Mycobacterium tuberculosis specific DNA fragment

FIELD OF INVENTION

This invention relates to a

Mycobacterium tuberculosis

specific DNA fragment containing IS like and repetitive sequences, a method of production of such DNA fragment and the use of such DNA fragment, for example, to rapidly diagnose

Mycobacterium tuberculosis

infection in clinical samples, and to identify clinical isolates of

Mycobacterium tuberculosis

. The DNA fragment may be used to determine information about the epidemiology of

Mycobacterium tuberculosis

infection. Specifically this invention relates to the use of sequence specific DNA fragments to diagnose

Mycobacterium tuberculosis

and strains of

Mycobacterium tuberculosis

. A purpose of the study of the epidemiology of tuberculosis is to distinguish the genetic diversity of the causative agent

Mycobacterium tuberculosis

and to obtain information about strain to strain variability. This can be achieved by molecular epidemiological methods including DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis. Such approaches, can aid the investigation of point source outbreaks, transmission, pathogenesis and may be employed as a marker of strain typing.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) is a major cause of infectious mortality. According to a recent WHO report, the number of deaths attributed to TB was larger number in 1995 than in any other year in history (Moran, N. 1996. WHO Issues Another Gloomy Report. Nature Medicine 4:377). Tuberculosis remains widespread worldwide and constitutes a major health problem particularly in developing countries. One third of the total world's population (nearly two billion people) is infected with

Mycobacterium tuberculosis

out of which 5 to 10% develop the disease. TB causes more than 3 million deaths per year and recently WHO has predicted that 30 million people will die of TB in the next ten years (Joint International Union Against Tuberculosis and World Health Organization Study Group. Tubercl 63:157-169, 1982). Tuberculosis is caused by a gram positive acid fact bacterium

Mycobacterium tuberculosis

or

M. bovis

, which are the tubercle bacilli of the family of Mycobacteriaceae.

M. bovis

is a species which causes tuberculosis in cattle and can be transmitted to humans and other animals in which it causes tuberculosis. At present nearly all tuberculosis in humans is caused by

Mycobacterium tuberculosis

. Infections occasionally result from other species of mycobacteria that are common environmental saprophytes. These species have been collectively termed as MOTT (Mycobacteria other than typical tubercle), environmental or tuberculoid bacilli. The difference between the two infections is that infection with

Mycobacterium tuberculosis

is always transmitted from host to host. In contrast, human beings infected with other mycobacteria rarely transmit the disease.

Hence the essential component of any tuberculosis control program is containment of the disease. Identification of infected individuals, especially those most likely to transmit viable bacilli, comes as a first priority in strategies for tuberculosis control. Early and timely diagnosis of tuberculosis is essential for identifying individuals carrying the bacilli. Therefore a need has arisen for a method of diagnosis of tuberculosis which is rapid, sensitive and specific. Routine diagnostic methods used for identification of

Mycobacterium tuberculosis

includes acid fast smear test in clinical samples like sputum, tests based on growth of bacilli of specific media and differential biochemical tests. The culture of mycobacteria from clinical samples is the most reliable and provides for definite diagnosis of tuberculosis. Although 100% specific, it takes six to eight weeks due to slow growth of organisms and further biochemical testing before identification can be made (Heifests, L B. and Good, R. C. 1994. Current Laboratory Methods for the Diagnosis of Tuberculosis. Tuberculosis Pathogenesis, Protection and Control (ed. B. R. Bloom) ASM Washington D.C., pp. 85-110).

Methods based on antigen and antibody detection in body fluids and more recently nucleic acid probes (sequence specific DNA fragments) have been developed as reagents for rapid diagnosis and monitoring of the epidemiology of tuberculosis (Young, D. B. and Mehlert, A. 1989, Serology of Mycobacteria: Characterization of Antigens Recognized by Monoclonal Antibodies. Rev. Infec. Dis. 12: S431-S435; Pfyfer, G. E., Kisling, P., Jahn, E. M. I., Martin, H., Salfinger; W. M. and Weber, R. 1996. Diagnostic Performance of Amplified

Mycobacterium Tuberculosis

Direct Test with Cerebrospinal Fluid, Other Non Respiratory and Respiratory Specimens, J. Clin. Microbiol. 34:834-841).

