WO2015083056A2

WO2015083056A2 - Genetic markers for diagnosis of tuberculosis caused by mycobacterium tuberculosis

Info

Publication number: WO2015083056A2
Application number: PCT/IB2014/066469
Authority: WO
Inventors: Syed Asad RAHMAN; Javeed AHMAD; Nasreen Z EHTESHAM; Seyed E HASNAIN
Original assignee: Europaisches Laboratorium fuer Molekularbiologie EMBL; KUSUMA SCHOOL OF BIOLOGICAL SCIENCES
Current assignee: Europaisches Laboratorium fuer Molekularbiologie EMBL; KUSUMA SCHOOL OF BIOLOGICAL SCIENCES
Priority date: 2013-12-03
Filing date: 2014-12-01
Publication date: 2015-06-11
Anticipated expiration: 2016-06-03
Also published as: US20160355872A1; EP3382040A3; US20200277657A1; EP3382040A2; US10597734B2; EP3077534A2

Description

GENETIC MARKERS FOR DIAGNOSIS OF TUBERCULOSIS CAUSED BY MYCOBACTERIUM

TUBERCULOSIS

Related Application

This application is related to and takes priority from the Indian Provisional Application

5572/CHE/2013 filed on December 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 a I, 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).

US5168039 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). US7638309 provides detection of mycobacteria in clinical specimens in the intergenic region between methyl mycoiic acid synthase genes mmaAl and mmaA2 and the flanking region in mmaAl 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 DMA. 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' AG CCAATTCCAG CACG ATGTCGCC 5'

(iii) 5' ATGCAGGTTGCGACTGTACACCCGG 3'

3' GGCCGCTCTTGTTCTTCGTCGGAT 5'

(iv) 5' TGTACCGCGTGCCCGACGATTTG 3'

3' AC AG G CAG CTA AC AG G G CGTCG G 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' AG CCAATTCCAG CACG ATGTCGCC 5'

(iii) 5' ATGCAGGTTGCGACTGTACACCCGG 3'

3' GGCCGCTCTTGTTCTTCGTCGGAT 5'

(iv) 5' TGTACCGCGTGCCCGACGATTTG 3'

3' AC AG G CAG CTA AC AG G G CGTCG G 5'

(v) a set of oligonucleotide primers complementary to (i), (ii), (iii) or (iv)

or

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 SSI at low and varied DNA template concentrations.

Fig. IB: Amplification of SS2 at low and varied DNA template concentrations.

Fig. 1C: Amplification of SS3 at low and varied DNA template concentrations.

Fig. ID: Amplification of SS4 at low and varied DNA template concentrations.

Fig. 2: Amplification of signature sequences SSI, SS2, SS3 and SS4 from patient sputum samples. Fig. 3: Amplification of signature sequences SSI 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 ulcernus 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'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 smegmatisand 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

Rvl507A 1 Hypothetical protein

Rvl509 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 VapBl

Rv0078B 2 Hypothetical protein

Rv0397A 2 Hypothetical protein

Rv0456B 2 Possible antitoxin MazEl

Rv0959A 2 Possible antitoxin VapB9

Rvl366A 2 Hypothetical protein

Rvl954A 2 Hypothetical protein

Rvl991A 2 Antitoxin MazE6

Rv2142A 2 Possible antitoxin ParD2

Rv2231A 2 Possible toxin VapC16

Rv2231B 2 Possible antitoxin VapB16

Rv2274A 2 Possible antitoxin MazE8

2 Acid and phagosome regulated

Rv2395A

protein A AprA

2 Acid and phagosome regulated

Rv2395B

protein B AprB

Rv2862A 2 Possible antitoxin VapB23

Rv3190A 2 Hypothetical protein 2 PE-PGRS family protein PE_PGRS49]

Rv3344c

[partial=5']

