SNP markers and combinations for diagnosis and genotyping brucella species in different samples

Abstract

The present disclosure relates to SNP points and their combinations used in a diagnostic panel, to diagnose and genotype Brucella species in different samples by using genomic DNA of a single sample with SNP markers and their combinations to determine which or which of the bacterial species is infected with high accuracy and in a short time by performing multiplex reactions, to both reduce the labour force, keep the cost low when multiple samples are used, and prevent false or negative results due to the high sensitivity of the system used.

Description

[0001] DESCRIPTION

[0002] SNP MARKERS AND COMBINATIONS FOR DIAGNOSIS AND GENOTYPING BRUCELLA SPECIES IN DIFFERENT SAMPLES

[0003] Field of the Invention

[0004] The present disclosure relates to SNP points and their combinations used in a diagnostic panel, to diagnose and genotype Brucella species in different samples by using genomic DNA of a single sample with SNP markers and their combinations to determine which or which of the bacterial species is infected with high accuracy and in a short time by performing multiplex reactions, to both reduce the labour force, keep the cost low when multiple samples are used, and prevent false or negative results due to the high sensitivity of the system used.

[0005] Prior Art

[0006] Today, the application and use of rapid diagnostic tools in the field of healthcare are of great importance in preventing the unnecessary application of antimicrobial agents. Microorganisms cause various diseases in humans and animals. Microorganisms are single-celled creatures that are too small to be seen with the naked eye. Bacteria, yeasts, moulds, algae, viruses, fungi and protozoa constitute the basic microorganisms. Definitive diagnosis of disease agents caused by microorganisms is carried out in the laboratory. For this reason, it is necessary to perform multifaceted examination of the samples brought to the laboratory. In these examinations, clinical findings and necropsy results are guiding. It is achieved by equipping clinicians with timely and accurate information and the use of rapid diagnostic tools that help them make informed decisions regarding the appropriate pharmacological intervention for patients. Diagnostic tools have the potential to facilitate the timely detection and diagnosis of antibiotic-resistant bacterial infections, facilitate disease surveillance and reduce disease transmission. It is imperative to consider pragmatic measures to accelerate the advancement and adoption of new diagnostic technologies. In addition, as the burden of antimicrobial resistance continues to increase worldwide, the use of rapid antimicrobial susceptibility testing is becoming an increasingly critical component of treatment planning. The development of diagnostic methods that can help to treat infection and identify non-infectious inflammatory conditions that do not require the use of antibiotics is of great importance, where it is important to reduce unnecessary antibiotic use. Diagnosis, typing, classification, determination of resistance and susceptibility of microorganisms, explanation of microorganism-host relationships, metagenomic studies on microbiome and microflora are increasing with bioinformatic tools and developing sequencing-genotyping technology. Bacterial infections are quite common in hospitalized children and adults. In a study by Vincent et al. examining 3147 patients in 198 intensive care units in 24 European countries, sepsis was observed in 37.4% of the patients, while 24.7% of the patients were reported to have sepsis during hospitalization. (Vincent et al., 2006) In the case of these individuals, infections are linked to higher morbidity, mortality and higher costs in healthcare expenditures (Vos et al., 2020; Lagu et al., 2012). Infection-related hazards also result in high antibiotic consumption; according to a global point prevalence survey, 70 per cent of all ICU patients receive at least one antibiotic on any given day, and this high antibiotic use is a direct consequence of infection-related hazards (Vincent et al., 2009). One of the most important steps in the clinical management of bacterial infections is to be able to quickly and accurately identify the disease-causing organism. In addition, as the burden of antimicrobial resistance continues to increase worldwide, the use of rapid antimicrobial susceptibility testing is becoming an increasingly critical component of treatment planning. It is of great importance to develop diagnostic methods that can help to treat infection and identify non-infectious inflammatory conditions that do not require antibiotic use, where it is important to reduce unnecessary antibiotic use (Denny et al., 2020; O'Neill 2014; Raudoniute et al., 2023; Cao et al., 2023). Brucellosis is a contagious, usually subacute and chronic infection caused by bacteria belonging to the Brucella genus in humans and animals. B. abortus and B. melitensis are the main agents of brucellosis in livestock farming. B. melitensis also causes the disease known as Malta fever in humans. Brucellosis is a bacterial disease caused by various species of Brucella that mostly infect cattle, pigs, goats, sheep and dogs. People usually contract the disease through direct contact with infected animals, by eating or drinking contaminated animal products, or by breathing airborne pathogens. Most cases occur through consumption of un pasteurized milk or cheese from infected goats or sheep. Brucellosis is one of the most common zoonoses transmitted by animals, and human brucellosis has serious public health consequences in endemic areas. The expansion of animal industries and urbanization and the lack of hygienic measures in animal husbandry and food processing partly explain why brucellosis remains a public health hazard. (WHO, 2020c). The main Brucellosis agents that cause infection in humans are Brucella abortus, B. melitensis, B. suis, B. ovis, and B. neotomae. The diagnosis of each Brucella species causing the infection is made in separate reactions for each sample, and multiple infection cases cannot be practically considered and may be overlooked. This situation causes loss of labor, financial loss in multiple samples, and false negative results, especially in the initial stages of infection and in multiple infections. There are classical serological, biochemical and culture-based tests as well as classical PCR and qPCR-based molecular methods used for disease control. However, since the current classical methods have to be applied separately for each bacterial species, they are not profitable in terms of labour and cost, especially for mass testing and reference laboratories where the sample diagnosis rate is high. It may cause different infections in the same sample to be missed. In addition, the sensitivity of these classical methods is low, some methods take a long time to apply and do not allow the detection of the causative agent, especially in the early stages of some infections.