The DNA probes utilize a wide array of sequences from

Mycobacterium tuberculosis

ranging from whole genomic DNA, to a single copy sequence, and to repetitive DNA elements. When evaluated directly on clinical samples they have proved to be highly specific, sensitive and dramatically reduce the time for diagnosis of tuberculosis (Kiehn, T. E. 1993. The Diagnostic Mycobacteriology Laboratory of the 1990's. Clin. Infect. Dis. 17 (suppl.2) S447-S454).

Several sequence specific probes have been used as targets for identification of

Mycobacterium tuberculosis

by amplification of specific sequences by PCR.

IS6110 is an IS element present in members of

Mycobacterium tuberculosis

complex (

Mycobacterium tuberculosis, M. bovis, M. africanum

, and

M. microti

).

Different regions of IS6110 have been amplified using different sets of primers for PCR based diagnosis like 123 base pair (bp) or 245 bp region (Einsenach, K. D., Cave, M. D., Bates, J. H. and Crawford, J. T. 1990. Polymerase chain reaction amplification of repetitive DNA sequence specific for

Mycobacterium tuberculosis

. J. Infect. Dis. 161:977-981; Kolk, A. H. J., Schuitema, A. R. J., Kuijper, S., VanLeeuwen J., Hermans, P. W. M., Van Embden, J. D. A. Hartskeerl, R. A. 1992, Detection of

Mycobacterium tuberculosis

in clinical samples by using polymerase chain reaction and a non radioactive detection system. J. Clin. Microbiol. 30:2567-2575).

IS6110 has some disadvantages. Several

Mycobacterium tuberculosis

strains with one copy or no copy of IS6110 have been reported (Sahadevan, R., Narayanan, S. Paramsivam, C. N., Prabhakar, R. and Narayanan, P. R. 1995. Restriction fragment length polymorphism typing of clinical isolates of

Mycobacterium tuberculosis

from patients with pulmonary tuberculosis in Madras, India by use of direct repeat probe. J. Clin. Microbiol. 33:3037-3039: Van Soolingen, D., Dehass, P. E. W., Hermans, P. W. M. Groenen, P. M. A. and Van Embden, J. D. A. 1993. Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of

Mycobacterium tuberculosis

. J. Clin. Microbiol. 31:1987-1995). Thus the repertoire of

Mycobacterium tuberculosis

strains present all over the world may not be selected/amplified using a single repetitive element or one DNA probe specific to

Mycobacterium tuberculosis

. The search for newer DNA probes is a constant requirement.

OBJECTS OF THE INVENTION

It is an object of this invention to detect

Mycobacterium tuberculosis

in a clinical sample.

It is another object of this invention to describe a DNA fragment containing IS like sequence and repetitive sequences.

It is a further object of this invention to use a DNA fragment containing IS like sequence and repetitive sequences to detect

Mycobacterium tuberculosis.

It is yet another object of this invention to describe a method for production of a DNA fragment containing IS like sequence and repetitive sequences.

It is another object of this invention to use a 1291 base pair sequence having an IS like sequence and repetitive sequences or a portion or fragment of the 1291 base pair sequence to detect

Mycobacterium tuberculosis.

It is a further object of this invention to construct a sequence specific DNA probe for the identification of

Mycobacterium tuberculosis

present in clinical samples.

It is another object of this invention to develop a sequence specific DNA probe for identification of clinical isolates of

Mycobacterium tuberculosis

in order to distinguish it from other mycobacteria which may be present in the clinical samples.

It is yet another object of this invention to provide a sequence specific DNA probe for monitoring the epidemiology of tuberculosis.

It is further object of this invention to provide a sequence specific DNA probe for the detection of polymorphism in clinical isolates of

Mycobacterium tuberculosis.