2 PE-PGRS family protein PE_PGRS56]

Rv3512

[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, Rvl991A, 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 (Rvl507A, Rvl509, Rv0078B, Rv2645, Rv0397A, Rvl366A, Rvl954A, 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 (Rvl507A and Rvl509) and two Class 2 genes (Rvl954A 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 Rvl507A >Rvl507A Gene length: CLASS 1 hypothetical ATG CAATCAG GTCAA AATATCCTCG CCA AGGT 504bp,

protein ATGTAATTTGATTGAACAATCGCGACTTTCTTC Protein

AACG CG GTGTCTCCAATTTAG AATAACAAATA length: 167aa

CGTCGCGCCCGCGACAG CTCCG CTG G AG CG A GTTCAAGCGATTCTGCGACATATTCAATATGG TG CTCG GGAAGGCCAGG ATG GGCCGCGACCC G G G G CGTCCG GTG CG CG ATG A ACGTCG CATC GTCTCCTGTGAGATAATTGCATCCGATCATAT AG G G CTG G CTG CG G CTAG GTTG CTG G C A A A A AG ATATCG CG G CCG ATCCG 1 1 1 1 G 1 1 1 1 GT

CTTG ATG ATC A A ATCCG CTTCCGTTC ACG AG A TCG ATTCCTG GTCTTCCCCCAG CGTCGCG ATG TCGATAGGTGTCGCGCTTTGTTCGTACCCGCA CTACG CGGCGGCGAG A ACCTCG CCACCG A AT CGGGATTGGGGGGAGGATACCACTCGGTCGA GGCCCGTCACCGGCCTTCTAGCGGGTTG

Rvl509Essen >Rvl509 Gene length: CLASS 1 tial GTGTTTGCGTTGAGTAATAATCTGAACCGTGT 882bp,

hypothetical G A ACG C ATG CATG G ATG G ATTCCTTG CCCGTA Protein

protein TCCG CTC AC ATGTTG ATG CG C ACG CG CC AG A A length: 293aa

TTG CGTTCACTGTTCG ATACG ATG G CG G CCG A G G CCCG ATTTG C ACG CG ACTG G CTGTCCG AG G ACCTCG CG CG GTTG CCTGTCG GTG C AG CATT GCTGGAAGTGGGCGGGGGGGTACTTCTGCTC AGCTGTCAACTGGCGGCGGAGGGATTTGACA TC ACCG CC ATCG AGCCGACGGGTGAAGG 1 1 1 1

G G C A AGTTC AG AC AG CTTG G CG AC ATCGTG C TG G A ATTG G CTG C AG C ACG ACCCACC ATCG C G CCATG CAAGGCGGAAG ACTTTATTTCCG AG

A AG CG GTTCG ACTTCG CCTTCTCG CTG A ATGT G ATG GAG CAC ATCG ACCTTCCG G ATG AG G C A GTC AG G CG G GTATCG G A AGTG CTG A AACCG G G G G CCAGTTACCACTTCCTGTG CCCG AATTAC GTATTCCCGTACG AACCG CATTTCAATATCCCA AC ATTCTTC ACC A A AG AG CTG AC ATG CCG G GT G ATG CG ACATCG C ATCG AG G G CA ATACG G G C ATG G ATG ACCCG A AG G G AGTCTG G CGTTCG C TC A ACTG G ATTACG GTTCCC A AG GTG A A ACG C TTTGCGGCGAAGGATGCGACGCTGACCTTGC GCTTCCACCGTGCAATGTTGGTATGGATGCTG G A ACG CG CG CTG ACG G ATA AG G A ATTCG CTG GTCG CCG G G CAC A ATG G ATG GTCG CTG CTATT CG CTCG G CG GTG A A ATTG CGTGTG C ATC ATCT G G C AG G CTATGTTCCCG CTACG CTG CAG CCC A TC ATG G ATGTG CG G CTA ACG A AG AG GTA A

Rvl954a >gi 1448814763:2201231-2201623 Gene length: CLASS 2

Hypothetica l Mycobacterium tu bercu losis H37Rv 303bp,

protei n complete genome Protein

TGGTATAAGCTGG 1 1 1 1 AGACGAAAAGGACC length :

CCACCTCG G G GTCTG ATG GCCAGGGGCAGGG lOObp TCGTGTG CATTG G GG ATG CAG GTTG CG ACTG TACACCCG GCGTGTTCCG CG CG ACAG CGG GT G G G ATG CCG GTG CTG GTG GTC ATCG AGTCTG G G AC AG G AG GTG ATC AG ATG G CTCGTA A AG C TACGTCCCCG G GTA AG CCG G CTCCG ACGTCG G G AC AGTATCG CCCGGTTGGCGGTGG C A ACG AG GTG ACCGTTCCG A AG G G AC ACCGTCTG CC TCCCTCGCCCAAGCCCGGTCAGAAGTGGGTG