[0007] Devices and methods for antibiotic susceptibility testing, which is seen in the state of the art, are described in the document numbered CA3111751A1. The disclosure generally relates to molecular diagnostic devices configured to amplify a single nucleotide polymorphism (SNP) locus and to indicate the presence or absence of a target allele by discriminating between two or more allelic variants of the SNP. In some embodiments, molecular diagnostic devices can detect SNPs associated with resistance or susceptibility to antibiotic treatment of bacterial infections at the point of care. In other aspects, the disclosure provides methods of treatment for diseases or disorders (e.g., bacterial infections) for which treatment is directed by the presence or absence of an allele at a SNP locus as determined by such molecular diagnostic devices. A sample preparation module configured to receive a biological sample, wherein the biological sample comprises a polynucleotide. Polynucleotide comprises a reagent module comprising a primer set targeting a single nucleotide polymorphism (SNP) locus; an amplification module comprising a reaction volume and a heater, an amplification solution comprising the reaction volume and primer set configured to receive the biological sample. The heater is configured to deliver thermal energy to the reaction volume to amplify the polynucleotide to produce an output containing a target amplicon containing the SNP locus. A detection module configured to receive the target amplicon contains a probe designed to bind to the SNP locus of the target amplicon if the SNP locus contains a target allele. The aim is to minimise binding of the target amplicon to the SNP locus if the SNP locus contains an alternative allele.

[0008] The document numbered CN107400708A, which appears in the prior art, describes the application of XRCC1 gene polymorphism to the diagnostic efficacy of rheumatoid arthritis. It is the application of XRCC1 gene polymorphism to the diagnostic efficacy of rheumatoid arthritis and the application of SNP loci of XRCC1 gene to the preparation of a detection kit to diagnose rheumatoid arthritis susceptibility. The correlation between the polymorphism of locus 641 of the XRCC1 gene and rheumatoid arthritis was discovered for the first time. A method and a kit to detect rheumatoid arthritis susceptibility risk via SNP have been introduced.

[0009] Document numbered WO2014190394A1, which appears in the state of the art, describes microbial indicators and their uses. Methods to identify and / or classify microbes using one or more single nucleotide polymorphisms (SNPs) in the 16S ribosomal RNA of prokaryotes and / or one or more SNPs in the 5.8S ribosomal RNA of eukaryotes. Also described are probes, primers, and kits useful in these methods. Methods for diagnosing sepsis based on these SNPs are also described.

[0010] Document numbered CN1847852A, which appears in the prior art, describes the rapid mononucleotide polymorphism test paper strip and the detection method. It describes a method for detecting two polymorphism-based SNPs using nucleic acid test paper strips. It relates to the method of detecting various polymorphism-based SNPs by nucleic acid test paper strip and the application of detection methods in molecular diagnosis of SNP-related human genetic diseases, detection of drug-resistant gene mutation of bacteria, viruses and other pathogenic microbes, detection and screening of drug reaction difference in human body, screening and typing of SNP as human health genetic basis. It is a method for rapid detection of single base mutations in a nucleic acid sequence using amplification techniques, single nucleotide extension techniques, and nucleic acid strip detection techniques. Test strips for rapid nucleic acid diagnosis are used in the field of rapid detection and typing of single nucleotide polymorphisms. It also relates to the use of a kit for the rapid detection of single nucleotide polymorphisms in the nucleic acid sequence.