It is still another object of this invention to provide an IS element for transposition, mutagenesis and gene inactivation in mycobacteria for the investigation of pathogenesis, virulence and vaccine development.

SUMMARY OF THE INVENTION

This invention relates to

Mycobacterium tuberculosis

specific DNA fragment containing IS like sequence and repetitive sequences, a method of production of such DNA fragment and the use of such DNA fragment, for example, to rapidly diagnose

Mycobacterium tuberculosis

infection in clinical samples, to identify clinical isolates of

Mycobacterium tuberculosis

and to track the epidemiology of

Mycobacterium tuberculosis

infection. Specifically this invention relates to the use of sequence specific DNA fragment to diagnose tuberculosis and strains of

Mycobacterium tuberculosis

. The

Mycobacterium tuberculosis

specific DNA fragment consists of 1291 base pair (bp) sequence which contains an IS like element and several repeat DNA sequences.

When this DNA fragment is incubated with clinical samples which may contain mycobacteria of interest, the presence of

Mycobacterium tuberculosis

can be detected by specificity of hybridization. The DNA primers designed from the nucleotide sequence of this 1291 bp fragment may be incubated with clinical samples to amplify the DNA of

Mycobacterium tuberculosis

present in the clinical samples. Hence if the samples contain

Mycobacterium tuberculosis

it may be diagnosed specifically. The DNA fragment when hybridized with genomic DNA of clinical isolates of

Mycobacterium tuberculosis

by Southern hybridization, specifically hybridizes with

Mycobacterium tuberculosis

isolates and displays polymorphism. Hence, the fragment can be used in the specific diagnosis of

Mycobacterium tuberculosis

infections and to determine information about the epidemiology of

Mycobacterium tuberculosis

infections.

Mycobacteria can be detected in samples of sputum, cerebrospinal fluid, pleural fluid, urine, ascitic fluid, gastric samples, bronchial lavage, pericardial fluid, pus or lymph node aspirate.

One way to produce the

Mycobacterium tuberculosis

specific DNA fragment of the invention, is to use a genomic library of

Mycobacterium tuberculosis

DNA which may be constructed in a plasmid or phage lambda gt11 vector. The library of the genomic DNA fragments may be screened with genomic DNA of

Mycobacterium tuberculosis

as a probe to select the fragments which rapidly hybridize to

Mycobacterium tuberculosis

genomic DNA.

The detection of positive hybridization signals within 60 minutes may indicate that the clones may contain repeat sequences in the DNA fragment of

Mycobacterium tuberculosis.

The clones thus obtained may further be screened by DNA-DNA hybridization with genomic DNA of mycobacteria other than

Mycobacterium tuberculosis

used as probes. The clones not producing any hybridization signals may be selected as containing

Mycobacterium tuberculosis

specific DNA fragments.

The DNA fragment of this invention may be used by incubating it with samples which may contain the specific mycobacteria of interest (

Mycobacterium tuberculosis

). If

Mycobacterium tuberculosis

is present, then the DNA fragment will hybridize with the DNA of

Mycobacterium tuberculosis

which can be detected for example by dot blot or Southern hybridization assay.

The nucleotide sequence of the specific DNA fragment may be determined and DNA primers may be designed for specific amplification of

Mycobacterium tuberculosis

DNA. The specific DNA primers may be added into the clinical samples to amplify

Mycobacterium tuberculosis

DNA if present in the sample. The amplified product may be visualized by for example agarose gel electrophoresis and hybridization with the specific DNA fragment of the invention. The polymerase chain reaction (PCR) amplification as well as hybridization with the specific DNA fragment of the invention may indicate that the sample contains

Mycobacterium tuberculosis

and hence may be used in detection and diagnosis of tuberculosis. By hybridization with the specific DNA fragment of the invention, epidemiology of tuberculosis and

Mycobacterium tuberculosis

may be traced as the fragment contains several repeat sequences and thus strain polymorphism of

Mycobacterium tuberculosis

may be determined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG.

