded DNA as shown in SEQ ID No. 4, with nucleotide C at position 26 and T at position 108; Probe 5: Single-stranded DNA as shown in SEQ ID No. 5, with nucleotide T at position 32 and G at position 106; Probe 6: Single-stranded DNA as shown in SEQ ID No. 6, with nucleotide A at position 100 and nucleotide T at position 135; Probe 7: Single-stranded DNA as shown in SEQ ID No. 7, with nucleotide T at position 88 and C at position 139; Probe 8: Single-stranded DNA as shown in SEQ ID No. 8, with nucleotide 100 being G, nucleotide 161 being A, and nucleotide 194 being T; Probe 9: Single-stranded DNA as shown in SEQ ID No. 9, with nucleotide A at position 100 and nucleotide T at position 220; Probe 10: Single-stranded DNA as shown in SEQ ID No. 10, with nucleotide A at position 135 and nucleotide A at position 136; Probe 11: Single-stranded DNA as shown in SEQ ID No. 11, with nucleotide A at position 100 and nucleotide T at position 154; Probe 12: Single-stranded DNA as shown in SEQ ID No. 12, with nucleotide T at position 67 and C at position 250; Probe 13: Single-stranded DNA as shown in SEQ ID No. 13, with nucleotide C at position 100 and nucleotide A at position 136; Probe 14: Single-stranded DNA as shown in SEQ ID No. 14, with nucleotide G at position 31, A at position 105, and T at position 109; Probe 15: Single-stranded DNA as shown in SEQ ID No. 15, with nucleotide 100 being G and nucleotide 168 being T; Probe 16: Single-stranded DNA as shown in SEQ ID No. 16, with nucleotide A at position 100 and nucleotide T at position 114; Probe 17: Single-stranded DNA as shown in SEQ ID No. 17, with nucleotide T at position 8 and nucleotide A at position 87; Probe 18: Single-stranded DNA as shown in SEQ ID No. 18, with nucleotide A at position 100 and nucleotide T at position 188; Probe 19: Single-stranded DNA as shown in SEQ ID No. 19, with nucleotide A at position 100 and nucleotide T at position 169; Probe 20: Single-stranded DNA as shown in SEQ ID No. 20, with nucleotide A at position 62 and nucleotide G at position 89; Probe 21: Single-stranded DNA as shown in SEQ ID No. 21, with nucleotide 100 being T and nucleotide 224 being A; Probe 22: Single-stranded DNA as shown in SEQ ID No. 22, with nucleotide A at position 100 and nucleotide C at position 253; Probe 23: Single-stranded DNA as shown in SEQ ID No. 23, with nucleotide 100 being G and nucleotide 120 being T; Probe 24: Single-stranded DNA as shown in SEQ ID No. 24, with nucleotide 100 being T and nucleotide 101 being C; Probe 25: Single-stranded DNA as shown in SEQ ID No. 25, with nucleotide C at position 100 and G at position 197; Probe 26: Single-stranded DNA as shown in SEQ ID No. 26, with nucleotide 100 being T and nucleotide 241 being C; Probe 27: Single-stranded DNA as shown in SEQ ID No. 27, with nucleotide C at position 100 and T at position 229; Probe 28: Single-stranded DNA as shown in SEQ ID No. 28, with nucleotide C at position 61 and G at position 97; Probe 29: Single-stranded DNA as shown in SEQ ID No. 29, with nucleotide G at position 12 and nucleotide A at position 119; Probe 30: Single-stranded DNA as shown in SEQ ID No. 30, with nucleotide C at position 100 and nucleotide A at position 270; Probe 31: Single-stranded DNA as shown in SEQ ID No. 31, with nucleotide A at position 42, nucleotide A at position 83, and nucleotide A at position 84; Probe 32: Single-stranded DNA as shown in SEQ ID No. 32, with nucleotide A at position 28 and nucleotide A at position 171; Probe 33: Single-stranded DNA as shown in SEQ ID No. 33, with nucleotide A at position 40 and nucleotide T at position 178.

4. A non-disease diagnostic method for detecting or assisting in the detection of whether a test strain is Brucella mesenteriae, comprising the following steps: (A1) Perform whole-genome sequencing on the test strain; (A2) Align the sequencing results with each probe in the probe set described in claim 3; (A3) Based on the sequence alignment results of (A2), determine whether the test strain is Brucella meliottii as follows: if the sequencing results contain the probe sequence of the probe group, then the test strain is or is a candidate for Brucella meliottii; if the sequencing results do not contain the probe sequence of the probe group, then the test strain is or is not a candidate for Brucella meliottii.

