FIELD OF INVENTION
The present invention provides for identifying and detecting clinically and economically important Brucella species and their biovars. More specifically the invention provides for novel primers for identifying and detecting clinically and economically important Brucella species irrespective to their biovars. The present invention also provides for use of novel primers in monoplex and multiplex PCR methods for direct detection of clinically and economically important Brucella species irrespective to their biovars.
BACKGROUND OF THE INVENTION
The alpha2 proteobacterial Brucella genus consists of ten species based on preferential host specificity: viz., Brucella abortus (cattle). Brucella melitensis (goats). Brucella suis (swine). Brucella canis (dogs). Brucella ovis (sheep). Brucella neotomae (desert mice). Brucella microti (voles). Brucella pinnipedialis (seal). Brucella ceti (cetacean). Brucella inopinata (unknown) [1]. However, in rare cases, these species demonstrate varied host specificity for reasons unknown. Hence, the mechanism of Brucella host specificity is still unclear. At molecular stratum, these species share a high degree of homology among their genome organization rendering it challenging to differentiate them from each other [2].
Some of the species like B. abortus, B. melitensis, B. suis and B. canis cause infection in both animals and humans [3]. The syndromes caused by Brucella species are collectively termed as brucellosis. Brucellosis is still a major zoonotic disease challenged by several developing countries including India. With the lack of suitable therapy for this infection, culling of the infected animals has been followed in developed countries. However, this is banned in countries like India based on religious grounds. As a result, the encroachment of this disease is directly on the economic loss in terms of loss of animals, deterrent for trade, low productivity of food-animals and humans which threatens our social life, agricultural life and the civilization. As a result these species are also identified as bio-threat agents.
The poor disease control strategy, inadequate animal birth control program and lack of efficient detection kits in low income-countries has resulted in failure of eradication of this