1

. Screening of recombinant lambda gtll library of

Mycobacterium tuberculosis

with DIG-labeled denatured genomic DNA of

Mycobacterium tuberculosis

by DNA hybridization. Positive plaques gave hybridization signals within 60 min and appeared blue in color.

FIG.

2

. Agarose (1%) gel electrophoresis analysis of EcoRI digests of recombinant lambda clones. The DNA from recombinant phages was isolated, digested with EcoRI restriction enzyme and electrophoresed. Lambda HinDIII (BRL) was used a molecular weight marker. Lane 2: Clone C8; Lane 3: Clone C10; Lane 4: Clone C16; Lane 5: Clone C33; Lane 6: Clone C38; Lane 7: Clone C41; Lane 8: Clone C60; Lane 9: Clone C70; Lane 10: Lambda HindIII.

FIG.

3

. Southern hybridization of EcoRI digests of recombinant lambda clones with DIG-labeled denatured genomic DNA of

Mycobacterium tuberculosis

. The recombinant DNA clones shown on the gel is: Lane 1: Clone C70; Lane 2: Clone C60; Lane 3: Clone C41; Lane 4: Clone C38; Lane 5: Clone C33; Lane 6: Clone C16; Lane 7: Clone C10; Lane 8: Clone C8.

FIG.

4

. Restriction map of 4.2 kb DNA fragment of

Mycobacterium tuberculosis

from Clone C8. The DNA was mapped with restriction endonucleases.

FIG.

5

. Restriction map of 1291 bp StuI-StuI fragment of

Mycobacterium tuberculosis

from clone C8.

FIG.

6

. DNA hybridization of 1291 bp DNA fragment from recombinant clone CD8 with other mycobacterial DNA. Southern hybridization of EcoRI digests of chromosomal DNA of various mycobacterial species (Lanes 1-7) with DIG labeled denatured 1291 bp DNA fragment. Lane 1

: M smegmatis

; Lane 2

: M. phlei

; Lane 3

: M. avium intracellulare complex

; Lane 4

: M. scrofulaceum

; Lane 5

: M. fortuitum

; Lane 6

: M. kansasii

; Lane 7

: M. gordonae

; Lane 8

: M. asiaticum

; Lane 9

: M. aurum

; Lane 10

: M. chitae

; Lane 11

: M chelonae

; Lane 12

: M. xenopi

; Lane 13

: M. triviale

; Lane 14

: M. marinum

; Lane 15

: M. microti

; Lane 16

: M. flavescens

; Lane 17

: Mycobacterium tuberculosis.

FIG.

7

. Dot-blot hybridization of non-mycobacterial DNA with DIG-labeled denatured 1291 bp DNA fragment. 1

. Salmonella typhimurium

; 2

. Enterobacter alginii

; 3

. Klebsiella pneumoniae

; 4

. Pseudomonas aeruginosa

; 5

. Proteus vulgaris

; 6

. Staphylococcus aureus

; 7

. Escherichia coli

; 8. Edwardsiella sp.; 9

. Mycobacterium tuberculosis

; 10. Human placental DNA.

FIG.

8

. DNA hybridization of 1291 bp StuI-StuI fragment with StuI digested genomic DNA of clinical isolates of

Mycobacterium tuberculosis.

FIG.

9

. Nucleotide sequence of the 1291 bp StuI-StuI fragment of

Mycobacterium tuberculosis

(SEQ ID NO:1).

FIG.

10

. Deduced amino acid sequence from the nucleotide sequence (as in

FIG. 9

) of 1291 bp StuI-StuI DNA fragment of

Mycobacterium tuberculosis

SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to

Mycobacterium tuberculosis

specific DNA fragment, methods of producing such DNA fragment, characterization of such DNA fragment, nucleotide sequence of such DNA fragment and the use of the DNA fragment or portion thereof for the rapid diagnosis of

Mycobacterium tuberculosis

infection in clinical samples to identify clinical isolates of

Mycobacterium tuberculosis

and to monitor the epidemiology of

Mycobacterium tuberculosis

infection. Specifically, this invention relates to the use of sequence specific DNA fragment to diagnose tuberculosis and strains of

Mycobacterium tuberculosis.