[0011] In the state of the art, there is a patent application numbered TR 2016 / 08981 registered in the name of the inventors, and the invention relates to probes, SNP markers and SNP combinations used for the diagnosis of bacterial species. In the invention numbered 2016 / 08981, in particular, SNP Markers, SNP Marker combinations specific to bacterial species, which enable the identification of the inventive bacterial species at the species level, and probes that enable the determination of said SNP Markers are described, and in this technique, detection and diagnosis can be provided with the help of forward and reverse probes.

[0012] The invention differs from the cited existing techniques, and in particular from the application numbered 2016 / 08981, in that the invention enables the detection of SNPs different from the previous one with a single kit for multiple Brucella species without the need for reverse and forward probe.

[0013] Classical serological, biochemical and culture-based tests and classical PCR and qPCR-based molecular methods for disease control in the state of the art are described. However, since the current classical methods have to be applied separately for each bacterial species, they are not profitable in terms of labour and cost, especially for mass testing and reference laboratories where the sample diagnosis rate is high. It may cause different infections in the same sample to be missed. In addition, the sensitivity of these classical methods used in the art is low, some methods take a long time to apply and do not allow the detection of the causative agent, especially in the early stages of some infections. For this reason, an R&D study needs to be conducted in this field.

[0014] Object of the Invention

[0015] The main object of the present invention is to determine SNP markers and their combinations for the diagnosis and genotyping of Brucella species in different samples, to determine Single Nucleotide Polymorphisms (SNP) that will provide interspecies distinction in phylogenetically significant gene regions for the first time with developing software technologies, and to develop a bacterial diagnostic panel that can diagnose the selected bacterium or bacterial group with combinations of these points in multiple samples with high accuracy, regardless of the source, and to develop a software to carry out this bioinformatics process.

[0016] The object of the present invention is to determine which or which of the bacterial species is infected by using genomic DNA of a single sample in a short time with high accuracy by performing multiplex reactions, both to reduce the cost when working on multiple samples with less labour and to prevent false negative results due to the high sensitivity of the system used.

[0017] Another object of the present invention is to enable the operations to be performed simultaneously for the DNA of more than one sample rather than for the DNA of a single sample.

[0018] Another object of the present invention is to provide a diagnostic panel for the rapid, early and effective diagnosis of primary and secondary pathogens that can be encountered in samples from different sources on a single platform.

[0019] Another object of the present invention is to detect species-specific differences of different bacterial species in multiple samples, enabling simultaneous diagnosis with high accuracy and sensitivity.

[0020] The object of the present invention is to determine the SNPs detected for the discrimination of Brucella species, to determine the discrimination points on a species basis, to ensure that the determined SNP points create a genotype difference between the species and to determine the SNP numbers that enable the species to be separated from another species for each species in the species discrimination profiles between Brucella abortus, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis.

[0021] Another object of the present invention is to establish a database for gene sequences using the MySQL v8.0.32 platform, and to download and save 118.414 gene sequences of 23S, 16S and rpoB terms to the database by using the NCBI Nucleotide database query screen and query tags, and thus to enable the 23S, 16S and rpoB gene sequences to be used in the alignment phase. The object of the present invention is to develop a local alignment tool by using a dynamic programming technique to dynamically perform sequence alignments at the species and genus level and by developing a strategy to avoid biases due to data density.

[0022] Another object of the present invention is to develop it in a way that includes multiple sequence alignments, from entering the query into the detection panel tool to SNP detection.

[0023] The object of the present invention is that the multiple sequence alignment process, which is one of the processes used in the detection panel, has the following steps: assigning all aligned sequences to equivalent lengths, preparing a common template from the nucleotide profile representing the species, where intra-species polymorphisms are defined as 'X' on the sequence, from the sequences of different strains belonging to the same species, in order to increase the accuracy and precision of sequence alignments and to reduce bias due to data density, performing alignment by taking the 90% consensus sequence (consensus sequence) formed after the sequences are aligned within the type as a target.