severe zoonotic disease. As a result, there is an immediate need for detection and prevention of spread of the disease by implementing rapid and proper diagnostic procedures.
The diagnostic procedures should possess the property of accurate identification of each Brucella species irrespective of biovars and differentiation of the species from each other. This intensive and specific diagnostic procedure is of great importance for epidemiological study, disease control strategies and clinical purposes. High degree of similarity in genome content among these species promotes a challenging task to achieve this objective.
Till date, conventional method is more reliable in identification and differentiation of Brucella species. The method includes isolation and purification of presumptive culture, followed by Gram’s staining, phenotypical and biochemical characterization including CO2, H2S test, dye sensitivity test, phage sensitivity test and nitrate reduction test [4, 5]. Automated and semi-automated biochemical strips like API20NE are also available. However, these methods are highly time consuming, projects need for isolated and pure presumptive culture, extremely trained and skilled manpower. These methods pose a great risk to the lab personnel also.
PCR assay for the simultaneous detection and differentiation of Brucella species has played an important and dynamic role in the past several decennary. These methods are becoming very important tools for the identification of Brucella, at the species level and recently also at the biovar level. These techniques require no isolated and/or pure culture and thus minimum biological containment and can provide results in a very short time. PCR-based methods are more useful and practical than conventional methods used to identify Brucella species.
The first Brucella multiplex PCR assay (AMOS) differentiating the 5 important species i.e.,
B. abortus, B. melitensis, B. ovis, B. suis and B. canis was developed in the year of 1994 by
Bricker BJ et al. The method had employed 5 different primers to differentiate the species from each other. However, the method was limited to identify selected biovars among the
species. It could identify biovars 1, 2 and 4 of B. abortus; biovars 1, 2 and 3 of B. melitensis; and only biovar 1 of each B. suis and B. ovis. This method was found to be reliable and produced 100% result when compared with other conventional biotyping methods on identifying field samples [6].
In a study, Darla R. Ewalt and Betsy J Bricker have employed three more additional primers to advance the AMOS PCR for simultaneous detection of B. abortus vaccine strain S19 and
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RB-51 [7]. In yet another study by Alain A. Ocampo-Sosa et al [8] the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars by using one more additional primer was facilitated. The new advanced AMOS PCR was evaluated on several field samples and ascertained to be useful and authentic for rapid as well as simultaneous detection and differentiation of Brucella species.
Le´niaFerra˜o-Beck et al [9] also tried to develop a multiplex PCR distinguishing B. suis biovars 1, 2 and 3 exploiting a single nucleotide polymorphism in Omp2b gene of B. suis. Nevertheless, the strategy failed to distinguish it properly, as the biovars 2 and 3 according to AMOS PCR and PCR RFLP method based on omp31 and omp25 genes, produced the same pattern as biovars 1 of this new strategy, confining the usefulness of this strategy of distinguishing B. suis biovar 1, 2 and 3.
A new perspective came into the function when a new multiplex PCR known as Bruce-ladder was developed identifying all 6 terrestrial species, 3 marine species viz. B. neotomae, B.
pinnipedialis, B. ceti and vaccine strains i.e., B. abortus S19, RB-51 and B. melitensis Rev 1
[10]. Further, the method was improved and the PCR assay could identify and differentiate all nine Brucella species including marine species [11].
A set of 19 primers could differentiate all the marine species along with differentiating B. abortus biovar 1, 2, 4 from biovars 3, 5, 6, 9; furthermore, it differentiated among B. suis biovar 1, biovars 3 and 4, and biovars 2 and 5 [12]. Yet this strategy produced similar amplicon pattern of B. canis second group with B. suis biovars 3 and 4.
Though found promising and reliable, the available multiplex PCR formats fail to facilitate the simultaneous identification of species in a single lane or in single sample analysis of electrophorosed agarose gel. The banding pattern of each species has to be analysed thoroughly for interpretation of the results. This necessitates skilled personnel for the interpretation of the results as well.
Real-time PCR is another advancement in the field of detection since this method takes very less time, sensitive and even more need not go for gel electrophoresis. The method facilitates a broad range of samples to be analyzed including Brucella culture [13], serum samples [14], blood samples from infected animals or human and paraffin embedded tissue samples [15]. The technique is beneficial for the identification of Brucella at species level. Three different
formats for rapid detection of B. abortus, B. melitensis and B. suis was developed
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successfully. All three methods were found to be extremely specific as well as sensitive when evaluated on field samples. Like AMOS-PCR for the detection of Brucella species, these strategies take the benefit of insertional sequence 711 to design the forward primer and the reverse primer for each strategy and was species specific.
A real-time PCR method with syber green targeting the bcsp 31 was compared with a PCR-enzyme linked immunosorbent assay. The real time PCR method being highly sensitive could detect 10 fg DNA from serum samples [14].
In addition, a TaqMan probed real-time PCR was developed to differentiate inactive, seropositive, and active human brucellosis in serum samples. This strategy was also able to detect 10 fg DNA into the serum samples [16].
Several other real-time PCRs were developed in the past decades targeting 16S-23S ITS, omp25and omp31 regions [15]. All these methods were very sensitive and specific in detecting Brucella at species level. Sometimes the same primers were even evaluated for conventional PCR method providing satisfactory results. Even so, real-time PCR needs highly skilled personnel for execution of the work and the reagents are too costly for performing detection test on day to day basis in a diagnostic laboratory with large number of samples, in developing countries like India.
Nevertheless, there is still a great deal of work required for verification, validation, establishment of standard positive and negative controls, internal control, reagents, and quality assurance before any of these methods may be used in routine laboratory testing for brucellosis.
SUMMARY OF THE INVENTION
In one aspect of the present invention the novel nucleotide SEQ ID No. 11-20 for the detection and/or identification of various species and/or biovars of Brucella genus have been provided. The nucleotide sequence can be used in a PCR based method or DNA based systems for the detection and/or identification of various species and/or biovars of Brucella genus.
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In another aspect of the present invention the PCR based methods comprise but are not limited to monoplex, multiplex PCR immune-PCR, real-time PCR and/or loop-mediated isothermal amplification PCR. The DNA based systems comprise but are not limited to /fluorescent/chemiluminescent DNA probe based blotting, DNA-DNA hybridization, DNA-RNA hybridization systems. The nucleotide sequences are useful for the detection and identification of Brucella species in clinical sample collected from humans or non-humans.
In yet another aspect of the present invention, set of primers of Brucella species and their biovars having SEQ ID Nos. 23-32 have been provided; wherein the species of Brucella
genus and their biovars are selected from B.abortus, B.melitensis, B.ovis, B.suis and B.canis.
The set of primers are useful for detection and identification of Brucella species in clinical samples collected from humans or non-humans; the primers are characterized as following:
(a) the primer set have SEQ ID Nos. 23-24 are Forward and Reverse primers and
enables identification of B.abortus;
(b) the primer set have SEQ ID Nos. 25-26 are Forward and Reverse primers and
enables identification of B.melitensis;
(c) the primer set have SEQ ID Nos. 27-28 are Forward and Reverse primers and
enables identification of B.ovis;
(d) the primer set have SEQ ID Nos. 29-30 are Forward and Reverse primers and enables identification of B.suis; and
(e) the primer set have SEQ ID Nos. 31-32 are Forward and Reverse primers and enables identification of B.canis.
In another aspect of the present invention the set of primers enable identification of specific
amplicon that is specific Brucella species and rescue amplicon that is common non-specific Brucella species but is absent in specific Brucella species. The said primers can be used in both monoplex and multiplex PCR methods for detection of Brucella species or biovars
In yet another aspect of the present invention, a PCR method for differential detection and identification of specific DNA of Brucella species or biovars using set of primer has been disclosed, said method comprising the steps of: isolating of DNA from Brucella species or biovars; amplifying the isolated DNA of step (a) using set of primers having SEQ ID Nos.
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23-31; and obtaining specific DNA amplicon and rescue DNA amplicon of Brucella species or biovars. The set of primers used in the PCR method are characterized as following:
(a) the primer set have SEQ ID Nos. 23 and 24 are Forward and Reverse primers
that enable identification of B.abortus;
(b) the primer set have SEQ ID Nos. 25 and 26 are Forward and Reverse primers
that enable identification of B.melitensis;
(c) the primer set have SEQ ID Nos. 27 and 28 are Forward and Reverse primers that enable identification of B.ovis;
(d) the primer set have SEQ ID Nos. 29 and 30 are Forward and Reverse primers that enable identification of B.suis; and
(e) the primer set have SEQ ID Nos. 31 and 32 are Forward and Reverse primers that enable identification of B.canis.
In yet another aspect of the present invention the set of primers used in the PCR method results in DNA amplicons or DNA amplification characterized as following:
(a) the primer set have SEQ ID Nos. 23 and 24 result in specific amplicon of
B.abortus having a size of 1154bp and rescue amplicon of B.melitensis, B.ovis, B.suis and B.canis having a size of 1478 bp;
(b) the primer set have SEQ ID Nos. 25 and 26 result in specific amplicon of
B.melitensis having a size of 745 bp and rescue amplicon of B.abortus, B.ovis, B.suis and B.canis having a size of 977 bp;
(c) the primer set have SEQ ID Nos. 27 and 28result in specific amplicon of
B.ovis having a size of 446 bp and rescue amplicon of B.abortus, B.melitensis, B.suis and B.canis having a size of 606 bp;
(d) the primer set have SEQ ID Nos. 29 and 30 result in specific amplicon of
B.suis having a size of 290 bp and rescue amplicon of B.abortus, B.melitensis, B.ovis and B.canis having a size of 382 bp; and
(e) the primer set have SEQ ID Nos. 31 and 32 result in specific amplicon of
B.canis having a size of 224 bp and rescue amplicon of B.abortus, B.melitensis, B.ovis and B.suis having a size of 521 bp.
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In yet another aspect of the present invention PCR method detects and identifies Brucella species in clinical samples collected from humans or non-humans. The PCR method comprise of monoplex, multiplex PCR immuno-PCR, real-time PCR and/or loop-mediated isothermal amplification PCR for detection and identification of Brucella species or biovars. Monoplex and/or multiplex PCR based detection system for detection of Brucella species and differentiation of Brucella species at stratum of biovars using at least a primer or a primer set as herein disclosed. The DNA-based detection systems for detection of Brucella species and their biovars using nucleotide sequences have SEQ ID No. 11-20 has been defined, the DNA-based detection systems comprise of including fluorescent/chemiluminescent DNA probe based blotting, DNA-DNA hybridization, DNA-RNA hybridization systems.
In yet another aspect of the present invention, a method for detection of species and biovars of Brucella genus using nucleotide sequences 11-20 has been defined, said method comprising steps of: carrying out in silico comparative genomic comparison of Brucella species to identify unique specific and rescue amplicons; (b) using nucleotide sequences have SEQ ID No. 11-20 as primer sequences in a PCR based method to identify presence or absence of specific and rescue amplicon of step (a) in species and biovars of Brucella genus; and (c) identifying and differentiating specific species of Brucella genus irrespective of their biovars.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Multiple sequence alignment of B. melitensis (NC_007618), B. ovis (NC_009505),
B. suis (CP002997) and B. canis (CP000872) nucleotide sequences possessing unique intergenic nucleotide sequence with that of B. abortus (NC_016795) lacking the unique intergenic nucleotide sequence indicated by dash.
Figure 2. Multiple sequence alignment of B. abortus (NC_016795), B. ovis (NC_009505), B.
suis (CP002997) and B. canis (CP000872) nucleotide sequences possessing unique intergenic nucleotide sequence with that of B. melitensis (NC_007618) lacking the unique intergenic nucleotide sequence indicated by dash.
Figure 3. Multiple sequence alignment of B. abortus (NC_016795), B. melitensis (NC_007618), B. suis (CP002997) and B. canis (CP000872) nucleotide sequences
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possessing unique intergenic nucleotide sequence with that of B. ovis (NC_009505)lacking the unique intergenic nucleotide sequence indicated by dash.
Figure 4. Multiple sequence alignment of B. abortus (NC016795), B. melitensis (NC_007618), B ovis (NC_009505) and B. canis (CP000872) nucleotide sequences possessing unique intergenic nucleotide sequence with that of B. suis (CP002997)lacking the unique intergenic nucleotide sequence indicated by dash.
Figure 5. Multiple sequence alignment of B. abortus (NC016795), B. melitensis (NC_007618), B ovis (NC_009505) and B. suis (CP002997) nucleotide sequences possessing unique intergenic nucleotide sequence with that of B. canis (CP000872) lacking the unique intergenic nucleotide sequence indicated by dash.
Figure 6. Pictorial illustration of the primer designing strategy for specific detection of B. abortus, B. melitensis, B. ovis, B. suis and B. canis.
Figure 7. Pictorial representation of PCR amplicon sizes expected from each primer pair of the present invention. . B abortus specific primer set amplifies a 1154 bp product with B. abortus and
1478 bp product with other four species namely B melitensis, B suis, B. ovis
and B. canis. . B melitensis specific primer set amplifies a 745 bp product with B melitensis
and 977 bp product with other four species namely B abortus, B suis, B. ovis
and B. canis. . B ovis specific primer set amplifies a 446 bp product with B ovis and 606 bp
product with other four species namely B. abortus, B. melitensis, B. suis and B.
canis. . B suis specific primer set amplifies a 290 bp product with B. suis and 383 bp
product with other four species namely B. abortus, B. melitensis, B. canis and
B. ovis. . B canis specific primer set amplifies a 224 bp product with B. canis and 521
bp product with other four species namely B. abortus, B. melitensis, B. suis
and B. ovis.
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. The IAC primer set amplifies 808 bp of pUC19 plasmid, yielding a product of 847 bp (inclusive of the 5’ B. abortus specific flanking regions of the primer pair).
Figure 8.Agarose gel electrophoretic analysis of monoplex PCR format for specific detection of Brucella abortus 544, Brucella melitensis 16M, Brucella ovis ATCC25840, Brucella suis1330 and Brucella canis ATCC 23365. Lane 1 and 7. 50 bp DNA ladder; Lane 2.B. abortus specific amplicon (1154 bp) with B. abortus specific primer; Lane 3.B. melitensis specific amplicon (745 bp) with B melitensis specific primer; Lane 4.B. ovis specific amplicon (446 bp) with B ovis specific primer; Lane 5.B. suis specific amplicon (290 bp) with B. suis specific primer; Lane 6. B canis specific amplicon (224 bp) with B. canis specific primer; Lane 8.B. abortus rescue amplicon (1478 bp) with primers of the present study except B abortus specific primer; Lane 9.B. melitensis rescue amplicon (977 bp) with primers of the present study except B. melitensis specific primer; Lane 10. B ovis rescue amplicon (606 bp) with primers of the present study except B. ovis specific primer; Lane 11. B. suis rescue amplicon (383 bp) with primers of the present study except B suis specific primer; Lane 12. B. canis rescue amplicon (521 bp) with primers of the present study except B. canis specific primer.
Figure 9. Agarose gel electrophoretic analysis of PCR format using Brucella abortus 544, Brucella melitensis 16M, Brucella ovis ATCC 25840, Brucella suis1330 and Brucella canis ATCC 23365genomic DNA as template and one pair of primer per reaction. Lane 1 and 7. 50 bp DNA ladder; Lane 2. Specific and rescue amplicon resulted from B. abortus specific primer pair (1154 bp and 1478 bp); Lane 3. Specific and rescue amplicon resulted from B. melitensis specific primer pair(745 bp and 977 bp); Lane 4. Specific and rescue amplicon resulted from B. ovis specific primer pair(446 bp and 606 bp); Lane 5. Specific and rescue amplicon resulted from B suis specific primer pair(290 bp and 383 bp); Lane 6. Specific and rescue amplicon resulted from B. canis specific primer pair(224 bp and 521 bp), Lane 7. 50 bp DNA ladder.
Figure 10.Agarose gel electrophoretic analysis of multiplex PCR assay optimized by incorporation of an internal amplification control (847 bp). All five novel primer pairs were involved in the PCR reaction. Lane 1 and 10. 50 bp DNA ladder; Lane
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2 and 9. PCR using B. abortus isolate DFRL1, B. melitensis isolate DFRL1, Brucella ovis ATCC 25840, Brucella suis 1330 and Brucella canis ATCC 23365
genomic DNA as template DNA; Lane 3. PCR using Brucella abortus 544 genomic DNA as template DNA; Lane 4.PCR using Brucella melitensis 16Mgenomic DNA as template DNA; Lane 5.PCR using Brucella ovis ATCC 25840 genomic DNA as template DNA; Lane 6.PCR using Brucella suis1330 genomic DNA as template DNA; Lane 7.PCR using Brucella canis ATCC 23365
genomic DNA as template DNA; Lane 8.PCR without any genomic DNA.
Figure 11. Figure illustrating the interpretation of integrative multiplex PCR formats for specific detection of Brucella species.
DETAILED DESCRIPTION
While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the drawings, graphs and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.
The graphs, tables, figures and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
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The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by “comprises... a” does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.
The terms, “alone or in combination” or any other variations thereof, are intended to described and/or cover a non-exclusive inclusion, wherein the molecules or the oligonucleotides exist individually or together with any one or all of the other oligonucleotides.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
It must be noted that, as used in the specification/description and the appended claims and examples, the singular forms “a”, “an” and “the” may include plural referents unless the context clearly dictates otherwise.
Ranges may be expressed herein as from “about” one particular value, and or “to about” another particular value. When such a range is expressed, another aspect includes from the one particular value and or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about”, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint
As used herein the term, “Species” in the context of the present invention means group strains of Brucella with approximately 90% or greater DNA-DNA homology.
As used herein the term, “Biovars/Biovar” in the context of the present invention means group of Brucella strains that are distinguishable from other strains of the same species
including B. abortus, B. melitensis, B. ovis, B. suis and B. canis on the basis of their
physiological characteristics.
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As used herein the term, “Brucella Species” in the context of the present invention means
group strains of Brucella including B. abortus, B. melitensis, B. ovis, B. suis and B. canis with
approximately 90% or greater DNA-DNA homology.
As used herein the term, “Brucella Biovar/s” in the context of the present invention means
strains of Brucella species including B. abortus, B. melitensis, B. ovis, B. suis and B. canis
that are distinguishable from each other on the basis of their physiological characteristics.
As used herein the term, “Brucella species and/or biovars” in the context of the present invention means group of Brucella strains irrespective of species and biovars of B. abortus,
B. melitensis, B. ovis, B. suis and B. canis.
As used herein the term, “Simultaneous Identification” in the context of the present invention means identification of any strain of B. abortus, B. melitensis, B. ovis, B. suis and B. canis at
a time in a single PCR reaction.
As used herein the term, “Differential Identification” in the context of the present invention means distinguishable identification of each Brucella species namely B. abortus, B. melitensis, B. ovis, B. suis and B. canis based on each species specific PCR amplicon size.
As used herein the term, “Simultaneous and Differential Identification” in the context of the
present invention means distinguishable identification of each Brucella species namely B.
abortus, B. melitensis, B. ovis, B. suis and B. canis based on each species specific PCR
amplicon size at same time in a single PCR reaction.
As used herein the term, “In-Silico” in the context of the present invention means
comparative genomic analysis of B. abortus, B. melitensis, B. ovis, B. suis and B. canis using
computer simulation and whole genome sequence data.
As used herein the term, “Differential Sequence/Differentially present sequences/ differentially present unique sequences/Target sequences/non-conserved sequences” in the
context of the present invention means the stretch of nucleotide sequences (Differential
Sequence/Differentially present sequences/ differentially present unique sequences)which are
uniquely distributed in the selected length (Target sequence) of genomic DNA of B. abortus,
B. melitensis, B. ovis, B. suis and B. canis making PCR based differential identification of each Brucella species.
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As used herein the term, “Conserved Sequence/s” in the context of the present invention means a sequence/s of nucleotides in DNA that is identical across B. abortus, B. melitensis,
B. ovis, B. suis and B. canis.
As used herein the terms, “Specific Brucella Species and/or Biovars” in the context of the
present invention means those Brucella species and/or biovars which comprise of specific
amplicon and has absence of rescue amplicon.
As used herein the terms, “Rescue Brucella species/Common Brucella Species/non-specific
Brucella species and/or Biovars” in the context of the present invention means those Brucella species and/or biovars which comprise of rescue amplicon and has absence of specific
amplicon. The terms “Rescue Brucella species/Common Brucella Species/non-specific
Brucella species and/or Biovars”, as used in the context of the present invention have been
used interchangeably and are meant to have the same definition, meaning and functions
As used herein the terms, “Amplicon/DNA Amplicon/DNA Amplification/DNA Amplicon sequence/DNA Amplification sequence” in the context of the present invention means a
unique DNA sequence which when amplified by a novel nucleotide sequence or nucleotide
PCR primers as herein described. The terms “Amplicon/DNA Amplicon/DNA
Amplification/DNA Amplicon sequence/DNA Amplification sequence”, as used in the context
of the present invention have been used interchangeably and are meant to have the same definition, meaning and functions.
As used herein the terms, “Specific Amplicon/Specific DNA Amplicon/Specific DNA
Amplification Sequences” in the context of the present invention means a specific DNA
sequence or Amplicon or DNA Amplicon sequence or DNA Amplification sequence, which is amplified by any one of the novel nucleotide sequence or specific primers as herein described, that detects and/or identifies and/or belongs to a specific Brucella species or their biovars or biovar thereof but is absent in the rescue or non-specific or common Brucella
species or their biovars or biovar thereof. The terms “Specific Amplicon/Specific DNA Amplicon/Specific DNA Amplification”, as used in the context of the present invention have
been used interchangeably and are meant to have the same definition, meaning and functions. The specific amplicon/s, are PCR amplification gel bands which represent or remain confined to a specific Brucella species. These amplicons were identifiable or detected only with a specific primer set belonging to that specific Brucella species.
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As used herein the term, “Rescue Amplicon/Rescue DNA Amplicon/Common Amplicon” in
the context of the present invention means a specific DNA sequence or Amplicon or DNA Amplification sequence or DNA Amplicon sequence, which is amplified by any of the novel specific primers as herein described, that is not a Specific Amplicon and is absent in the Specific Brucella species but present to the rescue or common Brucella species. The terms
“Rescue Amplicon/Rescue DNA Amplicon/Common Amplicon”, as used in the context of the
present invention have been used interchangeably and are meant to have the same definition, meaning and functions. The rescue amplicon/s, are PCR amplifications gel bands which represent or are common to the common or rescue Brucella species i.e. these amplicons were absent in specific Brucella species. Accordingly, the specific amplicons allowed the recognition of specific Brucella species, that was absent for rescue/common Brucella species, whereas rescue amplicon/s were absent in the specific Brucella species but present in rescue/common Brucella species.
As used herein the term “Monoplex PCR”, in the context of the present invention means a polymerase chain reaction where one specific set of primer is included in a single reaction mixture. The primers designed herein are specific for their target nucleotide sequences and efficiently result in specific amplicon upon performing PCR.
As used herein the term “Multiplex PCR” in the context of the present invention means is a polymerase chain reaction where more than one primer set is included in a single reaction mixture. The primers designed herein are specific for their target nucleotide sequences and efficiently anneal to the template DNA under similar PCR conditions and prevent cross-priming and non-specific annealing.
As used herein the term, “An internal amplification control/IAC” in the context of the
present invention means is a non-target DNA sequence present in the same sample reaction tube which is co-amplified simultaneously with the target sequence. In a PCR with an IAC, a control signal will always be produced when there is presence or absence of target sequence. When neither IAC signal nor target signal is produced, the PCR has failed. Thus, when a PCR-based method is designed for detection and diagnostic purpose, an IAC will rule out false-negative results.
As used herein the term, “Non-humans” in the context of the present invention means animals other than humans which includes cattle, domesticated animals or the like.
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As used herein the term, “Samples/Clinical Samples/Specimen/Clinical Specimen” in the
context of the present invention means non-infectious or infectious human or animal materials including, but not limited to, excreta, secreta, tissue and tissue fluids, blood or any FDA-approved pharmaceuticals or materials that are blood products, excreta, secreta, tissue
and tissue fluids or the like. The terms “Samples/Clinical Samples/Specimen/Clinical
Specimen”, as used in the context of the present invention have been used interchangeably and are meant to have the same definition, meaning and functions. The “Samples/Clinical Samples/Specimen/Clinical Specimen are materials as herein described which have been
isolated or extracted and used outside the human or non-human body and further used for
only in vitro analysis and purpose.
The present invention provides for novel nucleotide sequences for the detection and/or identification of various species and/or biovars of Brucella genus. Significantly it has been found in the present invention that novel nucleotide sequences function in such a manner so as to enable detection and/or identification of various species and/or biovars of Brucella genus using various PCR based methods or DNA-based detection and/or identification systems or methods.
In one aspect the present invention provides for novel nucleotide sequences which can be used in or by variety of conventional methods for detection and/or identification of various species and/or biovars of Brucella genus. Another aspect of the present invention provides for novel nucleotide sequences which enable their use and/or application in improving the conventional methods for detection and/or identification of various species and/or biovars of
Brucella genus.
Another aspect of the present invention provides for novel nucleotide sequences which alone or in combination can be used as novel primers for detection and/or identification of various species and/or biovars of Brucella genus. In yet another aspect the present invention provides for novel nucleotide sequences which when used as set of novel primers in PCR methods for detection and/or identification of various species and/or biovars of Brucella genus.
Another aspect of the present invention provides for use of novel PCR primers which can be used in detection and/or identification of various species and/or biovars of Brucella genus by using simultaneous and differential PCR methods.
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In another aspect the present invention provides for novel differentiation monoplex and multiplex PCR methods using novel primers/nucleotide sequences for the detection and/or identification of various species and/or biovars of Brucella genus.
Yet another aspect of the present invention provides for novel nucleotide sequences for detection and/or identification of various species and/or biovars of Brucella genus in DNA-based systems. Another aspect of the present invention provides for novel DNA-based systems which comprise novel nucleotides sequences as herein described for the detection and/or identification of various species and/or biovars of Brucella genus.
In another aspect the present invention provides for DNA-based detection systems that comprise of including fluorescent/chemiluminescent DNA probe based blotting, DNA-DNA hybridization and DNA-RNA hybridization systems which uses the novel nucleotides for the detection and/or identification of various species and/or biovars of Brucella genus.
The most commonly and/or clinically known species and likewise their biovars of the
Brucella genus belong to B. abortus, B. melitensis, B. ovis, B. suis and B. canis species. Thus
the novel nucleotides of the present invention enable identification and/or detection of one or more species and/or biovars of Brucella genus by the methods as herein described. In one aspect the present invention provides novel nucleotides which function as novel primers for detection and/or identification of one or more species and/or biovars of Brucella genus.
It has been found in the present invention that the unique features or attributes of the novel nucleotide sequences or the primers as herein described lies in identification of such truncated conserved regions of specific Brucella species or biovars that are specific as well as confined to a specific species or biovars of the Brucella genus, as well as conserved regions common to those common or rescue Brucella species or biovars. The nucleotide sequences thus (a) identify specific amplicons present in a specific Brucella species or biovars but are absent in other common/rescue Brucella species or biovars; and (b) identify rescue amplicons present in rescue/common Brucella species but absent in specific Brucella species.
The manner by which the nucleotides or primers of the present invention function is discussed as below that clearly reflect the unique and unexpected attributes of the nucleotide sequences or primers as herein described:
17