The invention relates to production and identification of a DNA fragment which contains an IS like sequence and various repeat sequences unique to

Mycobacterium tuberculosis

, which is the major etiologic agent of tuberculosis. In particular, it is based on the isolation of

Mycobacterium tuberculosis

specific DNA fragment containing repetitive sequences using genomic DNA of

Mycobacterium tuberculosis

as a probe. Repetitive sequences are those which are present in several copies in the genome and give positive hybridization with genomic DNA probe within a short detection period.

As used herein IS like element is intended to refer to an insertion sequence. An insertion sequence is a small, transposable element in bacteria (a small transposon) that can insert into several sites in a genome. Insertion sequences function in the transposition of segments which they flank. They usually contain genes coding for proteins that function in their transposition but contain no other genes and have no other known effect than to function in transposition. The termini of each insertion sequence consist of inverted repeats.

Accordingly, the isolated DNA fragment of

Mycobacterium tuberculosis

comprises 1291 base pairs, the DNA fragment having following nucleotide sequence (SEQ ID NO:1:):

AGGCCTCGGT GACCGTGATC ATGTTGCCGC CGAAGGTCAT TACGTTGTGT ACGTCAATGA

CCATCTGCTC GTTGTTTATG GGGATGAATC GGGAGTGGTG ACCGAGAGAT CGATGGCGAA

TCTGGCCCTG GTTATCGCCC GCCACCAAGA AGCCATTGTT GAAGTCGCCC GTGTCGAAAG

CGCCGGTATT GACGTTGCCG GGATTGAAGA AGCCGGTGTT GGTGTCACCC GGGTTATAGC

TGCCGGTATT GGTGTCACCC ACGTTGAAGT TGCCGGTGTT GGTGTTACCG ACGTTGAAGC

CGCCGGTGTT GTAGCTGCCC GTGTTGTAGA AGCCCGTGTT GAAGTCGCCG GCGTTGAGGA

TGCCCGTGTT GTAGCTGCCA GCATTGAGGA TGCCGGTATT GTCGGTACCC GGGTTCCCGA

TACCCCAGTT CCCGGTGCCC GAGTTTGCGA TGCCGACGTT TCCGGTGCCC GCGTTGAAGA

TGCCAACGTT ATTGGTGCCC GAATTGAACA GGCCGCTGTT GCCGGTGCCC GAGTTCCAGC

CGCTAGCAAT ATTGAAGCCC TGCTGGTTGT CGCCGGACAG CCCGATGCCG ATGTTGTTGT

TGCCGGTGTT GGCGAACCCG ATGTTGTTGT TGCCGGTGTT GGCAAAGCCT TGGTTGAAGT

CGCCCGCGTT CCCGAAGCCG ACGTTGTAGT CGCCGACGTT TCCAAAACCG ATGTTGTAGA

TCCCGAGGTT TCCGGATCCG ATGTTGTAGT TTCCCAGGCT TCCGGAACCG ACATTGAATA

CTCCGATGTT TCCACTGCCG ATATTGAAGC TGCCGACGTT GCCGCTGCCC AAGATGTTTT

GGCTGCCGAG GTTGCCGCTG CCAAGGATGT TGAAGTCACC GACGTTTCCG CTGCCGAGAA

TGTTGTAATT GCCGATGTTG GCGTTGCCGA GAATGTTCAC GACGCCCCGG TTTGCCAGGC

CGAGATTGAA GACCGGTGGG CCACCGAAAA ATCCCGACAT GTTGCTTCCG GTGTTGAAGA

AGCCCGAGAT CAAGGCCGGC GTTGTGATGG CCACCAGGCT CATGTTGAAC AAACCCGATA

CGGTGTTGCC CGAGTTGATC ACGCCCGATA CCAGCACGCC CGCGTTTGCC AGGCCGGAGT

TACCGATGGC CCCCGACGAA GAGTGGAAGA AGCCAGAATT GTTGGCACCG GAGTTCAGGA

AGCCGGACGC GCTACCGGCA CCGCTGTTGA AGAATCCCGA CGACGGCGCA CTGGTCGAGT

TGAAGAAGCC GGGCTCCCGA AAATCAGGCC T

In another embodiment of the invention, there is a process for the production and identification of DNA fragment of