AACGTCGATCCGACGAAGAACAAGAGCGGCC G CG G CTG AG CTTGTG CCGTCG G G ATG G GTGT CGCACCGTCTCGGCGGGTCGC

Rv2231A >gi 1448814763x2506224-2505671 Gene length: CLASS 2

Mycobacterium tuberculosis H37Rv 426bp,

complete genome Protein

GCCGCGGCGAGCCGGTAGCAAAGCTTGTGCC length: 141aa

G CTG CATCCTC ATG AG ACTCG G CG GTTAG G C A

TTG ACC ATG G CGTGTACCG CGTG CCCG ACG AT

TTGGACGCTCCGTTGTCAGACGACGTGCTCGA

ACG CTTTC ACCG GTG A AG CG CTACCTC ATCG A

C ACCCACGTTTG G CTG CG G ATG CCGTC A ACG A

AACACGGGCGATTGTTCAGGACGTCCGCAAC

AG CATTCTCTTGTCG GCCG CCAGTG CCTGGGA

G ATCG CG ATC A ACTACCG CCTCGG C A AG CTCC

CGCCGCCCGAG CC ATCG G CCTCTTACGTG CCC

G ATCG A ATG CG CCG CTG CG G C ACGTCG CCG C

TGTCAGTTG ACCACGCACACACTG CG CACCG C

AG AG CTTCCG G ATCACC ATCG AC ATCCATTCG

ACCGTGTG CTC ATCG CCC AG G C AC AG CTG CTT

GGCCTGACGATCATCACCGCCGACGCCCTGTT

AG CTG CCTGTG ATGTCGCG GTTGTCG CCGCGT

AG AC A ACG CGTCG G CG GTG CTCTG G ATTCTTG

GCCCGCACACCG

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, SSI (Rvl507A), SS2 (Rvl509), 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. SSI (Rvl507A): SEQ ID NO: 1

>Rvl507A

AT GCAAT C AGGT CAAAAT AT CC T CGCC AAGGT AT GTAAT T T GAT T GAACAAT C GCGAC T T T C T T C AAC GC GGTGTCTCCAATTTAGAATAACAAATACGTCGCGCCCGCGACAGCTCCGCTGGAGCGAGTTCAAGCGATT CTGCGACATATTCAATATGGTGCTCGGGAAGGCCAGGATGGGCCGCGACCCGGGGCGTCCGGTGCGCGAT GAACGTCGCATCGTCTCCTG

SS2 (Rvl509): SEQ ID NO: 2

>gi | 448814763:1700212-1701093 Mycobacterium tuberculosis H37Rv complete genome

GTGTTTGCGTTGAGTAATAATCTGAACCGTGTGAACGCATGCATGGATGGATTCCTTGCCCGTATCCGCT CACATGTTGATGCGCACGCGCCAGAATTGCGTTCACTGTTCGATACGATGGCGGCCGAGGCCCGATTTGC ACGCGACTGGCTGTCCGAGGACCTCGCGCGGTTGCCTGTCGGTGCAGCATTGCTGGAAGTGGGCGGGGGG GTACTTCTGCTCAGCTGTCAACTGGCGGCGGAGGGATTTGACATCACCGCCATCGAGCCGACGGGTGAAG GT T T TGGCAAGT TCAGACAGCT T GGC GAC AT CGT GCTGGAAT T GGC T GC A SS3 (RV1954A): SEQ ID NO: 3

>gi 1448814763:2201277-2201579 Mycobacterium tuberculosis H37Rv complete genome

ATGGCCAGGGGCAGGGTCGTGTGCATTGGGGATGCAGGTTGCGACTGTACACCCGGCGTGTTCCGCGCGA CAGCGGGTGGGATGCCGGTGCTGGTGGTCATCGAGTCTGGGACAGGAGGTGATCAGATGGCTCGTAAAGC TACGTCCCCGGGTAAGCCGGCTCCGACGTCGGGACAGTATCGCCCGGTTGGCGGTGGCAACGAGGTGACC GTTCCGAAGGGACACCGTCTGCCTCCCTCGCCCAAGCCCGGTCAGAAGTGGGTGAACGTCGATCCGACGA