[0024] Point scanning was performed on rpoB sequences for which alignment studies were completed for Brucella species. Each nucleotide position of the obtained templates was compared with the equivalent positions of other types of templates in the claim set. As a result of the comparison, candidate points that can be used in differential diagnosis were determined and a difference matrix was created by calculating the identification power for each candidate point. In the binary combinations of the species for which no solution was found in the difference matrix, the discrimination potential of at least 1 and at most 1203 points was determined and 84 points were stored as candidate points. Then, a distinguishing point was determined for each location in the coverage area by calculating the strongest distinguishing points with minimum usage from the candidate points in the solution set. As a result of the process with the calculated position density, it was concluded that the discrimination of Brucella abortus, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis species could be realised with positions numbered 846, 771, 1779, 1507 and 2016. Brief Description of the Invention

[0025] The present invention relates to SNP markers and combinations thereof for the diagnosis and genotyping of Brucella species in different samples, which fulfil the above-mentioned requirements, eliminating all disadvantages and bringing some additional advantages.

[0026] Diagnosis of bacterial species is made separately using classical systems (current technical and literature studies). Different methods with different sensitivities can be used to diagnose each bacterial species, and in some infections, early diagnosis cannot be made and multiple infections may be overlooked. Different bacteria cause different infections. The gene sequences of each bacterium are not the same as those of another; they show species-specific differences. With the help of the invention, these unique differences of different bacterial species are detected in multiple samples, enabling simultaneous diagnosis with high accuracy and sensitivity.

[0027] Humans usually acquire brucellosis through direct contact with infected animals, by eating or drinking contaminated animal products or by inhaling airborne agents. Most cases occur through consumption of unpasteurized milk or cheese from infected goats or sheep. Brucellosis is one of the most common zoonoses transmitted by animals, and human brucellosis has serious public health consequences in endemic areas. The expansion of animal industries and urbanization and the lack of hygienic measures in animal husbandry and food processing partly explain why brucellosis remains a public health hazard (WHO, 2020c). The main Brucellosis agents that cause infection in humans are Brucella abortus, B. melitensis, B. suis, B. ovis, and B. neotomae.

[0028] With this discovery, the differences (SNPs) of Brucella species that are the subject of this application were determined for the first time in this study.

[0029] Another feature of the invention is that it makes it possible to analyse the target bacteria of the panel independently of the sample or tissue source (blood, FFPE tissues, fresh tissue, food sample, etc.) from which they are isolated.

[0030] A feature of the invention is the detection of specific probe sequences in each bacterium for diagnosis. The detection of probe sequences was determined for the first time in this study. During the multiple sequence alignment process, the first difficulty encountered with a large number of sequences is the mismatch of base pair lengths between a large number of sequences. For this purpose, the nucleotide sequences recorded in the database were first aligned according to their length, then the longest sequence was aligned with the next one and the length of the 2ndsequence was updated according to the alignment result. After this process, the length of all sequences is equalized with the longest sequence. A number of different strategies have been considered to align all of these sequences.

[0031] One of the objects of the present invention is to develop a SNP-based panel that will diagnose Brucella infection agents, five different bacterial pathogens that are the primary cause of 75% of bacterial infection-related deaths worldwide.

[0032] With the invention, it is also aimed to make a great contribution to public health through the development of diagnosis, treatment and protection methods against future pandemics and the rapidly advancing danger of antibiotic resistance with developing computer and health technologies.

[0033] The comparison of the solutions in the state of the art and the solution developed with the invention subject to this application is given in the table below.

[0034] Table a: Comparison of the state of the art with the invention in terms of features

[0035] The structural and characteristic features of the present invention will be understood clearly by the following drawings and the detailed description made with reference to these drawings and therefore the evaluation shall be made by taking these figures and the detailed description into consideration. Detailed Description of the Invention

[0036] For the analyses of the detection panel within the scope of the invention, an Intel Core i9-7900 X CPU 3.30 GHz processor, 64 GB RAM and 4 GeForce 1080 Ti GPU equipped computers installed in Altmba§ University Faculty of Engineering and Architecture, Department of Software Engineering were used. Local databases created throughout the study were created using Microsoft SQL V16.0 and MySQL v8.0.32 platforms. The ODOTool Strategy Based Local Alignment Tool developed within the scope of the invention was prepared on the Python v3.8.10 platform and utilized the Biopython vl.75 library (Cock et al., 2009). (Deoxyribo Nucleic Acid (DNA), Ribonucleic acid (RNA), Adenine (A), Cytosine (C), Thymine (C), Guanine (G).) Additionally, the C# v9.0 platform was also used in different stages of the study.

