Patent Publication Number: US-2020277657-A1

Title: Genetic markers for diagnosis of tuberculosis caused by mycobacterium tuberculosis

Description:
RELATED APPLICATION 
     This application is related to and takes priority from the Indian Provisional Application 5572/CHE/2013 filed on Dec. 3, 2013 and is incorporated herein in its entirety. 
     FIELD OF THE INVENTION 
     The application is related to novel signature sequences for diagnosis of  Mycobacterium tuberculosis  in clinical samples. These signature sequences have the ability to differentiate  Mycobacterium tuberculosis  DNA from other mycobacterial species by PCR with 100% specificity and very high sensitivity. 
     BACKGROUND OF THE INVENTION 
     Tuberculosis (TB) is a major global health problem with an alarming rate of mortality associated with it. It is one of the leading infectious diseases caused by bacteria taking one human life every 15-20 seconds globally. Estimates of 2011 reveal that there are almost 9 million new cases and 1.4 million TB deaths (Global Tuberculosis Report 2012, WHO 2013). The disease is caused by  Mycobacterium tuberculosis , a member of the genus  Mycobacterium , while in a few cases  Mycobacterium bovis  has been reported to be the causal organism. More than 100  mycobacterium  species are known and among them only a few are pathogenic for humans. 
     Conventional diagnostic methods include examination of sputum smear under a microscope for acid-fast mycobacteria and radiological reading of lungs. However, in most cases the sputum smear examination turns out to be negative for the bacteria due to early stages of infection and lung changes are not readily visible on an x-rays until several months into the infection. 
     Diagnosis of mycobacteria-related disease poses difficulties for several reasons which have been recognized by researchers and clinicians over the years. Firstly, these bacteria are in small numbers and are already at a contagious stage when they become detectable by conventional methods. Pulmonary disease caused by different mycobacteria are not readily detectable clinically or radiologically. Detection of organism in culture achieves 100% specificity but the growth of mycobacteria in culture takes about 3-6 weeks (Bates et al, Am. Respir. 134, 415-417, 1986) thus making the process time-consuming. In addition, repeated cultures may be required to ensure success. 
     Several molecular tests for tuberculosis have been developed in the past. These include the Gen-probe ‘Amplified  mycobacterium  direct test’ by Abbe et al (J. Clin. Mincrobiol. 31, 3270, 1993), ligase chain reaction (LCR) (J. Clin. Microbiol. 35, 2424, 1997), PCR based Roche Amplicor TB test (J. Clin. Microbiol. 33, 1832, 1995) and IS6110 based detection (J. Clin. Microbiol. 28, 2668, 1990). 
     U.S. Pat. No. 5,168,039 discloses the IS6110 based detection of  M. tuberculosis  wherein a repetitive DNA segment specific for members of  M. tuberculosis  complex is used for the diagnosis. While IS6110 based detection system has been shown to have high level of specificity, there are also reports on false positive detections of 3 to 20% making it unreliable (J. Clin. Microbiol. 32, 277, 1994). In addition, lack of IS6110 sequence in some  M. tuberculosis  strains may also limit the use of the same for diagnostic tests routinely (Tuber. Lung Dis. 76, 550, 1995). U.S. Pat. No. 7,638,309 provides detection of mycobacteria in clinical specimens in the intergenic region between methyl mycotic acid synthase genes mmaA1 and mmaA2 and the flanking region in mmaA1 and mmaA2 genes. 
     Thus, it appears that there is a paucity of simple, rapid and reliable tests that can specifically detect  M. tuberculosis  during early stages of the disease. The present invention has identified ‘signature sequences’ that can differentiate  M. tuberculosis  from a large number of other mycobacterial DNA. These ‘signature sequences’ are used in detection of early disease in clinical samples of patients. 
     SUMMARY OF THE INVENTION 
     The invention provides novel signature sequences for diagnosis of  Mycobacterium  species (sps) in clinical samples with 100% specificity and a very high degree of sensitivity. 
     In one aspect, the invention provides a nucleotide sequence capable of selectively detecting pathogenic  Mycobacterium  sps using oligonucleotide primers corresponding to the signature sequence selected from SEQ ID NO: 1, 2, 3 or 4. 
     In another aspect the invention provides a method of detecting pathogenic  mycobacterium  sps in a clinical sample, said method comprising the steps of: 
     a. removal of contaminants from the clinical sample by conventional methods; 
     b. extraction of genomic DNA from the contaminant-free clinical sample; 
     c. designing a set of specific oligonucleotide primers capable of specifically detecting SEQ ID NO: 1, 2, 3 or 4 for use in RT-PCR; 
     d. analyzing PCR product by electrophoresis or specific probe nucleotide sequence complementary to SEQ ID NO: 1, 2, 3 or 4. 
     The set of oligonucleotide primers of the invention are selected from 
     
       
         
           
               
               
            
               
                   
                 (i)    5′ ATGCAGGTTGCGACTGTACACCCGG 3′ 
               
               
                   
                        3′ GGCCGCTCTTGTTCTTCGTCGGAT 5′ 
               
               
                   
               
               
                   
                 (ii)   5′ GTGTTTGCGTTGAGTAATAATCTGAACCGTGT 3′ 
               
               
                   
                        3′ AGCCAATTCCAGCACGATGTCGCC 5′ 
               
               
                   
               
               
                   
                 (iii)  5′ ATGCAGGTTGCGACTGTACACCCGG 3′ 
               
               
                   
                        3′ GGCCGCTCTTGTTCTTCGTCGGAT 5′ 
               
               
                   