SS4 (Rv2231A): SEQ ID NO: 4

>gi 1448814763x2506161-2505736 Mycobacterium tuberculosis H37Rv complete genome

TTGACCATGGCGTGTACCGCGTGCCCGACGATTTGGACGCTCCGTTGTCAGACGACGTGCTCGAACGCTT TCACCGGTGAAGCGCTACCTCATCGACACCCACGTTTGGCTGCGGATGCCGTCAACGAAACACGGGCGAT TGTTCAGGACGTCCGCAACAGCATTCTCTTGTCGGCCGCCAGTGCCTGGGAGATCGCGATCAACTACCGC CTCGGCAAGCTCCCGCCGCCCGAGCCATCGGCCTCTTACGTGCCCGATCGAATGCGCCGCTGCGGCACGT CGCCGCTGTCAGTTGACCACGCACACACTGCGCACCGCAGAGCTTCCGGATCACCATCGACATCCATTCG ACCGTGTGCTCATCGCCCAGGCACAGCTGCTTGGCCTGA

For the purposes of PCR validation, the signature sequences SSI, 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 SSI, SS2, SS3 and SS4 and respective oligonucleotide primers

Signature

Prediction Sequences Sequence Description

Class (SS)

SSI from >Rvl507A NZE_Rvl954A_F CLASS 1

Rvl507A ATG C A ATC AG GTC A A A ATATCCTCG CC ATG C AG GTTG CG ACTGTA

AAGGTATGTAATTTGATTGAACAATCG CACCCGG

CGACTTTCTTCAACGCGGTGTCTCCAAT

Length = 25, Tm = 58.6,

TTAG A ATA AC A A ATACGTCG CG CCCG C

%G+C = 60

G AC AG CTCCG CTG G AG CG AGTTC A AG C

G ATTCTG CG AC ATATTC A AT ATG G TG CT NZE_Rvl954A_R

CGGGAAGGCCAGGATGGGCCGCGACC

GG CCG CTCTTGTTCTTCGT

CGGGGCGTCCGGTGCGCGATGAACGT

CGGAT

CG C ATCGTCTCCTG

Length = 24, Tm = 57.4,

%G+C = 58.3

Amplicon Size = ~280 bp

SS2 from >gi 1448814763:1700212-1701093 NZE_Rvl509_F CLASS 1

Mycobacterium tuberculosis H37Rv

GTGTTTGCGTTGAGTAAT Rvl509 complete genome AATCTGAACCGTGT

GTGTTTGCGTTGAGTAATAATCTGAACC Length - 32, Tm -

GTGTGAACG CATGCATG G ATG G ATTCC 57.5%G+C = 41

TTG CCCGTATCCG CTCACATGTTG ATG C

NZE_Rvl509_R

G C ACG CG CC AG A ATTG CGTTC ACTGTT

CG ATACG ATG G CG G CCG AG G CCCG ATT AG CC A ATTCCAG C ACG AT

TG C ACG CG ACTG G CTGTCCG AG G ACCT GTCGCC

CGCGCGGTTGCCTGTCGGTGCAGCATT

Length = 24, Tm = 58.8,

GCTGGAAGTGGGCGGGGGGGTACTTC

%G+C = 58.3

TG CTCAG CTGTC A ACTG GCGGCGGAGG

GATTTGACATCACCGCCATCGAGCCGA Amplicon Size = ~330bp

CGGGTGAAGG 1 1 1 1 GGCAAGTTCAGAC

AGCTTGGCGACATCGTGCTGGAATTGG

CTGCA

SS3 from >gi 1448814763:2201277-2201579 NZE_ Rvl954A_F CLASS 2

Mycobacterium tuberculosis H37Rv

Rvl954A ATG C AG GTTG CG ACTGTA

complete genome

CACCCGG

ATGGCCAGGGGCAGGGTCGTGTGCATT

Length = 25, Tm = 58.6,

GGGGATGCAGGTTGCGACTGTACACCC

%G+C = 60

GGCGTGTTCCGCGCGACAGCGGGTGG

GATGCCGGTGCTGGTGGTCATCGAGTC NZE_ Rvl954A_R

TGGGACAGGAGGTGATCAGATGGCTC GG CCG CTCTTGTTCTTCGT

GTA A AG CTACGTCCCCG G GTA AG CCG G CGGAT

CTCCG ACGTCGG G ACAGTATCG CCCG G

Length = 24, Tm = 57.4,

TTG GCG GTG G CAACG AG GTG ACCGTTC

%G+C = 58.3

CG AAG GGACACCGTCTG CCTCCCTCGC

CCA AG CCCG GTC AG A AGTG G GTG A AC GTCGATCCGACGA Amplicon Size = ~280 bp

SS4 from >gi 1448814763x2506161-2505736 NZE_Rv2231A_F CLASS 2

Mycobacterium tuberculosis H37Rv

Rv2231A