The novel nucleotide sequences or novel primers when used in the PCR methods as herein described enable PCR amplicons/PCR amplifications of different lengths and these PCR amplicons based on the amplification criterion by the novel set of primers are classified as
“Specific Amplicons” and “Rescue Amplicons”.
The novel nucleotides used as primer sequence or set of primer sequences are capable of
amplifying a specific amplicon with its respective specific Brucella species in a monoplex PCR and a rescue amplicon with remaining common or rescue Brucella species, if in case the sample contains any or all 4 species of Brucella apart from the target species. Further, each species specific primer was exploited in development of a monoplex PCR format for each species with specific amplifications of 1154 bp, 745 bp, 446 bp, 290 bp and 224 bp with B.
abortus, B. melitensis, B. ovis, B. suis and B. canis, respectively(Figure 8). However, when
the same 5 species-specific primer pairs were employed in PCR with simultaneously in a multiplex PCR for detection of each species, they resulted in a unique multiplex PCR format (Figure 10). This was because all the primers would anneal to the conserved regions flanking all 5 of nucleotide sequences Brucella genome, resulting 4 additional amplicons along with the species specific amplicon. Yet, seeking for the species-specific amplicon size for particular species was sufficient to identify it. To explain, seeking for amplicons of 1154 bp, 745 bp, 446 bp, 290 bp and 224 bp within that multiplex format was sufficient to identify the
species as B. abortus, B. melitensis, B. ovis, B. suis and B. canis, respectively. The rescue
amplicons would provide the additional information of presence of common or rescue Brucella species in the test sample and hence rules out the chance of having nonspecific amplification with other contaminating organisms. Nevertheless in a multiplex PCR in presence of all 5 Brucella species, an integrative format comprising of all the species specific amplicons was obtained for identification and differentiation of all 5 species together in single lane on gel electrophoresis technique. Further, these primers were employed in stabilization of conventional monoplex and multiplex PCR formats. In order to avoid false negative results, competitive internal amplification control (IAC) was also incorporated in the multiplex PCR format, by employing pUC 19 plasmid DNA. The stabilized monoplex and multiplex PCRs were evaluated for specificity with reference strains of the above mentioned Brucella species. As an outcome of this invention, disregard of biovars, highly specific PCR based detection format was developed that could successfully and differentially identify B.
abortus, B. melitensis, B. ovis, B. suis and B. canis.
18