               
               
                   
                 (iv)   5′ TGTACCGCGTGCCCGACGATTTG 3′ 
               
               
                   
                        3′ ACAGGCAGCTAACAGGGCGTCGG 5′ 
               
            
           
         
       
     
     (v) a set of oligonucleotide primers complementary to (i), (ii), (iii) or (iv) or 
     (vi) a set of oligonucleotide primers comprising of sequence containing any 10 consecutive bases from one of the sequences selected from SEQ ID NO: 1, 2, 3 or 4. 
     In yet another aspect, the invention provides a kit for the detection of pathogenic  mycobacterium  sps in clinical samples, said kit comprising set of oligonucleotide primers selected from 
     
       
         
           
               
               
            
               
                   
                 (i)    5′ ATGCAGGTTGCGACTGTACACCCGG 3′ 
               
               
                   
                        3′ GGCCGCTCTTGTTCTTCGTCGGAT 5′ 
               
               
                   
               
               
                   
                 (ii)   5′ GTGTTTGCGTTGAGTAATAATCTGAACCGTGT 3′ 
               
               
                   
                        3′ AGCCAATTCCAGCACGATGTCGCC 5′ 
               
               
                   
               
               
                   
                 (iii)  5′ ATGCAGGTTGCGACTGTACACCCGG 3′ 
               
               
                   
                        3′ GGCCGCTCTTGTTCTTCGTCGGAT 5′ 
               
               
                   
               
               
                   
                 (iv)   5′ TGTACCGCGTGCCCGACGATTTG 3′ 
               
               
                   
                        3′ ACAGGCAGCTAACAGGGCGTCGG 5′ 
               
            
           
         
       
     
     (v) a set of oligonucleotide primers complementary to (i), (ii), (iii) or (iv) or 
     (vi) a set of oligonucleotide primers comprising of sequence containing any 10 consecutive bases from one of the sequences selected from SEQ ID NO: 1, 2, 3 or 4. 
     Furthermore, the invention provides a method of detecting pathogenic  mycobacterium  sps in a clinical sample wherein the sample is isolated from individuals vaccinated against tuberculosis. It also provides a method of detecting pathogenic  mycobacterium  sps in a clinical sample wherein the sample is isolated from individuals treated against tuberculosis. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         FIG. 1A : Amplification of SS1 at low and varied DNA template concentrations. 
         FIG. 1B : Amplification of SS2 at low and varied DNA template concentrations. 
         FIG. 1C : Amplification of SS3 at low and varied DNA template concentrations. 
         FIG. 1D : Amplification of SS4 at low and varied DNA template concentrations. 
         FIG. 2 : Amplification of signature sequences SS1, SS2, SS3 and SS4 from patient sputum samples. 
         FIG. 3 : Amplification of signature sequences SS1 and SS3 from patient blood samples. 
     
    
    
     DETAILS OF THE INVENTION 
     The present invention relates to detection of pathogenic  Mycobacterium  species using signature sequences SEQ ID NO: 1, 2, 3 or 4 with a high degree of sensitivity and 100% specificity. 
     In one embodiment, the invention provides novel DNA diagnostic markers for specific detection of  Mycobacterium tuberculosis  which causes tuberculosis. 
     A three-pronged approach was carried out to identify novel DNA diagnostic marker for detection of pathogenic  mycobacterium  sps, especially,  Mycobacterium tuberculosis . First step provides an in-silico approach to identify and shortlist potential sequences of  Mycobacterium , unique and exclusive to pathogenic  mycobacterium  sps, especially  Mycobacterium tuberculosis . The criteria used for selection of the potential sequences are presented below which involves comparative proteomic analysis of 13  mycobacterium  species:
         i. Strict pathogens (the most virulent pathogens) such as  Mycobacterium tuberculosis, Mycobacterium laprae, Mycobacterium  ulcerous and  Mycobacterium bovis.      ii. Opportunistic pathogens, which belong to Non Tuberculous Mycobacteria (NTM) group, can cause pulmonary and other disseminated infections in immune compromised individuals (Infect. Genet. Evol. 12, 832, 2012; Appl. Environ. Microbiol. 79, 825, 2013).  Mycobacterium marinum, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium aviumpara  tuberculosis and  Mycobacterium abscessus , cause opportunistic pulmonary infection in human whereas  Mycobacterium avium  subspecies paratuberculosis (MAP), the third member of MAC is the suspected causative agent of Crohn&#39;s disease in human (Appl. Environ. Microbiol. 79, 825, 2013; Crit. Rev. Microbiol. 38, 52, 2012).   iii. Non-pathogenic group includes  Mycobacterium gilvum, Mycobacterium vanbaalenii, Mycobacterium smegmatis  and  Mycobacterium indicus pranii , which does not cause disseminated infections even in immune compromised individuals (BMC Microbiol. 10, 237, 2010; Infect. Genet. Evol., 12, 853, 2012; Br. J. Exp. Pathol. 52, 627, 1971).       

     The following bioinformatics process flow resulted in the identification of potential gene markers of the invention.
         a) Perform all against all NCBI BLAST (J Mol Biol., 215, 403, 1990) on the protein sequences from the selected genomes.   b) Perform all against all NCBI BLAST ((J Mol Biol., 215, 403, 1990) on the nucleotide sequences from the selected genomes.   C) Identify Protein Domains from Pfam (Comp. Genomics, 396, 43, 2007) and GENE-3D/CATH (Nucleic Acids Research, 40, D465, 2012) using InterPro (Nucleic Acids Research, 40, D306, 2012).   d) Classify sequences into three categories namely i) CLASS 1 and ii) CLASS 2 as potential hits and iii) rest as non-hits.       