complete genome

TGTACCG CGTG CCCG ACG

TTG ACCATG G CGTGTACCG CGTGCCCG ATTTG

ACGATTTGGACGCTCCGTTGTCAGACG

Length= 23, Tm = 59.1,

ACGTGCTCGAACGCTTTCACCGGTGAA

%G+C = 61

GCGCTACCTCATCGACACCCACGTTTGG

CTG CG G ATG CCGTC A ACG A A AC ACG G G

CGATTGTTCAGGACGTCCGCAACAGCA

NZE_Rv2231A_R

TTCTCTTGTCG GCCGCCAGTGCCTGGG

AG ATCG CG ATCAACTACCG CCTCGG CA AC AG G C AG CTA AC AG G G

AGCTCCCGCCGCCCGAG CC ATCG G CCT CGTCGG

CTTACGTG CCCG ATCG A ATG CG CCG CT

Length = 23, Tm = 57.1,

GCGGCACGTCGCCGCTGTCAGTTGACC

%G+C = 65

ACG C AC AC ACTG CGCACCGCAGAG CTT

Amplicon Size = ~390 bp

CCGGATCACCATCGACATCCATTCGACC

GTGTG CTCATCG CCC AG G C AC AG CTG C

TTGGCCTGAC

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 (50μΙ) consisted of lOxtaqPCR buffer, 0.5mmolMgCI2, 0.4mmol dNTP, 10 pmol forward and reverse primers respectively, 4% DMSO and lUtaq DNA

polymerase.The reaction conditions were the following: 95°C for 5minutes, followed by 35 cycles of 95°C for 30seconds, annealing temperature 50°C for 30seconds, 72° C for Iminute 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 mycobacteruim genus-specific primers of the 'signature sequences'. SSI 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 SSI, 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 <1 ng (lOOpg) DNA as shown in Fig. 1A, IB, 1C and ID for the four primers of the signature sequences SSI, 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 Helden et al (Isolation of DNA from Mycobacterium tuberculosis, Paul D. van Helden, Thomas C. Victor, Robin M. Warren, and Eileen G. van Helden)

The results show amplification of SSI and SS3 as seen in Fig. 3.

Sequence Listing

(1) GENERAL INFORMATION:

(iii) NUMBER OF SEQUENCES: 4

(2) INFORMATION FOR SEQ ID NO: 1

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 230 base pairs

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1, Rvl507A

1 ATGCAATCAG GTCAAAATAT CCTCGCCAAG GTATGTAATT TGATTGAACA ATCGCGACTT

61 TCTTCAACGC GGTGTCTCCA ATTTAGAATA ACAAATACGT CGCGCCCGCG ACAGCTCCGC

121 TGGAGCGAGT TCAAGCGATT CTGCGACATA TTCAATATGG TGCTCGGGAA GGCCAGGATG

181 GGCCGCGACC CGGGGCGTCC GGTGCGCGAT GAACGTCGCA TCGTCTCCTG

(2) INFORMATION FOR SEQ ID NO: 2

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 330 base pairs

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2, Rvl509

1 GTGTTTGCGT TGAGTAATAA TCTGAACCGT GTGAACGCAT GCATGGATGG ATTCCTTGCC 1 CGTATCCGCT CACATGTTGA TGCGCACGCG CCAGAATTGC GTTCACTGTT CGATACGATG21 GCGGCCGAGG CCCGATTTGC ACGCGACTGG CTGTCCGAGG ACCTCGCGCG GTTGCCTGTC 81 GGTGCAGCAT TGCTGGAAGT GGGCGGGGGG GTACTTCTGC TCAGCTGTCA ACTGGCGGCG 41 GAGGGATTTG ACATCACCGC CATCGAGCCG ACGGGTGAAG GTTTTGGCAA GTTCAGACAG 01 CTTGGCGACA TCGTGCTGGA ATTGGCTGCA