Thus the present invention also provides for novel monoplex and multiplex PCR methods of identifying and/or detecting any Brucella genus species or their related biovars.
The novel monoplex and multiplex PCR designs of the present invention exploit the specific, unique or conserved sequences which are differentially present in the species and/or biovars of Brucella genus but absent in other and also in intergenic and/or intragenic sequences of Brucella species. It has been found in the present invention that such differentially present unique sequences are embedded within conserved regions of species and/or biovars Brucella genus. The method/s of the present invention is unique in locating, isolating and identifying such target sequences for clinically and economically important species and/or biovars of
Brucella species.
In the present invention in silico method was used for comparative analysis of polymorphic sequences, which appeared to be differential nucleotide sequences. During the in silico analysis it was found that there were stretch of nucleotide sequences embedded within a conserved region of the genome of various species of Brucella genus. The said analysis also showed that the flanking conserved region around these embedded nucleotide sequences demonstrated 100% homology in all species of Brucella genus. In accordance with information retrieved from in silico analysis novel PCR primers were designed which could enable identification the unique nucleotide sequences of the various species of Brucella genus.
Accordingly, the main embodiment of the present invention provides for novel nucleotide sequences having SEQ ID No. 11-20.
Another embodiment of the present invention provides for novel nucleotide sequences as herein described for the detection and/or identification of various species and/or biovars of
Brucella genus.
Another embodiment of the present invention provides for novel nucleotide sequences as herein described, wherein the said nucleotide sequences can be used in a PCR based method or DNA based systems the detection and/or identification of various species and/or biovars of
Brucella genus.
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Another embodiment of the present invention provides for novel nucleotide sequences as herein described, wherein the PCR based methods comprise but are not limited to monoplex, multiplex PCR immuno-PCR, real-time PCR and/or loop-mediated isothermal amplification PCR.
Another embodiment of the present invention provides for novel nucleotide sequences as herein described, wherein the DNA based systems comprise but are not limited to fluorescent/chemiluminescent DNA probe based blotting, DNA-DNA hybridization, DNA-RNA hybridization systems.
Another embodiment of the present invention provides for novel nucleotide sequences as herein described useful for detection and identification of Brucella species in clinical samples.
Another embodiment of the present invention provides for novel nucleotide sequences as herein described, wherein the clinical samples are collected from humans or non-humans.
Another embodiment of the present invention provides for a set of Primers for detection of Brucella species and their biovars having SEQ ID Nos. 23-32.
Another embodiment of the present invention relates to the set of primers as herein described,
wherein the Brucella species and their biovars are selected from B.abortus, B.melitensis,
B.ovis, B.suis and B.canis.
Another embodiment of the present invention relates to the set of primers as herein described, wherein the said primers are useful for detection and identification of Brucella species in clinical samples.
Another embodiment of the present invention relates to the set of primers as herein described, wherein the clinical samples are collected from humans or non-humans.
Another embodiment of the present invention relates to the set of primers as herein described, wherein the said primers are characterized as following:
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(a) the primer set have SEQ ID Nos. 23-24 are Forward and Reverse primers and
enables identification of B.abortus;
(b) the primer set have SEQ ID Nos. 25-26 are Forward and Reverse primers and
enables identification of B.melitensis;
(c) the primer set have SEQ ID Nos. 27-28 are Forward and Reverse primers and
enables identification of B.ovis;
(d) the primer set have SEQ ID Nos. 29-30 are Forward and Reverse primers and enables identification of B.suis; and
(e) the primer set have SEQ ID Nos. 31-32 are Forward and Reverse primers and enables identification of B.canis.
Another embodiment of the present invention relates to the set of primers as herein described,
wherein the said primers enable identification of specific amplicon that is specific Brucella species and rescue amplicon that is common non-specific Brucella species but is absent in specific Brucella species.
Another embodiment of the present invention relates to the set of primers as herein described, wherein the said primers can be used in both monoplex and multiplex PCR methods for detection of Brucella species or biovars
Yet another embodiment of the present invention provides for a PCR method for differential detection and identification of specific DNA of Brucella species or biovars using set of primer as herein as described, said method comprising the steps of:
(a) isolating of DNA from Brucella species or biovars;
(b) amplifying the isolated DNA of step (a) using set of primers having SEQ ID Nos. 23-31; and
(c) obtaining specific DNA amplicon and rescue DNA amplicon of Brucella species or biovars.
Yet another embodiment of the present invention provides for a PCR as herein described, wherein the set of primers are characterized as following:
(a) the primer set have SEQ ID Nos. 23 and 24 are Forward and Reverse primers
that enable identification of B.abortus;
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(b) the primer set have SEQ ID Nos. 25 and 26 are Forward and Reverse primers
that enable identification of B.melitensis;
(c) the primer set have SEQ ID Nos. 27 and 28 are Forward and Reverse primers that enable identification of B.ovis;
(d) the primer set have SEQ ID Nos. 29 and 30 are Forward and Reverse primers that enable identification of B.suis; and
(e) the primer set have SEQ ID Nos. 31 and 32 are Forward and Reverse primers that enable identification of B.canis.
Yet another embodiment of the present invention provides for a PCR method as herein described, wherein the set of primers as herein described results in DNA amplicons or DNA amplification characterized as following:
(a) the primer set have SEQ ID Nos. 23 and 24 result in specific amplicon of
B.abortus having a size of 1154bp and rescue amplicon of B.melitensis, B.ovis, B.suis and B.canis having a size of 1478 bp;
(b) the primer set have SEQ ID Nos. 25 and 26 result in specific amplicon of
B.melitensis having a size of 745 bp and rescue amplicon of B.abortus, B.ovis, B.suis and B.canis having a size of 977 bp;
(c) the primer set have SEQ ID Nos. 27 and 28 result in specific amplicon of
B.ovis having a size of 446 bp and rescue amplicon of B.abortus, B.melitensis, B.suis and B.canis having a size of 606 bp;
(d) the primer set have SEQ ID Nos. 29 and 30 result in specific amplicon of
B.suis having a size of 290 bp and rescue amplicon of B.abortus, B.melitensis, B.ovis and B.canis having a size of 382 bp; and
(e) the primer set have SEQ ID Nos. 31 and 32 result in specific amplicon of
B.canis having a size of 224 bp and rescue amplicon of B.abortus, B.melitensis, B.ovis and B.suis having a size of 521 bp.
Yet another embodiment of the present invention provides for a PCR method as herein described, wherein the said method detects and identifies Brucella species in clinical samples.
Yet another embodiment of the present invention provides for a PCR method as herein described, wherein the clinical samples are collected from humans or non-humans.
22