     The above process flow resulted in the classification of the potential hits into class 1 and class 2. As per the present invention, class 1 are genes unique to the organism of interest based on the fact that they do not share protein domain and protein sequence identity of more than 20% and nucleotide sequence identity of more than 35% with any other organism in the selected organism list. In another embodiment, class 2 genes are those that do not share protein domain and protein sequence identity of more than 20% and nucleotide sequence identity of more than 35% with any other organism in the selected organism list. 
     Table 1 provides potential candidate genes carrying ‘signature sequences’ based on the bioinformatics process flow. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Potential “signature sequences” carrying candidate genes 
               
            
           
           
               
               
               
            
               
                 H37Rv Gene 
                   
                   
               
               
                 Identifiers 
                 Class 
                 H37Rv protein description 
               
               
                   
               
               
                 Rv1507A 
                 1 
                 Hypothetical protein 
               
               
                 Rv1509 
                 1 
                 Hypothetical protein 
               
               
                 Rv2645 
                 1 
                 Hypothetical protein 
               
               
                 Rv2653c 
                 1 
                 Possible PhiRv2 prophage protein 
               
               
                 Rv2654c 
                 1 
                 Possible PhiRv2 prophage protein 
               
               
                 Rv2658c 
                 1 
                 Possible prophage protein 
               
               
                 Rv0064A 
                 2 
                 Possible antitoxin VapB1 
               
               
                 Rv0078B 
                 2 
                 Hypothetical protein 
               
               
                 Rv0397A 
                 2 
                 Hypothetical protein 
               
               
                 Rv0456B 
                 2 
                 Possible antitoxin MazE1 
               
               
                 Rv0959A 
                 2 
                 Possible antitoxin VapB9 
               
               
                 Rv1366A 
                 2 
                 Hypothetical protein 
               
               
                 Rv1954A 
                 2 
                 Hypothetical protein 
               
               
                 Rv1991A 
                 2 
                 Antitoxin MazE6 
               
               
                 Rv2142A 
                 2 
                 Possible antitoxin ParD2 
               
               
                 Rv2231A 
                 2 
                 Possible toxin VapC16 
               
               
                 Rv2231B 
                 2 
                 Possible antitoxin VapB16 
               
               
                 Rv2274A 
                 2 
                 Possible antitoxin MazE8 
               
               
                 Rv2395A 
                 2 
                 Acid and phagosome regulated protein A AprA 
               
               
                 Rv2395B 
                 2 
                 Acid and phagosome regulated protein B AprB 
               
               
                 Rv2862A 
                 2 
                 Possible antitoxin VapB23 
               
               
                 Rv3190A 
                 2 
                 Hypothetical protein 
               
               
                 Rv3344c 
                 2 
                 PE-PGRS family protein PE_PGRS49] 
               
               
                   
                   
                 [partial = 5′] 
               
               
                 Rv3512 
                 2 
                 PE-PGRS family protein PE_PGRS56] 
               
               
                   
                   
                 [partial = 5′] 
               
               
                 Rv3599c 
                 2 
                 Hypothetical short protein 
               
               
                   
               
            
           
         
       
     
     In one aspect, the invention functionally characterizes the potential ‘signature sequences (SS)’-carrying candidate genes based on functional information retrieved from Tuberculist (Tuberculosis (Edinb) 91, 7, 2011) and TB database (Nucleic Acids Research, 37, D499, 2009). Accordingly, the potential signature sequences can be functionally characterized into the following groups:
         a. 9 (Rv0064A, Rv0456B, Rv0959A, Rv1991A, Rv2142A, Rv2231A, Rv2231B, Rv2274A and Rv2862A) fell into the toxin-antitoxin category.   b. 3 (Rv2653c, Rv2654c, Rv2658c) are possible prophages.   c. 2 (Rv3344c and Rv3512) belong to PE_PGRS family of proteins   d. 2 (Rv2645 and Rv2653c) are deleted (partially or completely) in one or more clinical isolates eliminating their use as diagnostic markers.   e. 9 (Rv1507A, Rv1509, Rv0078B, Rv2645, Rv0397A, Rv1366A, Rv1954A, Rv3599c, Rv3190A) are hypothetical proteins.   f. 2 (Rv2395A, Rv2395B) are acid and phagosome regulated proteins.       

     Based on the in-silico analysis, two Class 1 genes (Rv1507A and Rv1509) and two Class 2 genes (Rv1954A and Rv2231A) with homologs in  Mycobacterium bovis  BCG were selected as potential candidates (Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Nucleotide Sequences of Potential “signature sequences” carrying genes 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Prediction 
               
               
                 Gene Name 
                 Sequence 
                 Description 
                 Class 
               
               
                   
               
               
                 Rv1507A 
                 &gt;Rv1507A 
                 Gene length: 
                 CLASS 1 
               
               
                 hypothetical 
                 ATGCAATCAGGTCAAAATATCCTCGCCAAGG 
                 504 bp, 
                   
               
               
                 protein 
                 TATGTAATTTGATTGAACAATCGCGACTTTC 
                 Protein length: 
                   
               
               
                   
                 TTCAACGCGGTGTCTCCAATTTAGAATAACA 
                 167 aa 
                   
               
               
                   
                 AATACGTCGCGCCCGCGACAGCTCCGCTGGA 
                   
                   
               