[0037] Within the scope of the invention, 16S ribosomal DNA, 23S ribosomal DNA and RNA polymerase Beta subunit (rpoB) gene sequences were used to create bacterial diagnostic panels.

[0038] To download the bacterial gene sequences of the determined panel, a search was performed using NCBI, Nucleotide database query screen and query tags. A total of 118.414 gene sequences with 23S, 16S and rpoB terms belonging to 5 microorganisms at the genus and species level studied within the scope of the subject matter of the invention and the taxonomy, microbiome or those related to these microorganisms in a way that cannot be understood were downloaded. After data cleaning, a total of 3760 gene sequences, including 381 23S, 893 rpoB and 2486 16S, were used in marker detection analyses.

[0039] A database for gene sequences was established using the MySQL v8.0.32 platform. In the search conducted using the NCBI Nucleotide database query screen and query tags, 118.414 gene sequences containing the terms 23S, 16S and rpoB in the title were downloaded and saved in the database. Registration was completed by matching the information given in fasta format for each gene (gene sequence, accession number, molecule type, organism name, taxonomic class steps, sequencing platform, product, isolation source, culture collection information, serotype- biovar, binary organism name) with the information given during registration to the database (Species no, Sequence no, Input no,). Subsequently, genome sequences with a sequence length less than 1000 base pairs and greater than 5000 base pairs, coming from metagenomic studies although not on the desired organism list, containing different gene fragments, containing too many unreadable regions as 'N' (Nucleotide) were excluded from the analysis. After data cleaning, 23S, 16S and rpoB gene sequences were used in the alignment step.

[0040] Establishment of a diagnostic panel that enables the identification of Brucella abortus, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis species / genera at the species level and the identification of multiple bacterial pathogens or species / genera in a single sample to ensure species identification by creating genotype difference between species, characterized by comprising the process steps of; preparation of queries and downloading from open access database, recording the information that will cluster the data into the desired categories in the local database, aligning gene sequences and rearranging them in the database, identifying conserved and variational regions within aligned gene sequences, detection of SNPs within conserved regions, defining a combinatorial panel of detected SNPs that can be used in the diagnosis of Brucella species

[0041] In this scope, the Strategy-Based Local Alignment Tool was developed as a software that can perform the above-mentioned process steps and ODOTool, which was first mentioned in the article published in 2020 (llgurel et al., 2020a).

[0042] The probe and SNP combination detection panel for the diagnosis of Brucella species in multiple samples with a single panel enables simultaneous diagnosis with high accuracy and sensitivity by detecting these unique differences of different bacterial species in multiple samples.

[0043] The differences (SNPs) of Brucella species that are the subject of this application were determined for the first time in this study, and the probe sequences determined for each bacterium for diagnosis were determined for the first time in this study. It is possible to analyze the target bacteria of the panel, Brucella species, independently of the sample or tissue source from which they were isolated (blood, FFPE tissues, fresh tissue, food sample, etc.). Below is the list of Brucella species to be detected.

[0044] Sensitivity and specificity of the identified SNP biomarkers were investigated using the BLASTN tool. This verification analysis was conducted in two stages. In the first stage, the oligo sequence to be used in genotyping was scanned for genomes that belonged only to that organism and could not be used in SNP detection, and the intra-species polymorphism risk of the detected SNP was examined. In the second stage, the risk of cross-reactivity between other species in the same panel and the risk of similarity to a region found in all genomes was investigated.

[0045] Table 1. List of Brucella species distinguished in the panel

[0046] The species-based discrimination points of the SNPs detected to distinguish between five Brucella species are shown in Table 2. SNP points determined in this study create genotype differences between species.