Yet another embodiment of the present invention provides for a PCR method as herein described, wherein the said PCR methods comprise of monoplex, multiplex PCR immuno-PCR, real-time PCR and/or loop-mediated isothermal amplification PCR for detection and identification of Brucella species or biovars.
Another embodiment of the present invention provides for a use of set of primers as herein described for the detection and identification of Brucella species or biovars.
Another embodiment of the present invention provides for monoplex and/or multiplex PCR based detection system for detection of Brucella species and differentiation of Brucella species at stratum of biovars using at least a primer or a primer set as herein described.
Yet another embodiment of the present invention provides for DNA-based detection systems for detection of Brucella species and their biovars using set of primers as herein described.
Yet another embodiment of the present invention provides for DNA-based detection systems as herein described wherein the said DNA-based detection systems comprise of including fluorescent/chemiluminescent DNA probe based blotting, DNA-DNA hybridization, DNA-RNA hybridization systems.
Another embodiment of the present invention provides for novel monoplex and multiplex PCR methods for the detection and/or identification of various species and/or biovars of Brucella genus, wherein the monoplex and/or multiplex PCR methods use the set of primer sequences as herein described.
Yet another embodiment of the present invention provides for a method for detection of species and biovars of Brucella genus using nucleotide sequences as herein described, said method comprising steps of:
(a) carrying out in silico comparative genomic comparison of Brucella species to identify unique specific and rescue amplicons;
(b) using nucleotide sequences as claimed in claim 1 as primer sequences in a PCR based method to identify presence or absence of specific and rescue amplicon of step (a) in species and biovars of Brucella genus; and
23

(c) identifying and differentiating specific species of Brucella genus irrespective of their biovars.
The invention will now be explained with the help of following examples. However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the proportion of the ingredients and combinations.
EXAMPLES
EXAMPLE 1
Bacterial strains and media
Reference isolates of Brucella species were procured from Indian Veterinary Research Institute (IVRI), Bareilly, India as well isolated locally. The list of bacterial strains used in the present invention is tabulated in Table 1. Trypticase soy broth and Brucella selective agar was used to culture the bacteria.
Table 1: Bacterial isolates used in this study and their reactivity in the mPCR