               
                   
                 GCGAGTTCAAGCGATTCTGCGACATATTCAA 
                   
                   
               
               
                   
                 TATGGTGCTCGGGAAGGCCAGGATGGGCCGC 
                   
                   
               
               
                   
                 GACCCGGGGCGTCCGGTGCGCGATGAACGTC 
                   
                   
               
               
                   
                 GCATCGTCTCCTGTGAGATAATTGCATCCGA 
                   
                   
               
               
                   
                 TCATATAGGGCTGGCTGCGGCTAGGTTGCTG 
                   
                   
               
               
                   
                 GCAAAAAGATATCGCGGCCGATCCGTTTCTG 
                   
                   
               
               
                   
                 GTTTTGTCTTGATGATCAAATCCGCTTCCGT 
                   
                   
               
               
                   
                 TCACGAGATCGATTCCTGGTCTTCCCCCAGC 
                   
                   
               
               
                   
                 GTCGCGATGTCGATAGGTGTCGCGCTTTGTT 
                   
                   
               
               
                   
                 CGTACCCGCACTACGCGGCGGCGAGAACCTC 
                   
                   
               
               
                   
                 GCCACCGAATCGGGATTGGGGGGAGGATACC 
                   
                   
               
               
                   
                 ACTCGGTCGAGGCCCGTCACCGGCCTTCTAG 
                   
                   
               
               
                   
                 CGGGTTG 
                   
                   
               
               
                   
               
               
                 Rv1509 
                 &gt;Rv1509 
                 Gene length: 
                 CLASS 1 
               
               
                 Essential 
                 GTGTTTGCGTTGAGTAATAATCTGAACCGTG 
                 882 bp, 
                   
               
               
                 hypothetical 
                 TGAACGCATGCATGGATGGATTCCTTGCCCG 
                 Protein length: 
                   
               
               
                 protein 
                 TATCCGCTCACATGTTGATGCGCACGCGCCA 
                 293 aa 
                   
               
               
                   
                 GAATTGCGTTCACTGTTCGATACGATGGCGG 
                   
                   
               
               
                   
                 CCGAGGCCCGATTTGCACGCGACTGGCTGTC 
                   
                   
               
               
                   
                 CGAGGACCTCGCGCGGTTGCCTGTCGGTGCA 
                   
                   
               
               
                   
                 GCATTGCTGGAAGTGGGCGGGGGGGTACTTC 
                   
                   
               
               
                   
                 TGCTCAGCTGTCAACTGGCGGCGGAGGGATT 
                   
                   
               
               
                   
                 TGACATCACCGCCATCGAGCCGACGGGTGAA 
                   
                   
               
               
                   
                 GGTTTTGGCAAGTTCAGACAGCTTGGCGACA 
                   
                   
               
               
                   
                 TCGTGCTGGAATTGGCTGCAGCACGACCCAC 
                   
                   
               
               
                   
                 CATCGCGCCATGCAAGGCGGAAGACTTTATT 
                   
                   
               
               
                   
                 TCCGAGAAGCGGTTCGACTTCGCCTTCTCGC 
                   
                   
               
               
                   
                 TGAATGTGATGGAGCACATCGACCTTCCGGA 
                   
                   
               
               
                   
                 TGAGGCAGTCAGGCGGGTATCGGAAGTGCTG 
                   
                   
               
               
                   
                 AAACCGGGGGCCAGTTACCACTTCCTGTGCC 
                   
                   
               
               
                   
                 CGAATTACGTATTCCCGTACGAACCGCATTT 
                   
                   
               
               
                   
                 CAATATCCCAACATTCTTCACCAAAGAGCTG 
                   
                   
               
               
                   
                 ACATGCCGGGTGATGCGACATCGCATCGAGG 
                   
                   
               
               
                   
                 GCAATACGGGCATGGATGACCCGAAGGGAGT 
                   
                   
               
               
                   
                 CTGGCGTTCGCTCAACTGGATTACGGTTCCC 
                   
                   
               
               
                   
                 AAGGTGAAACGCTTTGCGGCGAAGGATGCGA 
                   
                   
               
               
                   
                 CGCTGACCTTGCGCTTCCACCGTGCAATGTT 
                   
                   
               
               
                   
                 GGTATGGATGCTGGAACGCGCGCTGACGGAT 
                   
                   
               
               
                   
                 AAGGAATTCGCTGGTCGCCGGGCACAATGGA 
                   
                   
               
               
                   
                 TGGTCGCTGCTATTCGCTCGGCGGTGAAATT 
                   
                   
               
               
                   
                 GCGTGTGCATCATCTGGCAGGCTATGTTCCC 
                   
                   
               
               
                   
                 GCTACGCTGCAGCCCATCATGGATGTGCGGC 
                   
                   
               
               
                   
                 TAACGAAGAGGTAA 
                   
                   
               
               
                   
               
               
                 Rv1954a 
                 &gt;gi|448814763:2201231-2201623 
                 Gene length: 
                 CLASS 2 
               
               
                 Hypothetical 
                   Mycobacterium tuberculosis  H37Rv 
                 303 bp, 
                   
               
               
                 protein 
                 complete genome 
                 Protein length: 
                   
               
               
                   
                 TGGTATAAGCTGGTTTTAGACGAAAAGGACC 
                 100 bp 
                   
               
               
                   
                 CCACCTCGGGGTCTGATGGCCAGGGGCAGGG 
                   
                   
               
               
                   