[0047] Table 2. Genotype Combination Table The rpoB sequences, whose alignment studies were completed, were subjected to point scanning. Each nucleotide position of the obtained templates was compared with the equivalent positions of other types of templates in the claim set. As a result of the comparison, candidate points that can be used in differential diagnosis were determined and a difference matrix was created by calculating the identification power for each candidate point. In the binary combinations of the species for which no solution was found in the difference matrix, the discrimination potential of at least 1 and at most 1203 points was determined and 84 points were stored as candidate points. The number of binary combinations checked at this stage is 85.113. Then, a distinguishing point was determined for each location in the coverage area by calculating the strongest distinguishing points with minimum usage from the candidate points in the solution set.

[0048] As a result of the calculated position density process, it was concluded that the separation of these 5 species could be achieved at positions 846, 771, 1779, 1507 and 2016. The scanning of the obtained in siiico results was first performed with Jalview. Jalview images of the distinctions on the RNA Polymerase Beta Subunit of five species are given. (Figure - 1)

[0049] In the inter-species discrimination profiles given below, the SNP number that distinguishes each species from the opposite species is given in Table 3.

[0050] Table 3. Interspecies Discrimination Matrix

[0051] Table 4. Nucleotide sequences to be used in determining genotypes

[0052] Table 5. Separation matrix of the five species on the RNA Polymerase Beta subunit In order to show the distinction between species of the obtained SNPs, a speciesdiscrimination matrix was created and the species that were distinguished by the SNPs that would provide the distinction were determined and are given in Table 6.

[0053] Table 6. Discrimination matrix of five species for SNPs on the RNA Polymerase Beta subunit

[0054] The RNA Polymerase Beta subunit gene sequence is a valuable gene for bacterial identification studies as it is highly conserved and can be used to distinguish between closely related species. This is particularly important in the context of antibiotic resistance, where incorrect or delayed diagnosis can have serious consequences for patient outcomes. In general, the implementation of these measures represents an important step forward in the field of bacterial diagnostics and has the potential to have a significant impact. In addition to the importance of accurate diagnosis, understanding the genetic diversity within bacterial species is also crucial.

[0055] In the study conducted with the NCBI BLAST tool, the NCBI nucleotide database was scanned and biomarkers that could diagnose Brucella abortus, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis were investigated. As a result of BLAST validations, it was determined that these 4 points were usable in the panel. It is a BLASTN tool that allows the investigation of the sensitivity and specificity of the identified SNP 1, SNP 2, SNP 3, SNP 4, SNP 5 biomarkers. Jalview is a tool that allows viewing and editing of multiple sequence alignments.

[0056] With the results obtained, within the scope of the invention, a diagnostic panel has been created to discriminate SNP points obtained as a result of sequencing for the diagnosis of Brucellosis. Thus, discrimination between species was made possible by directly determining SNP markers, combinations and points without the need for probes used in previous techniques.

Claims

CLAIMS1. Diagnostic panel for the identification of genetic markers and the identification of Brucella abortus, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis species / genera at the species level in the detection panel that enables the identification of more than one bacterial pathogen or species distinction in a single sample and the identification of species by creating genotype difference between species, characterized in that; SNP points and SNP point locations, which enable the determination of species distinction of Brucella species are as follows,2. The SNPs or combinations of SNPs according to claim 1, characterised in that; five species are said SNP points on the RNA Polymerase Beta subunit of the discrimination matrix.

3. The SNP or combination of SNPs according to any one of the preceding claims, characterised in that; the nucleotide sequences enabling the identification of SNPs are as follows;4. The SNP or combination of SNPs according to any one of the preceding claims, characterised in that; the following discrimination matrices are used to distinguish between Brucella species:

5. The SNP or combination of SNPs according to any one of the preceding claims, characterised in that; a Blast tool that allows the investigation of the sensitivity and specificity of the mentioned SNP biomarkers.

6. The SNP or combination of SNPs according to any one of the preceding claims, characterised in that; a Jalview tool that allows viewing and editing of the mentioned SNP multiple sequence alignments.

7. Establishment of a diagnostic panel that enables the identification of Brucella abortus, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis species / genera at the species level and the identification of multiple bacterial pathogens or species / genera in a single sample to ensure species identification by creating genotype difference between species, characterized by comprising the process steps of; preparation of queries and downloading from open access database, recording the information that will cluster the data into the desired categories in the local database,aligning gene sequences and rearranging them in the database, identifying conserved and variational regions within aligned gene sequences, detection of SNPs within conserved regions, - defining a combinatorial panel of detected SNPs that can be used in the diagnosis of Brucella species.

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