Organism

Source

PCR

Brucella Isolate

B. abortus isolate DFRL1 Clinical +
B. abortus isolate DFRL2 Clinical +
B. abortus isolate DFRL3 Clinical +
B. abortus isolate DFRL4 Clinical +
B. abortus isolate DFRL5 Clinical +
B. abortus isolate DFRL6 Clinical +
B. abortus isolate DFRL7 Clinical +
B. abortus isolate DFRL8 Clinical +
B. abortus isolate DFRL9 Clinical +
B. abortus isolate DFRL10 Clinical +
B. abortus isolate DFRL11 Clinical +
B. abortus isolate DFRL12 Clinical +
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B. abortus isolate DFRL13 Clinical +
B. melitensis isolate DFRL1 Clinical +
B. ovis ATCC25840 NVSL, USA +
B. suis1330 IVRI +
B. canis ATCC23365 NVSL, USA +
EXAMPLE 2
In silico comparative genomic analysis for determination of unique nucleotide
sequences
The strains of Brucella species and their respective genome sequences employed for this analysis include Brucella abortus A13334 (NC_016795 and NC_016777), Brucella melitensis ATCC 23457 (NC_012441 and NC_012442), Brucella ovis ATCC 25840 (NC_009505 and NC_009504), Brucella suis 1330 (NC_017251 and NC_017250), Brucella suisATCC23445(NC_010169 and NC_010167), Brucella suis bv. 1 str. S2 (CP006961 and CP006962), Brucella suis VBI22(NC_016797 and NC_016775), and Brucella canis ATCC
23365 (CP000872 and CP000873). Briefly, the whole genome sequence of the selected Brucella species was divided into fragments of 1000 bp by the implementation of a script written in the Perl programming language. Resultant nucleotide fragments of each species (for example Brucella abortus) were aligned against the genomic sequences of all the other biovars within the species using the BLASTN program. The fragments that matched all biovars with an E-value less than 10-200 were considered as highly conserved and thus were selected for further studies. The protocol was repeated with the rest four species of interest. Thus determined conserved sequences from each species of Brucella were further aligned against the genomic sequences of rest other four species in order to evaluate their uniqueness. This analysis revealed polymorphic distribution of certain unique nucleotide sequences in the 5 species analyzed and they had a striking pattern of distribution among them. To explain, a nucleotide sequence is present in 4 species with 100 % identity whereas completely absent in the 5th species. However, the flanking regions of that nucleotide sequence are highly identical
in all 5 species. For example, a 1000 bp fragment of B. abortus was present in B. melitensis, B. suis, B. ovis and B. canis with an additional 324 bp of novel and unique interrupting nucleotide stretch within the fragment (that was strikingly absent in B. abortus) making the fragment 1324 bp in length. Identical pattern was observed in rest four species of Brucella. These unique intergenic and/or intragenic sequences were analyzed for conservation and
25

variation by multiple sequence alignment using CLUSTALW tool available on EMBL-EBI website (http://www.ebi.ac.uk/Tools/msa/clustalw2/) (Figures 1-5). This striking organization of genome favored in development of the novel detection strategy, exploiting the flanking conserved sequences as primer annealing region to yield differential amplicons among Brucella species.
EXAMPLE 3
Conservation and Variation analysis
In order to examine their universality and applicability, the unique nucleotide sequences (SEQ ID 1 to 10) were aligned against several strains of the five species of interest as well as their respective biovars available on NCBI and Broad Institute Brucella databases. The list of strains/biovars analyzed for conservation and variation is tabulated in Table 2. It was noticed that the B. abortus specific sequence was 100 % conserved in all strains and biovars referred. Similar observations were recorded in case of B. melitensis, B. ovis and B. canis. However, BLASTN analysis revealed that biovar 3, 4 and 5 of B. suis were identical to that of B. abortus, B. melitensis, B. ovis and B. canis. On the other hand, biovar 1 and 2 ofB. suis were unique and differentiable from rest other species.
Table 2. List of bacterial genome sequences used in comparative genomic analysis

EXAMPLE 4
Design of Nucleotide Sequences
In the present invention, oligonucleotide sequences or nucleotide sequences were designed using Gene Runner 3.0 software (http://www.generunner.net/) (Table 3). The nucleotide sequences were designed with a notion of obtaining specific yet easily distinguishable and interpretable amplifications in identical PCR condition and thus deployable in development of novel monoplex and multiplex PCR formats. In silico analysis of the designed oligonucleotide sequences for specificity was performed by using BLASTN program.
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Table 3: Designed Nucleotide Sequences
Example 5: Design of primers
The oligonucleotide or nucleotide sequences designed in the present invention and discussed in Example 3 above were further used for designing primers. The strategy of designing primers in the proposed invention is illustrated in Figure 6. The primers were designed with a notion of obtaining specific yet easily distinguishable and interpretable amplifications in identical PCR condition and thus deployable in development of novel monoplex and multiplex PCR formats. In silico analysis of the designed primers for specificity was performed by using BLASTN program.
In order to prevent false negative results due to the interference of inhibitory substances in test samples, competitive internal amplification controls (lAC) was developed and included in the present invention. The lAC primer set i.e., SEQ ID No. 21 and SEQ ID No. 22 was designed such that it had 5' overhanging ends identical to the primer sequence ofB. abortus specific primer pair (italicized) and 3' ends complementary to pUC19 plasmid sequence. The primer sequence of the 3' end (bold and underlined) was designed so as to amplify 808 bp of pUC19 plasmid, yielding a product of 847 bp (inclusive of the 5' B. abortus specific flanking regions of the primer pair).

The novel primer sequences as shown in Table 4were designed based on information available from the oligonucleotide or nucleotide sequences in Table 3. Each oligonucleotide sequence or nucleotide sequence of Table 3 was assigned or designated as set of primers or primers comprising as forward and reverse primer and belonged to a specific species of Brucella genus as described in Table 4.
The PCR amplicon sizes resulting from each primer pair is represented in Figure 7, 8 and 9. The Table 4 provides for the primers used in the PCR of the present invention.
It was found in the present invention that (a) The B. abortus specific primer set i.e., SEQ ID No. 23 and SEQ ID No. 24 amplifies a 1154 bp product with B. abortus and 1478 bp product with other four species namely B. melitensis, B. suis, B. ovis and B. canis. B. melitensis,(h) specific primer set i.e., SEQ ID No. 25 and SEQ ID No. 26 amplifies a 745 bp product with B. melitensis and 977 bp product with other four species namely B. abortus, B. suis, B. ovis and B. canis. B. ovis; (c)specific primer set i.e., SEQ ID No. 27 and SEQ ID No. 28amplifies a 446 bp product with B. ovis and 606 bp product with other four species namely B. abortus, B. melitensis, B. suis and B. canis. B. suis; (d)specific primer set i.e., SEQ ID No. 29 and SEQ ID No. 30 amplifies a 290 bp product with B. suis and 383 bp product with other four species namely B. abortus, B. melitensis, B. canis and B. ovis. B. canis and (e)specific primer

set i.e., SEQ ID No. 31 and SEQ ID No. 32 amplifies a 224 bp product with B. canis and 521 bp product with other four species namely B. abortus, B. melitensis, B. suis and B. ovis.
EXAMPLE 6
Preparation of genomic DNA
Genomic DNA from the bacteria listed in Table 1 was isolated using phenol-chloroform method as described by Sambrook et al. Briefiy, 3 ml of overnight culture was pelleted and re-suspended in 200 |il of TE buffer. Each suspension was subjected to lysozyme and proteinase K treatment, followed by phenol-chloroform extraction. The resulting genomic DNA in aqueous phase was precipitated using absolute ethanol and 5 M ammonium acetate. The DNA precipitate was pelleted by centrifugation at maximum rpm, washed twice with 70% ethanol and air dried. The dried DNA pellet was re-suspended in 100 |il of Tris-Cl buffer and stored at -20°C until used. Before use DNA was diluted in sterile water to an approximate concentration of 50 ng/|il.
Monoplex PCR amplification
For initial standardization of the detection system, monoplex PCR was performed using the primer sets and respective genomic DNA as template. Each 25 |iL PCR reaction contained the following: 1 U Ta^polymerase, 250 |iM of each deoxynucleotide triphosphate, IX PCR buffer (including 1.5 mM MgCb), 10 pmol of each oligonucleotide primer, and 25 ng of template DNA. Thermal cycling was carried out in Eppendorf thermal cycler (Eppendorf, USA) for 35 cycles of 30 s at 94 °C, annealing for 30 s at 56 °C, and extension at 72 °C for 1 min, with a final 8 min extension at 72 °C. Approximately 2 |il of each PCR product was visualized by agarose gel electrophoresis. The template DNA of each species was used with its respective specific primer in each monoplex PCR. The result of each monoplex PCR (Figure 8) is as follows:
- Monoplex PCR using B. abortus544 as template DNA yielded an amplicon of 1154bp in presence of and its specific primer.
- Monoplex PCR using B. melitensislSM as template DNA yielded an amplicon of 745bp in presence of and its specific primer.
- Monoplex PCR using B. suis\330 as template DNA yielded an amplicon of 290 bp in presence of and its specific primer.