                 TCGTGTGCATTGGGGATGCAGGTTGCGACTG 
                   
                   
               
               
                   
                 TACACCCGGCGTGTTCCGCGCGACAGCGGGT 
                   
                   
               
               
                   
                 GGGATGCCGGTGCTGGTGGTCATCGAGTCTG 
                   
                   
               
               
                   
                 GGACAGGAGGTGATCAGATGGCTCGTAAAGC 
                   
                   
               
               
                   
                 TACGTCCCCGGGTAAGCCGGCTCCGACGTCG 
                   
                   
               
               
                   
                 GGACAGTATCGCCCGGTTGGCGGTGGCAACG 
                   
                   
               
               
                   
                 AGGTGACCGTTCCGAAGGGACACCGTCTGCC 
                   
                   
               
               
                   
                 TCCCTCGCCCAAGCCCGGTCAGAAGTGGGTG 
                   
                   
               
               
                   
                 AACGTCGATCCGACGAAGAACAAGAGCGGCC 
                   
                   
               
               
                   
                 GCGGCTGAGCTTGTGCCGTCGGGATGGGTGT 
                   
                   
               
               
                   
                 CGCACCGTCTCGGCGGGTCGC 
                   
                   
               
               
                   
               
               
                 Rv2231A 
                 &gt;gi|448814763:c2506224-2505671 
                 Gene length: 
                 CLASS 2 
               
               
                   
                   Mycobacterium tuberculosis  H37Rv 
                 426 bp, 
                   
               
               
                   
                 complete genome 
                 Protein length: 
                   
               
               
                   
                 GCCGCGGCGAGCCGGTAGCAAAGCTTGTGCC 
                 141 aa 
                   
               
               
                   
                 GCTGCATCCTCATGAGACTCGGCGGTTAGGC 
                   
                   
               
               
                   
                 ATTGACCATGGCGTGTACCGCGTGCCCGACG 
                   
                   
               
               
                   
                 ATTTGGACGCTCCGTTGTCAGACGACGTGCT 
                   
                   
               
               
                   
                 CGAACGCTTTCACCGGTGAAGCGCTACCTCA 
                   
                   
               
               
                   
                 TCGACACCCACGTTTGGCTGCGGATGCCGTC 
                   
                   
               
               
                   
                 AACGAAACACGGGCGATTGTTCAGGACGTCC 
                   
                   
               
               
                   
                 GCAACAGCATTCTCTTGTCGGCCGCCAGTGC 
                   
                   
               
               
                   
                 CTGGGAGATCGCGATCAACTACCGCCTCGGC 
                   
                   
               
               
                   
                 AAGCTCCCGCCGCCCGAGCCATCGGCCTCTT 
                   
                   
               
               
                   
                 ACGTGCCCGATCGAATGCGCCGCTGCGGCAC 
                   
                   
               
               
                   
                 GTCGCCGCTGTCAGTTGACCACGCACACACT 
                   
                   
               
               
                   
                 GCGCACCGCAGAGCTTCCGGATCACCATCGA 
                   
                   
               
               
                   
                 CATCCATTCGACCGTGTGCTCATCGCCCAGG 
                   
                   
               
               
                   
                 CACAGCTGCTTGGCCTGACGATCATCACCGC 
                   
                   
               
               
                   
                 CGACGCCCTGTTAGCTGCCTGTGATGTCGCG 
                   
                   
               
               
                   
                 GTTGTCGCCGCGTAGACAACGCGTCGGCGGT 
                   
                   
               
               
                   
                 GCTCTGGATTCTTGGCCCGCACACCG 
               
               
                   
               
            
           
         
       
     
     In a most preferred aspect, the signature sequences were designed keeping in view the diagnostic tool of RT PCR. These were short sequences amenable for PCR amplification from selected genes. The specific signature sequences, SS1 (Rv1507A), SS2 (Rv1509), SS3 (RV1954A) and SS4 (Rv2231A) of the invention are provided below. Homology search using NCBI nucleotide BLAST against the genus  Mycobacterium  was conducted on these signature sequences to confirm their uniqueness. 
     
       
         
           
               
            
               
                 SS1 (Rv1507A): 
               
               
                 &gt;Rv1507A 
               
               
                 SEQ ID NO: 1 
               
               
                 ATGCAATCAGGTCAAAATATCCTCGCCAAGGTATGTAATTTGATTGAACA 
               
               
                   
               
               
                 ATCGCGACTTTCTTCAACGCGGTGTCTCCAATTTAGAATAACAAATACGT 
               
               
                   
               
               
                 CGCGCCCGCGACAGCTCCGCTGGAGCGAGTTCAAGCGATTCTGCGACATA 
               
               
                   
               
               
                 TTCAATATGGTGCTCGGGAAGGCCAGGATGGGCCGCGACCCGGGGCGTCC 
               
               
                   
               
               
                 GGTGCGCGATGAACGTCGCATCGTCTCCTG 
               
               
                   
               
               
                 SS2 (Rv1509): 
               
               
                 &gt;gi|448814763:1700212-1701093  Mycobacterium   
               
               
                   tuberculosis  H37Rv complete genome 
               
               
                 SEQ ID NO: 2 
               
               
                 GTGTTTGCGTTGAGTAATAATCTGAACCGTGTGAACGCATGCATGGATGG 
               
               
                   
               
               
                 ATTCCTTGCCCGTATCCGCTCACATGTTGATGCGCACGCGCCAGAATTGC 
               
               
                   
               
               
                 GTTCACTGTTCGATACGATGGCGGCCGAGGCCCGATTTGCACGCGACTGG 
               
               
                   