Mycobacterium tuberculosis

comprising 1291 base pairs. One of the processes comprises the following steps:

(a) constructing a library of

Mycobacterium tuberculosis

in a vector including phage lambda gt11 or a plasmid;

(b) screening the genomic library of

Mycobacterium tuberculosis

with genomic DNA of

Mycobacterium tuberculosis

as probe to select DNA fragments capable of specifically and rapidly hybridizing to

Mycobacterium tuberculosis

genomic DNA;

(c) labeling the probe with a label such as digoxigenin, biotin, radiolabelled with

32

P or alkaline phosphatase;

(d) isolating repetitive DNA fragments with positive hybridization signals ranging between 10 to 60 minutes; and

(e) further screening of the clones isolated above for validation by DNA-DNA hybridization with genomic DNA of mycobacteria other than

Mycobacterium tuberculosis

as probe to confirm that the clone selected above do not produce any hybridization signals with DNA of mycobacteria other than

Mycobacterium tuberculosis.

The DNA fragment identified in this process can be used for the detection of

Mycobacterium tuberculosis

by incubating the isolated DNA fragment with samples which may contain specific mycobacteria (

Mycobacterium tuberculosis

) and detecting the presence or absence of

Mycobacterium tuberculosis.

If DNA of

Mycobacterium tuberculosis

is present then the DNA fragment will hybridize with the DNA of

Mycobacterium tuberculosis.

To produce a sequence specific DNA fragment of

Mycobacterium tuberculosis

, a genomic library of

Mycobacterium tuberculosis

DNA may be constructed in plasmid or phage lambda gt11 vector. The library of genomic fragments may be screened with genomic DNA of

Mycobacterium tuberculosis

as a probe to screen and select DNA fragments which rapidly hybridize to

Mycobacterium tuberculosis

genomic DNA. This may provide DNA fragments which hybridize very rapidly to

Mycobacterium tuberculosis

DNA. The DNA fragment may be incubated with clinical samples suspected of containing

Mycobacterium tuberculosis

of interest or may be incubated with

Mycobacterium tuberculosis

after it is cultured. If

Mycobacterium tuberculosis

is present, then the specific DNA fragment will hybridize with the DNA of

Mycobacterium tuberculosis

and can be detected by, for example, dot blot or Southern hybridization assay.

The nucleotide sequence of the specific DNA fragment may be determined and specific DNA primers may thus be designed. The so designed primers may be added to the clinical samples. If

Mycobacterium tuberculosis

is present in the clinical samples, the genomic DNA homologous to the specific DNA fragment may be amplified by PCR. The amplified product may be visualized by for example agarose gel electrophoresis and hybridization. The PCR amplification as well as hybridization with the specific DNA fragment of the invention may indicate that the sample contains

Mycobacterium tuberculosis

and hence may be used in detection and diagnosis of tuberculosis. In addition, the specific DNA fragment of the invention may be used to monitor the epidemiology of tuberculosis and strain polymorphism of

Mycobacterium tuberculosis

may be determined.

The method of this invention allows for rapid and specific diagnosis of tuberculosis and

Mycobacterium tuberculosis

infection. The assessment of the diagnosis with the DNA probe of this invention is accurate because the DNA probe owing to its sequence specificity only provides for hybridization and detection of

Mycobacterium tuberculosis

which indicates that the sample contains the mycobacteria of interest i.e.

Mycobacterium tuberculosis.

The inventors have determined the nucleotide sequence of the DNA fragment of this invention which is 1291 bp long. The nucleotide sequence of the DNA fragment shows several interesting features including the presence of repeat sequences and an IS like sequence with an open reading frame. The IS like sequence is characterized by the presence of two inverted repeats flanked with direct repeat GTT on either side. GTT is a direct repeat which is located at 458 to 460 and at 1193 to 1195.