- Monoplex PCR using B. canis ATCC 23365 as template DNA yielded an amplicon of 224 bp in presence of and its specific primer.
- Monoplex PCR using B. ov/'5ATCC25840 as template DNA yielded an amplicon of 446 bp in presence of and its specific primer.
Characterization of the PCR products
In order to evaluate the authenticity the designed pimer sets, the amplified products were sequenced and confirmed. Briefly, the PCR amplified products were purified from agarose gel, sequenced and nucleotide sequence obtained was matched with that of the reference strain available on NCBI website. Complete identity of the amplified products with their specific genes was observed
Multiplex PCR assay
25 ng of genomic DNA of each 5rwce//a species namely 5. abortusSAA, B. melitensislSM, B. ovis ATCC 25840, B. suis 1330 and 5. canis ATCC 23365 along with 1:9000 dilution of lAC DNA was subjected to each multiplex PCR in a 50 |il reaction mixture containing Ix PCR buffer, 1.5 mM MgCb, 200 mM concentration of each deoxynucleotide triphosphate, 10 pmol of each oligonucleotide primer and 1 U of Taq polymerase (Sigma Aldrich, India). Amplification was carried out as follows: initial denaturation at 94 °C for 10 min; 30 cycles of 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1.30 min; and a final elongation step at 72 °C for 8 min. Amplicons were visualized after running at 100 Vfor 1 h on a 2 % agarose gel containing ethidium bromide (Figure 10).
Interpretation of multiplex PCR format
The multiplex PCR described in the proposed invention includes a total of 6 pairs of primer -one each for the 5 species oi Brucella namely, B. abortus, B. melitensis, B. ovis, B. suis and B. canis and one pair for internal amplification control. Each primer had the capability of revealing the presence of all 5 species - by yielding a specific amplicon of a particular size with its respective species and a rescue amplicon longer than specific amplicon, in size, with rest other 4 species, making them to fall in a separate group altogether. For example, the primer specific for B. abortus, resulted in a 1154bp amplicon with B. abortus, whereas a 1478 bp amplicon with other 4 species namely B. melitensis, B. suis, B. ovis and B. canis. The interpretation of the mPCR assay described here is illustrated in Figure 11.

Example 7:
The invention is further explained with the help of following exemplified models 1-12 that clearly recites the unique and unexpected finding of the use or the application of the nucleotide sequences as primer sequences or set of primer sequences for detection and/or identification of various species and/or biovars oi Brucella genus. However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the proportion of the ingredients and combinations.
Model 1. If the mPCR reaction is performed with a test sample contaminated with all 5 Brucella species namely B. abortus, B. melitensis, B. suis, B. canis and B. ovis, the result will be as follows:
- B. abortus specific primer yields amplicon of 1154 bp with B. abortus and 1748 bp with other 4 species
- B. melitensis specific primer 745 bp with B. melitensis and yields 977 bp with other 4 species
- B. suis specific primer yields 290 bp with B. suis and 383 bp with other 4 species
- B. canis specific primer yields 224 bp with B. canis and 521 bp with other 4 species
- B. ovis specific primer yields 446 bp with B. ovis and 606 bp with other 4 species and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 2. If the mPCR reaction is performed with a test sample contaminated with B. abortus the result will be as follows:
- B. abortus specific primer yields amplicon of 1154 bp with B. abortus
- B. melitensis specific primer yields 977 bp with B. abortus
- B. suis specific primer yields 383 bp with B. abortus
- B. canis specific primer yields 521 bp with 5. abortus
- B. ovis specific primer yields 606 bp with B. abortus and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 3. If the mPCR reaction is performed with a test sample contaminated with B. melitensis the result will be as follows:
- B. abortus specific primer yields amplicon of 1478 bp with B. melitensis
- B. melitensis specific primer yields 745 bp with B. melitensis

-B. suis specific primer yields 383 bp with 5. melitensis
- B. canis specific primer yields 521 bp with 5. melitensis
- B. ovis specific primer yields 606 bp with B. melitensis and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 4. If the mPCR reaction is performed with a test sample contaminated with B. ovis the result will be as follows:
- B. abortus specific primer yields amplicon of 1478 bp with B. ovis
- B. melitensis specific primer yields 977 bp with B. ovis
- B. suis specific primer yields 383 bp with B. ovis
- B. canis specific primer yields 521 bp with 5. ovis
- B. ovis specific primer yields 446 bp with B. ovis and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 5. If the mPCR reaction is performed with a test sample contaminated with B. suisthe result will be as follows:
- B. abortus specific primer yields amplicon of 1478 bp with B. suis
- B. melitensis specific primer yields 977 bp with B. suis
- B. suis specific primer yields 290 bp with B. suis
- B. canis pecific primer yields 521 bp with B. suis
- B. ovis specific primer yields 606 bp with B. suis and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model6. If the mPCR reaction is performed with a test sample contaminated with B. canis the result will be as follows:
- B. abortus specific primer yields amplicon of 1478 bp with B. canis
- B. melitensis specific primer yields 977 bp with B. canis
- B. suis specific primer yields 383 bp with B. canis
- B. canis specific primer yields 224 bp with B. canis
- B. ovis specific primer yields 606 bp with B. canis and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 7. If the mPCR reaction is performed with a test sample contaminated with B. abortus and B. melitensis the result will be as follows:

- B. abortus specific primer yields amplicons of 1154 bp with B. abortus DNA and 1478 bp with B. melitensis
- B. melitensis specific primer yields 745 bp with B. melitensis and 977 bp with B. abortus
- B. suis specific primer yields 383 bp with both B. abortus and B. melitensis
- B. canis specific primer yields 521 bp with both B. abortus and B. melitensis
- B. ovis specific primer yields 606 bp with both B. abortus and B. melitensis
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 8. If the mPCR reaction is performed with a test sample contaminated with B. abortus and B. ovis the result will be as follows:
- B. abortus specific primer yields amplicons of 1154 bp with B. abortus DNA and 1478 bp with 5. ovis
- B. melitensis specific primer yields 977 bp with both B. abortus and B. ovis
- B. ^w/.s'specific primer yields 383 bp with both B. abortus and B. ovis
- B. canis specific primer yields 521 bp with both B. abortus and B. ovis
- B. ovis specific primer yields 446 bp with B. ovis and 606 bp with B. abortus
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 9. If the mPCR reaction is performed with a test sample contaminated with B. ovis and B. suis the result will be as follows:
- B. abortus specific primer yields 1478 bp with both B. ovis and B. suis
- B. melitensis specific primer yields 946 bp with both B. ovis and B. suis
- B. suis specific primer yields 290 bp with B. suis and 383 bp with B. ovis
- B. canis specific primer yields 521 bp with both B. ovis and B. suis
- B. ovis specific primer yields 446 bp with B. ovis and 606 bp with B. suis and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 10. If the mPCR reaction is performed with a test sample contaminated with B. suis and B. canis the result will be as follows:
- B. abortus specific primer yields 1478 bp with both B. suis and B. canis
- B. melitensis specific primer yields 946 bp with both B. suis and B. canis
- B. suis specific primer yields 290 bp with B. suis and 383 bp with B. canis
- B. canis specific primer yields 224 bp with B. canis and 521 bp with B. suis
- B. ovis specific primer yields 606 bp with both B. suis and B. canis and

- lAC specific primer yields 847 bp amplification with lAC DNA
Model 11. If the mPCR reaction is performed with a test sample contaminated with B. abortus, B. melitensis and B. ovis the result will be as follows:
- B. abortus specific primer yields amplicons of 1154 bp with B. abortus DNA and 1478 bp with B. melitensis and B. ovis
- B. melitensis specific primer yields 745 bp with B. melitensis and 977 bp with B. abortus and 5. ovis
- B. ^w/.s'specific primer yields 290 bp with B. suis and 383 bp with B. canis
- B. canis specific primer yields 224 bp with B. canis and 521 bp with B. suis
- B. ovis specific primer yields 446 bp with B. ovis and 606 bp with B. abortus and B. melitensis and
- lAC specific primer yields 847 bp amplification with lAC DNA
Model 12. If the mPCR reaction is performed with a test sample is not contaminated with any Brucella species the result will be as follows:
- None of the Brucella species specific primers yield any amplification lAC specific primer yields 847 bp amplification with lAC DNA
The results of Modelsl-12 have been depicted in Figure 11 and also diagrammatically explained and represented in Figure 7.
Thus each primer pair had a capability of detecting specific Brucella species and differentiates it from other 4 species. This species dependent differential amplification was noticed in case of all specific primers, wherein the primer specific species would result in an amplicon different from the amplicon size yielded by remaining 4 species.