               
               
                 CTGTCCGAGGACCTCGCGCGGTTGCCTGTCGGTGCAGCATTGCTGGAAGT 
               
               
                   
               
               
                 GGGCGGGGGGGTACTTCTGCTCAGCTGTCAACTGGCGGCGGAGGGATTTG 
               
               
                   
               
               
                 ACATCACCGCCATCGAGCCGACGGGTGAAGGTTTTGGCAAGTTCAGACAG 
               
               
                   
               
               
                 CTTGGCGACATCGTGCTGGAATTGGCTGCA 
               
               
                   
               
               
                 SS3 (RV1954A): 
               
               
                 &gt;gi|448814763:2201277-2201579  Mycobacterium   
               
               
                   tuberculosis  H37Rv complete genome 
               
               
                 SEQ ID NO: 3 
               
               
                 ATGGCCAGGGGCAGGGTCGTGTGCATTGGGGATGCAGGTTGCGACTGTAC 
               
               
                   
               
               
                 ACCCGGCGTGTTCCGCGCGACAGCGGGTGGGATGCCGGTGCTGGTGGTCA 
               
               
                   
               
               
                 TCGAGTCTGGGACAGGAGGTGATCAGATGGCTCGTAAAGCTACGTCCCCG 
               
               
                   
               
               
                 GGTAAGCCGGCTCCGACGTCGGGACAGTATCGCCCGGTTGGCGGTGGCAA 
               
               
                   
               
               
                 CGAGGTGACCGTTCCGAAGGGACACCGTCTGCCTCCCTCGCCCAAGCCCG 
               
               
                   
               
               
                 GTCAGAAGTGGGTGAACGTCGATCCGACGA 
               
               
                   
               
               
                 SS4 (Rv2231A): 
               
               
                 &gt;gi|448814763:c2506161-2505736  Mycobacterium   
               
               
                   tuberculosis  H37Rv complete genome 
               
               
                 SEQ ID NO: 4 
               
               
                 TTGACCATGGCGTGTACCGCGTGCCCGACGATTTGGACGCTCCGTTGTCA 
               
               
                   
               
               
                 GACGACGTGCTCGAACGCTTTCACCGGTGAAGCGCTACCTCATCGACACC 
               
               
                   
               
               
                 CACGTTTGGCTGCGGATGCCGTCAACGAAACACGGGCGATTGTTCAGGAC 
               
               
                   
               
               
                 GTCCGCAACAGCATTCTCTTGTCGGCCGCCAGTGCCTGGGAGATCGCGAT 
               
               
                   
               
               
                 CAACTACCGCCTCGGCAAGCTCCCGCCGCCCGAGCCATCGGCCTCTTACG 
               
               
                   
               
               
                 TGCCCGATCGAATGCGCCGCTGCGGCACGTCGCCGCTGTCAGTTGACCAC 
               
               
                   
               
               
                 GCACACACTGCGCACCGCAGAGCTTCCGGATCACCATCGACATCCATTCG 
               
               
                   
               
               
                 ACCGTGTGCTCATCGCCCAGGCACAGCTGCTTGGCCTGA 
               
            
           
         
       
     
     For the purposes of PCR validation, the signature sequences SS1, SS2, SS3 and SS4 were selected and oligonucleotide primers were designed to generate corresponding specific PCR amplification products. Table 3 provides the set of designed oligonucleotide primers. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Signature sequences SS1, SS2, SS3 and  
               
               
                 SS4 and respective oligonucleotide primers 
               
            
           
           
               
               
               
               
            
               
                 Signature 
                   
                   
                   
               
               
                 Sequences 
                   
                   
                 Prediction 
               
               
                 (SS) 
                 Sequence 
                 Description 
                 Class 
               
               
                   
               
               
                 SS1 from 
                 &gt;Rv1507A 
                 NZE_Rv1954A_F 
                 CLASS 1 
               
               
                 Rv1507A 
                 ATGCAATCAGGTCAAAATATCCTCGCC 
                 ATGCAGGTTGCGA 
                   
               
               
                   
                 AAGGTATGTAATTTGATTGAACAATCG 
                 CTGTACACCCGG 
                   
               
               
                   
                 CGACTTTCTTCAACGCGGTGTCTCCAA 
                 Length = 25,  
                   
               
               
                   
                 TTTAGAATAACAAATACGTCGCGCCCG 
                 Tm = 58.6, 
                   
               
               
                   
                 CGACAGCTCCGCTGGAGCGAGTTCAAG 
                 % G + C = 60 
                   
               
               
                   
                 CGATTCTGCGACATATTCAATATGGTG 
                 NZE_Rv1954A_R 
                   
               
               
                   
                 CTCGGGAAGGCCAGGATGGGCCGCGAC 
                 GGCCGCTCTTGTT 
                   
               
               
                   
                 CCGGGGCGTCCGGTGCGCGATGAACGT 
                 CTTCGTCGGAT 
                   
               
               
                   
                 CGCATCGTCTCCTG 
                 Length = 24,  
                   
               
               
                   
                   
                 Tm = 57.4, 
                   
               
               
                   
                   
                 % G + C = 58.3 
                   
               
               
                   
                   
                 Amplicon  
                   
               
               
                   
                   
                 Size = ~280 bp 
                   
               
               
                   
               
               
                 SS2 from 
                 &gt;gi|448814763:1700212-1701093 
                 NZE_Rv1509_F 
                 CLASS 1 
               