A section or fragment of the 1291 base pair sequence can also be used to detect

Mycobacterium tuberculosis.

The deduced amino acid sequence from the nucleotide sequence described in

FIG. 9

of the 1291 bp DNA fragment is shown in FIG.

10

.

I. Construction of a recombinant expression library of

Mycobacterium tuberculosis

DNA.

A recombinant DNA expression library of

Mycobacterium tuberculosis

DNA may be constructed using lambda gt11 vector. The library may be constructed with

Mycobacterium tuberculosis

genomic DNA fragments in such a way that all protein coding sequences would be represented and expressed (Young, R. A., B. R. Bloom, C. M. Grosskinsky, J. Ivyani, D. Thomas and R. W. Davis, Proceedings of the National Academy of Sciences, USA, 82:2583-2587 (1985)).

Lambda gt11 is a bacteriophage vector which is capable of driving the expression of foreign insert DNA with

E. coli

transcription and translation signals. Lambda gt11 expresses the insert DNA as a fusion protein connected to the

E. coli

betagalactosidase polypeptide. Lambda gt11 and the

E. coli

strain used (Y1088 and Y1990) have been described previously (Young, R. A. and R. Davis. Proceedings of the National Academy of Sciences, USA, 80:1194-1198, 1983). The techniques of these publications are incorporated herein by reference. The library contained in this manner has a titer of 1×10

10

pfu/ml. It contains approximately 60% recombinants with an average size of 4 kb.

II. Screening of the lambda gt11

Mycobacterium tuberculosis

library with genomic DNA of

Mycobacterium tuberculosis

as probe

Genomic DNA from

Mycobacterium tuberculosis

(

Mycobacterium tuberculosis

H37Rv and confirmed Indian clinical isolates) was used as probe to screen the

Mycobacterium tuberculosis

recombinant DNA library. This work is described below and with specific reference to isolation of repetitive sequences.

The chromosomal DNA from

Mycobacterium tuberculosis

was labeled with digoxigenin and used as a probe. Screening of the lambda genomic library was performed as described in (Sambrook, H., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor, N.Y.).

Briefly lambda gt11 recombinants were arrayed on lawn of

E. coli

Y1088. The phages were grown and blotted onto nitrocellulose paper and probed with DIG labeled denatured chromosomal DNA from

Mycobacterium tuberculosis

by DNA hybridization. The signal producing plaques were detected with anti-DIG-alkaline phosphatase labeled antibodies according to the manufacturer's protocol (Boehringer Mannheim, Germany). DNA antibody conjugates were detected by alkaline phosphatase reaction with Nitro Blue tetrazolium (NBT) and 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP). The positive hybridization was detected as blue plaque spots. Such signal producing clones were isolated in screen of approximately 6×10

4

recombinant plaques within 60 minutes of detection time (FIG.

1

).

III. Probing of the Arrays of lambda gt11 DNA clones with chromosomal DNA as probe

0.3 ml of a saturated culture of Y1088 was mixed with recombinant phage (6,000 plaque forming units, pfu) and incubated at 36° C. for 10 minutes. The mixture was added to 3 ml of molten LB soft agar and poured onto 90 mm plates containing 1.5% LB agar and allowed to harden at room temperature for 10 min. The plates were incubated at 37° C. for period at which point clear plaques approximately 1 mm in diameter were visible. The plates were then overlaid with nitrocellulose filters. Subsequent processing of filters for detection of clones containing repetitive sequences was identical to the procedures described for screening of lambda gt11 library with DNA probes (Davis, L. G., M. D. Dibner and J. F. Battey. Basic Methods in Molecular Biology, Elsevier. 1986). The nonradioactive DNA labeling and detection system of Boehringer Mannheim, Germany was used for labeling, hybridization and detection during screening. The amplified library was plated to an approximate 6×10

3

pfu per plate using

E. coli

Y1088 as host strain. Of total 60,000 recombinant plaques, 14 plaques were picked up as a result of tertiary screening with DIG-labeled denatured chromosomal DNA from

Mycobacterium tuberculosis

as a probe. These fourteen plaques gave positive signals within 60 minutes.