5. A non-disease diagnostic method for detecting or assisting in the detection of whether the Brucella bacteria to be tested is Brucella mesenteriae, comprising the following steps: (C1) Detect all nucleotide polymorphisms in the 33 SNP sites described in claim 1 in the genome of the Brucella bacteria to be tested: (C2) Based on the results obtained in (C1), determine whether the Brucella to be tested is Brucella ovis as follows: if the nucleotide polymorphisms of the 33 SNP sites match the corresponding Brucella ovis criteria, then the Brucella to be tested is Brucella ovis; if none of the nucleotide polymorphisms of the 33 SNP sites match the corresponding Brucella ovis criteria, then the Brucella to be tested is not Brucella ovis. The criteria for determining Brucella adenosine mononucleosis in sheep are as follows: (a1) The criteria for determining the presence of Brucella ovis in relation to SNP11 and SNP12 are as follows: the nucleotide at the position of SNP11 is T and the nucleotide at the position of SNP12 is A. (a2) The criteria for determining the presence of Brucella aureus in sheep for SNP21, SNP22 and SNP23 are as follows: the nucleotide at the position of SNP21 is T, the nucleotide at the position of SNP22 is G and the nucleotide at the position of SNP23 is T. (a3) The criteria for determining the presence of Brucella aureus in sheep for SNP31 and SNP32 are as follows: the nucleotide at the position of SNP31 is A and the nucleotide at the position of SNP32 is G; (a4) The criteria for determining the presence of Brucella aureus in sheep for SNP41 and SNP42 are as follows: the nucleotide at the position of SNP41 is C and the nucleotide at the position of SNP42 is T. (a5) The criteria for determining the presence of Brucella ovis in relation to SNP51 and SNP52 are as follows: the nucleotide at the position of SNP51 is T and the nucleotide at the position of SNP52 is G. (a6) The criteria for determining the presence of Brucella ovis in relation to SNP61 and SNP62 are as follows: the nucleotide at the position of SNP61 is A and the nucleotide at the position of SNP62 is T. (a7) The criteria for determining the presence of Brucella ovis in relation to SNP71 and SNP72 are as follows: the nucleotide at the position of SNP71 is T and the nucleotide at the position of SNP72 is C. (a8) The criteria for determining the presence of Brucella aureus in sheep for SNP81, SNP82 and SNP83 are as follows: the nucleotide at the position of SNP81 is G, the nucleotide at the position of SNP82 is A and the nucleotide at the position of SNP83 is T. (a9) The criteria for determining the presence of Brucella ovis in relation to SNP91 and SNP92 are as follows: the nucleotide at the position of SNP91 is A and the nucleotide at the position of SNP92 is T. (a10) For SNP101 and SNP102, the criteria for determining the sheep Brucella to be tested are: the nucleotide at the position of SNP101 is A, and the nucleotide at the position of SNP102 is A; (a11) The criteria for determining the presence of Brucella aureus in sheep for SNP111 and SNP112 are as follows: the nucleotide at the position of SNP111 is A and the nucleotide at the position of SNP112 is T. (a12) For SNP121 and SNP122, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP121 is T and the nucleotide at the position of SNP122 is C; (a13) The criteria for determining the presence of Brucella ovis in relation to SNP131 and SNP132 are as follows: the nucleotide at the position of SNP131 is C and the nucleotide at the position of SNP132 is A. (a14) For SNP141, SNP142 and SNP143, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP141 is G, the nucleotide at the position of SNP142 is A and the nucleotide at the position of SNP143 is T. (a15) The criteria for determining the presence of Brucella ovis in relation to SNP151 and SNP152 are as follows: the nucleotide at the position of SNP151 is G and the nucleotide at the position of SNP152 is T. (a16) The criteria for determining the presence of Brucella aureus in sheep for SNP161 and SNP162 are as follows: the nucleotide at the position of SNP161 is A and the nucleotide at the position of SNP162 is T. (a17) For SNP171 and SNP172, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP171 is T, and the nucleotide at the position of SNP172 is A; (a18) For SNP181 and SNP182, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP181 is A, and the nucleotide at the position of SNP182 is T; (a19) The criteria for determining the presence of Brucella ovis in relation to SNP191 and SNP192 are as follows: the nucleotide at the position of SNP191 is A and the nucleotide at the position of SNP192 is T. (a20) For SNP201 and SNP202, the criteria for determining the sheep Brucella to be tested are: the nucleotide at the position of SNP201 is A, and the nucleotide at the position of SNP202 is G; (a21) For SNP211 and SNP212, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP211 is T, and the nucleotide at the position of SNP212 is A; (a22) The criteria for determining the presence of Brucella ovis in relation to SNP221 and SNP222 are as follows: the nucleotide at the position of SNP221 is A and the nucleotide at the position of SNP222 is C. (a23) For SNP231 and SNP232, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP231 is G, and the nucleotide at the position of SNP232 is T; (a24) For SNP241 and SNP242, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP241 is T and the nucleotide at the position of SNP242 is C; (a25) For SNP251 and SNP252, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP251 is C and the nucleotide at the position of SNP252 is G; (a26) For SNP261 and SNP262, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP261 is T and the nucleotide at the position of SNP262 is C; (a27) For SNP271 and SNP272, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP271 is C, and the nucleotide at the position of SNP272 is T; (a28) For SNP281 and SNP282, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP281 is C, and the nucleotide at the position of SNP282 is G; (a29) The criteria for determining the presence of Brucella ovis in relation to SNP291 and SNP292 are as follows: the nucleotide at the position of SNP291 is G and the nucleotide at the position of SNP292 is A. (a30) For SNP301 and SNP302, the criteria for determining the presence of Brucella aureus in sheep are as follows: the nucleotide at the position of SNP301 is C, and the nucleotide at the position of SNP302 is A; (a31) For SNP311, SNP312 and SNP313, the criteria for determining the sheep Brucella to be tested are: the nucleotide at the position of SNP311 is A, the nucleotide at the position of SNP312 is A and the nucleotide at the position of SNP313 is A; (a32) The criteria for determining the presence of Brucella ovis in relation to SNP321 and SNP322 are as follows: the nucleotide at the position of SNP321 is A and the nucleotide at the position of SNP322 is A. (a33) The criteria for determining the presence of Brucella ovis in relation to SNP331 and SNP332 are as follows: the nucleotide at the position of SNP331 is A and the nucleotide at the position of SNP332 is T.

6. A probe set for identifying or assisting in the identification of Brucella melanogaster, as described in claim 3.

7. A reagent or kit for identifying or assisting in the identification of Brucella mellitus, comprising the probe set as described in claim 6.

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