               
                 Rv1509 
                   Mycobacterium   tuberculosis  H37Rv 
                 GTGTTTGCGTTGA 
                   
               
               
                   
                 complete genome 
                 GTAATAATCTGAA 
                   
               
               
                   
                 GTGTTTGCGTTGAGTAATAATCTGAAC 
                 CCGTGT 
                   
               
               
                   
                 CGTGTGAACGCATGCATGGATGGATTC 
                 Length = 32,  
                   
               
               
                   
                 CTTGCCCGTATCCGCTCACATGTTGAT 
                 Tm = 57.5 
                   
               
               
                   
                 GCGCACGCGCCAGAATTGCGTTCACTG 
                 % G + C = 41 
                   
               
               
                   
                 TTCGATACGATGGCGGCCGAGGCCCGA 
                 NZE_Rv1509_R 
                   
               
               
                   
                 TTTGCACGCGACTGGCTGTCCGAGGAC 
                 AGCCAATTCCAGC 
                   
               
               
                   
                 CTCGCGCGGTTGCCTGTCGGTGCAGCA 
                 ACGATGTCGCC 
                   
               
               
                   
                 TTGCTGGAAGTGGGCGGGGGGGTACTT 
                 Length = 24,  
                   
               
               
                   
                 CTGCTCAGCTGTCAACTGGCGGCGGAG 
                 Tm = 58.8, 
                   
               
               
                   
                 GGATTTGACATCACCGCCATCGAGCCG 
                 % G + C = 58.3 
                   
               
               
                   
                 ACGGGTGAAGGTTTTGGCAAGTTCAGA 
                 Amplicon  
                   
               
               
                   
                 CAGCTTGGCGACATCGTGCTGGAATTG 
                 Size = ~330 bp 
                   
               
               
                   
                 GCTGCA 
                   
                   
               
               
                   
               
               
                 SS3 from 
                 &gt;gi|448814763:2201277-2201579 
                 NZE_Rv1954A_F 
                 CLASS 2 
               
               
                 Rv1954A 
                   Mycobacterium   tuberculosis  H37Rv 
                 ATGCAGGTTGCGA 
                   
               
               
                   
                 complete genome 
                 CTGTACACCCGG 
                   
               
               
                   
                 ATGGCCAGGGGCAGGGTCGTGTGCATT 
                 Length = 25,  
                   
               
               
                   
                 GGGGATGCAGGTTGCGACTGTACACCC 
                 Tm = 58.6, 
                   
               
               
                   
                 GGCGTGTTCCGCGCGACAGCGGGTGGG 
                 % G + C = 60 
                   
               
               
                   
                 ATGCCGGTGCTGGTGGTCATCGAGTCT 
                 NZE_Rv1954A_R 
                   
               
               
                   
                 GGGACAGGAGGTGATCAGATGGCTCGT 
                 GGCCGCTCTTGTT 
                   
               
               
                   
                 AAAGCTACGTCCCCGGGTAAGCCGGCT 
                 CTTCGTCGGAT 
                   
               
               
                   
                 CCGACGTCGGGACAGTATCGCCCGGTT 
                 Length = 24,  
                   
               
               
                   
                 GGCGGTGGCAACGAGGTGACCGTTCCG 
                 Tm = 57.4, 
                   
               
               
                   
                 AAGGGACACCGTCTGCCTCCCTCGCCC 
                 % G + C = 58.3 
                   
               
               
                   
                 AAGCCCGGTCAGAAGTGGGTGAACGTC 
                 Amplicon  
                   
               
               
                   
                 GATCCGACGA 
                 Size = ~280 bp 
                   
               
               
                   
               
               
                 SS4 from 
                 &gt;gi|448814763:c2506161-2505736 
                 NZE_Rv2231A_F 
                 CLASS 2 
               
               
                 Rv2231A 
                   Mycobacterium   tuberculosis  H37Rv 
                 TGTACCGCGTGCC 
                   
               
               
                   
                 complete genome 
                 CGACGATTTG 
                   
               
               
                   
                 TTGACCATGGCGTGTACCGCGTGCCCG 
                 Length = 23,  
                   
               
               
                   
                 ACGATTTGGACGCTCCGTTGTCAGACG 
                 Tm = 59.1, 
                   
               
               
                   
                 ACGTGCTCGAACGCTTTCACCGGTGAA 
                 % G + C = 61 
                   
               
               
                   
                 GCGCTACCTCATCGACACCCACGTTTG 
                 NZE_Rv2231A_R 
                   
               
               
                   
                 GCTGCGGATGCCGTCAACGAAACACGG 
                 ACAGGCAGCTAAC 
                   
               
               
                   
                 GCGATTGTTCAGGACGTCCGCAACAGC 
                 AGGGCGTCGG 
                   
               
               
                   
                 ATTCTCTTGTCGGCCGCCAGTGCCTGG 
                 Length = 23,  
                   
               
               
                   
                 GAGATCGCGATCAACTACCGCCTCGGC 
                 Tm = 57.1, 
                   
               
               
                   
                 AAGCTCCCGCCGCCCGAGCCATCGGCC 
                 % G + C = 65 
                   
               
               
                   
                 TCTTACGTGCCCGATCGAATGCGCCGC 
                 Amplicon  
                   
               
               
                   
                 TGCGGCACGTCGCCGCTGTCAGTTGAC 
                 Size = ~390 bp 
                   
               
               
                   
                 CACGCACACACTGCGCACCGCAGAGCT 
                   
                   
               
               
                   