REFERENCES
1. O'Callaghan D, Whatmore AM. Brucella genomics as we enter the multi-genome era. Brief Funct Genomics. 2011 Nov; 10(6):334-41.
2. Gandara B, Merino AL, Rogel MA, Martinez-Romero E. Limited genetic diversity of Brucella spp. J ClinMicrobiol. 2001 Jan; 39(l):235-40. 2
3. Pappas G., Akritidis N., Bosilkovski M. &Tsianos E. (2005). - Brucellosis. N. Engl. I Med., 352, 2325-2336. 3
4. Corbel, 1989: Corbel Michael J. Microbiology of the Genus Brucella. 53 -72./«; Young Edward J, Corbel Michael ] Brucellosis: Clincial and Laboratory Aspects\9S9. CRC Press, Inc., Boca Raton, Florida. 4
5. Laboratory techniques in brucellosis. Alton GG, Jones LM, Pietz DE MonogrSer World Health Organ. 1975; (55): 1-163.
6. Baily GG, Krahn JB, Drasar BS, Stoker NG Detection of Brucella melitensis and Brucella abortus by DNA amplification. J TropMedHyg. 1992; 95:271-5.
7. Romero C, Gamazo C, Pardo M, Lopez-Goni I. Specific detection oi Brucella DNA hy FCR. J ClinMicrobiol. 1995; 33:615-7.
8. Rijpens NP, Jannes G, Van Asbroeck M, Rossau R, Herman LM. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Appl Environ Microbiol. 1996; 62:1683-8.
9. Bricker BJ, Ewalt DR, MacMillan AP, Foster G, Brew S. Molecular characterization of Brucella strains isolated from marine mammals. J ClinMicrobiol. 2000;38:1258-62.
10. Keid LB, Soares RM, Vasconcellos SA, Chiebao DP, Salgado VR, Megid J, et al. A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches. Theriogenology. 2007; 68:1260-70.
ll.Henault S, Calvez D, Thiebaud M, Bouliere M, Garin-Bastuji B. Validation of a nested-PCR based on the IS6501/711 sequence for the detection of Brucella in animal samples. Proceedings of the Brucellosis 200 International Research Conference 91 including the 53rd Brucellosis Research Conference; 2000 Sep 7-9; Nimes, France. Paris: INRA; 2000.
12. Bogdanovich T, Skumik M, Lubeck PS, Ahrens P, Hoorfar J. Validated 5' nuclease PCR assay for rapid identification of the genus Brucella. J ClinMicrobiol. 2004; 42:2261-3.

13. Leal-Klevezas DS, Martinez-Vazquez 10, Lopez-Merino A, Martinez- Soriano JP. Single-step PCR for detection of Brucella spp. from blood and milk of infected ammah. JClinMicroMol. 1995; 33:3087-90.
14. Imaoka K, Kimura M, Suzuki M, Kamiyama T, Yamada A. Simultaneous detection of the genus Brucella by combinatorial PCR. Jpn J Infect Dis. 2007; 60:137-9.
15. Vizcaino N, Caro-Hernandez P, Cloeckaert A, Fernandez-Lago L. DNA polymorphism in the omp25/omp31 family oiBrucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infect. 2004; 6:821-34.
16. Fekete A, Bantle JA, Hailing SM, Stich RW. Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary pnmtx?,. J Bacterial. 1992; 174:7778-83.
17. Bricker et al., 1994. Bricker BJ, Hailing SM. Differentiation oiBrucella abortus hw. 1, 2, and 4, Brucella melitensis. Brucella ovis, and Brucella suishv. 1 by PCR. J ClinMicroMol. 1994; 32(11): 2660 - 2666.
18. Ewalt DR and Bricker BJ. 2000. Ewalt DR, Bricker BJ. Validation of the abbreviated 5rwce//a AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51.J ClinMicroMol. 2000; 38(8): 3085 - 3086.
19. Ocampo-Sosa AA, Agiiero-Balbin J, Garcia-Lobo JM. Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar3. Vet Microbiol. 2005;110:41-51. 7
20. Ferrao-Beck L, Cardoso R, Munoz PM, de Miguel MJ, Albert D, Ferreira AC, et al. Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine. Vet Microbiol. 2006; 115:269-77.
21. Lopez-Gofii I., et al. 2008. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J. Clin. Microbiol. 46: 3484-3487.
22. Mayer-Scholl A., Draeger A., Gollner C, Scholz H. C, Nockler K. 2010. Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. J. Microbiol. Methods 80: 112-114.
23. Huber B, Scholz HC, Lucero N, Busse HJ. Development of a PCR assay for typing and subtyping of Brucella species. Int J Med Microbiol. 2009; 299:563-73.

24. Redkar R, Rose S, Bricker B, DelVecchio V. Real-time detection of Brucella abortus. Brucella melitensis and Brucella suis. Mol Cell Probes. 2001; 15:43-52.
25. Queipo-Ortuno MI, Colmenero JD, Reguera JM, Garcia-OrdonezMA , Pachon ME, Gonzalez M, et al. Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. ClinMicrobiol Infect. 2005; 11:713-8.
26. Kattar MM, Zalloua PA, Araj GF, Samaha-Kfoury J, Shbaklo H, Kanj SS, et al. Development and evaluation of real-time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human brucellosis. DiagnMicrobiol Infect Dis. 2007; 59:23-32.
27. Queipo-Ortuno MI, Colmenero JD, Bravo MJ, Garcia-Ordonez MA, Morata P. Usefulness of a quantitative real-time PCR assay using serum samples to discriminate between inactive, serologically positive and active human brucellosis. ClinMicrobiol Infect. 2008; 14:1128-34.

We Claim:
1. Novel nucleotide sequences having SEQ ID No. 11-20.
2. The novel nucleotide sequences as claimed in claim 1 for the detection and/or identification of various species and/or biovars oi Brucella genus.
3. The novel nucleotide sequences as claimed in claims 1-2, wherein the nucleotide sequences can be used in a PCR based method or DNA based systems for the detection and/or identification of various species and/or biovars oi Brucella genus.

4. The novel nucleotide sequences as claimed in claims 1-3, wherein the PCR based methods comprise but are not limited to monoplex, multiplex PCR immuno-PCR, real-time PCR and/or loop-mediated isothermal amplification PCR.
5. The novel nucleotide sequences as claimed in claims 1-3, wherein the DNA based systems comprise but are not limited to fluorescent/chemiluminescent DNA probe based blotting, DNA-DNA hybridization, DNA-RNA hybridization systems.
6. A Set of Primers for detection oi Brucella species and their biovars having SEQ ID
Nos. 23-32.
7. The set of primers as claimed in claim 6, wherein the species oi Brucella genus and their biovars are selected from B.abortus, B.melitensis, B.ovis, B.suis and B.canis.
8. The set of primers as claimed in claims 6-7, wherein the set of primers enable identification of specific amplicon that is specific Brucella species and rescue amplicon that is common non-specific Brucella species but is absent in specific Brucella species.
9. The set of primers as claimed in claims 6-8, wherein the said primers can be used in both monoplex and multiplex PCR methods for detection of Brucella species or biovars

10. A PCR method for differential detection and identification of specific DNA of
Brucella species or biovars using set of primer as claimed in claims 5 to 8, said
method comprising the steps of:
(a) isolating of DNA from Brucella species or biovars;
(b) amplifying the isolated DNA of step (a) using set of primers having SEQ ID Nos. 23-31;and
(c) obtaining specific DNA amplicon and rescue DNA amplicon of Brucella species or biovars
11. A method for detection of species and biovars of Brucella genus using nucleotide
sequences as claimed in claim 1, said method comprising steps of
(a) carrying out in silico comparative genomic comparison of Brucella species
to identify unique specific and rescue amplicons;
(b) using nucleotide sequences as claimed in claim 1 as primer sequences in a
PCR based method to identify presence or absence of specific and rescue amplicon of step (a) in species and biovars of Brucella genus; and
(c) identifying and differentiating specific species of Brucella genus
irrespective of their biovars.