(2) INFORMATION FOR SEQ ID NO: 3

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 280 base pairs

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3, RV1954A ATGGCCAGGG GCAGGGTCGT GTGCATTGGG GATGCAGGTT GCGACTGTAC ACCCGGCGTG

1 TTCCGCGCGA CAGCGGGTGG GATGCCGGTG CTGGTGGTCA TCGAGTCTGG GACAGGAGGT21 GATCAGATGG CTCGTAAAGC TACGTCCCCG GGTAAGCCGG CTCCGACGTC GGGACAGTAT 81 CGCCCGGTTG GCGGTGGCAA CGAGGTGACC GTTCCGAAGG GACACCGTCT GCCTCCCTCG 41 CCCAAGCCCG GTCAGAAGTG GGTGAACGTC GATCCGACGA

(2) INFORMATION FOR SEQ ID NO: 4

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 389 base pairs

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4, Rv2231A

TTGACCATGG CGTGTACCGC GTGCCCGACG ATTTGGACGC TCCGTTGTCA GACGACGTGC

1 TCGAACGCTT TCACCGGTGA AGCGCTACCT CATCGACACC CACGTTTGGC TGCGGATGCC21 GTCAACGAAA CACGGGCGAT TGTTCAGGAC GTCCGCAACA GCATTCTCTT GTCGGCCGCC 81 AGTGCCTGGG AGATCGCGAT CAACTACCGC CTCGGCAAGC TCCCGCCGCC CGAGCCATCG 41 GCCTCTTACG TGCCCGATCG AATGCGCCGC TGCGGCACGT CGCCGCTGTC AGTTGACCAC 01 GCACACACTG CGCACCGCAG AGCTTCCGGA TCACCATCGA CATCCATTCG ACCGTGTGCT 61 CATCGCCCAG GCACAGCTGC TTGGCCTGA

Claims

Claims We claim:

1. 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.

2. 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;

complementary to SEQ ID NO: 1, 2, 3 or 4.

3. The method of claim 2 wherein the set of oligonucleotide primers are selected from

(i) 5' ATGCAGGTTGCGACTGTACACCCGG 3'

3' GGCCGCTCTTGTTCTTCGTCGGAT 5'

(ii) 5' GTGTTTGCGTTGAGTAATAATCTGAACCGTGT 3'

3' AG CCAATTCCAG CACG ATGTCGCC 5'

(iii) 5' ATGCAGGTTGCGACTGTACACCCGG 3'

3' GGCCGCTCTTGTTCTTCGTCGGAT 5'

(iv) 5' TGTACCGCGTGCCCGACGATTTG 3'

3' AC AG G CAG CTA AC AG G G CGTCG G 5'

(v) a set of oligonucleotide primers complementary to (i), (ii), (iii) or (iv)

or

4. A kit for 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' AG CCAATTCCAG CACG ATGTCGCC 5'

(iii) 5' ATGCAGGTTGCGACTGTACACCCGG 3'

3' GGCCGCTCTTGTTCTTCGTCGGAT 5'

(iv) 5' TGTACCGCGTGCCCGACGATTTG 3'

3' AC AG G CAG CTA AC AG G G CGTCG G 5'

(v) a set of oligonucleotide primers complementary to (i), (ii), (iii) or (iv)

or

5. A method of detecting pathogenic mycobacterium sps in a clinical sample as in claim 2 wherein the sample is isolated from individuals vaccinated against tuberculosis.

6. A method of detecting pathogenic mycobacterium sps in a clinical sample as in claim 2 wherein the sample is isolated from individuals treated against tuberculosis.