                 TCCGGATCACCATCGACATCCATTCGA 
                   
                   
               
               
                   
                 CCGTGTGCTCATCGCCCAGGCACAGCT 
                   
                   
               
               
                   
                 GCTTGGCCTGAC 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment, pathogenic  mycobacterium  sps can be detected with 100% specificity following PCR using DNA isolated from clinical samples from patients who presented with clinical symptoms of the disease. In another embodiment, pathogenic  mycobacterium  sps is also detected using the above method in clinical samples isolated from individuals vaccinated against tuberculosis. In yet another embodiment, pathogenic  mycobacterium  sps is also detected using the above method in clinical samples isolated from individuals treated against tuberculosis. 
     Pathogenic  mycobacterium  sps, as provided in the invention, includes  Mycobacterium tuberculosis  and  Mycobacterium bovis . More specifically, pathogenic  mycobacterium  sps represents  Mycobacterium tuberculosis , the TB causing bacterium. 
     Clinical samples, as meant here, includes specimens such as blood, sputum, cerebrospinal fluid, gastric lavage, tissue biopsies and the likes thereof. PCR product can be easily visualized by any conventional method that can be readily recognized by a person skilled in the art such as electrophoresis. 
     Following Examples serve as a tool to illustrate the invention. However, it should in no way be considered to be limiting the invention. 
     Example 1 
     Determination of Specificity and Sensitivity of Signature Sequences 
     Genomic DNA for PCR Amplification 
     Genomic DNA of  Mycobacterium tuberculosis  and 13 other mycobacterial species were used for testing the specificity of signature sequences using PCR. These include,  M. avium  subspecies paratuberculosis,  M. smegmatis  (ATCC19420),  M. vaccae, M. marinum  (ATCC927),  M. chelonae  (ATCC14472),  M. flavescens  (ATCC14474),  M. fortuitum  (ATCC6481),  M. kansasii  (ATCC12478),  M. bovis  (ATCC27294),  M. bovis  (BCG),  M. avium  (ATCC25291),  M. gastri, M. indicuspranii.    
     PCR Reaction 
     The PCR reaction mixture (50 μl) consisted of 10×taqPCR buffer, 0.5 mmolMgCl2, 0.4 mmol dNTP, 10 pmol forward and reverse primers respectively, 4% DMSO and 1Utaq DNA polymerase. The reaction conditions were the following: 95° C. for 5 minutes, followed by 35 cycles of 95° C. for 30 seconds, annealing temperature 50° C. for 30 seconds, 72° C. for 1 minute and finally 72° C. for 10 minutes. All PCR products were electrophoresed on 2% agarose gel with ethidium bromide staining. 
     The “signature sequences” were tested for their ability to differentiate  Mycobacterium tuberculosis  DNA from a large number of other mycobacterial DNA in PCR using primers complementary to these “signature sequences” as shown in Table 3. For this purpose, chromosomal DNA extracted from 13  mycobacterium  species including human genomic DNA were tested by  mycobacterium  genus-specific primers of the ‘signature sequences’. SS1 and SS2 were negative for all 13  mycobacterium  species tested whereas SS3 and SS4 show positive PCR results only when  M. bovis  BCG genomic DNA was used as template. 
     Table 4 summarizes the specificity data resulting from PCR using specific primers of signature sequences SS1, SS2, SS3 and SS4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Specific amplification of signature sequences from 
               
               
                   Mycobacterium tuberculosis  and  M. bovis  BCG 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Specimen 
                 SS1 
                 SS2 
                 SS3 
                 SS4 
               
               
                   
                   
               
               
                   
                 
                   M. tuberculosis 
                 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                   M. bovis  BCG 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. avium 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. smegmatis 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. vaccae 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. avium 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. chelonae 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. flori 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. fortuitum 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. kansasi 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. bovis 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. marinum 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. gastri 
                 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 MIP 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 MAP 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 
                   M. leprae 
                 
               
               
                   
                 Human Genome 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                   
               
            
           
         
       
     
     Furthermore, sensitivity analysis revealed that the signature sequences were highly sensitive in being able to detect &lt;1 ng (100pg) DNA as shown in  FIGS. 1A, 1B, 1C and 1D  for the four primers of the signature sequences SS1, SS2, SS3 and SS4 respectively. 
     Example 2 
     Evaluation of  Mycobacterium tuberculosis -Specific Primer Pair Using Clinical Samples
 
A) Amplification of Signature Sequences from Patient Sputum Samples
 
     Sputum samples were processed by the Universal Sample Processing (USP) method for DNA extraction as described by Chakravorty et al (J Clin Microbiol 43, 4357, 2005). DNA was isolated from the USP sediments by boiling in the presence of five volumes of solution containing 10% Chelex-100 resin, 0.03% triton X-100, and 0.3% Tween 20. The isolated DNA was stored at 20° C. and used for PCR assay. 
     PCR reaction was carried out using specific primers as given in Table 3. 
     The results show amplification of signature sequences in patient sputum sample ( FIG. 2 ) demonstrating the diagnostic utility of the signature sequences for detecting pathogenic  Mycobacterium tuberculosis.    
     B) Amplification of Signature Sequences from Patient Blood Samples 
     DNA from blood samples of tuberculosis patients were isolated as per the protocol described in van Heiden et al (isolation of DNA from  Mycobacterium tuberculosis , Paul D. van Heiden, Thomas C. Victor, Robin M. Warren, and Eileen G. van Holden) 
     The results show amplification of SS1 and SS3 as seen in  FIG. 3 .