The recombinant plaques containing repetitive sequences specific to

Mycobacterium tuberculosis

were isolated by subtractive screening of 14 recombinant plaques with denatured chromosomal DNA from mycobacterial strains other than

Mycobacterium tuberculosis

by DNA hybridization. This resulted in isolation of eight recombinant plaques which hybridized with

Mycobacterium tuberculosis

but not with denatured genomic DNA from

M. aviumintracellulare, M. fortuitum, M. chelonae, M. smegmatis

and

M. phlei

. These recombinant plaques were purified, the phage DNA was isolated and digested with EcoRI restriction enzyme to determine the size of the insert DNA in the recombinant plaques (FIG.

2

and Table I). The other set was subjected to Southern blotting and hybridization using DIG labeled denatured genomic DNA from

Mycobacterium tuberculosis

. Positive hybridization was detected within 60 minutes (FIG.

3

).

All eight clones contained inserts of different sizes and hybridized with chromosomal DNA of

Mycobacterium tuberculosis

. The insert from clone C8 showed positive hybridization within 10 minutes of detection and was selected for detailed study.

TABLE I

Recombinant clone

insert fragments (kb)

C8

4.2* kb

C10

3.8* kb

C16

4.2* kb

C33

2.7* kb

C38

3.0* kb

C41

2.4, 1.6*, 1.1, 0.7 kb

C60

3.8, 2.8, 0.76*, 0.64 kb

C70

2.4*, 1.6, 1.1, 0.7 kb

*The fragment(s) which gave positive hybridization signal with DIG-labeled denatured genomic DNA of Mycobacterium tuberculosis within 10-60 min.

IV. Recombinant DNA manipulation

The clone C8 was selected. It contained an insert of 4.2 kb which gave strong hybridization signal within 10 minutes with DIG-labeled denatured chromosomal DNA from

Mycobacterium tuberculosis

. The 4.2 kb DNA fragment from clone C8 was isolated by restriction digestion and subsequent elution of 4.2 kb fragment from the agarose gel. The 4.2 kb fragment was purified and subcloned into pUC 13 plasmid vector. The resulting recombinant plasmid was designated as pC8.

V. Localization of repetitive sequences within 1291 bp StuI-StuI DNA Fragment

The restriction map of the 4.2 kb DNA fragment was determined (FIG.

4

). It contained one site each for BamHI, SalI and two sites each for StuI and KpnI restriction enzymes.

The repetitive sequences within the 4.2 kb DNA fragment were further localized by hybridization of BamHI, SalI, StuI and KpnI restricted fragments of 4.2 kb DNA fragment with DIG-labeled denatured genomic DNA of

Mycobacterium tuberculosis.

The repetitive sequences were localized within StuI-StuI fragment present within 4.2 kb DNA fragment cloned in this invention. The detailed restriction map of StuI-StuI fragment was deduced from the nucleotide sequence of 1291 base pair and confirmed by subsequent digestion with restriction enzymes (FIG.

5

).

VI. Hybridization of 1291 bp StuI-StuI DNA fragment with different mycobacterial strains and other pathogens

The genomic DNA from different mycobacteria and other pathogens was isolated and hybridized with DIG-labeled 1291 bp DNA fragment of pC8 by Southern hybridization as well as dot blot hybridization. As shown in

FIG. 6

, the fragment did not hybridize in Southern hybridization to the following mycobacterial strains:

M. smegmatis

M. phlei

M. fortuitum

M. chelonae

M. flavescens

M. chelonae

M. trivale

M. duvali

M. marinum

M. gordonae

M. kansasii

M. avium

M. intracellulare

M. scrofulaceum

M. gordonae

M. xenopi

M. aurum

M. microti

M. szulgai

The genomic DNA from the above mentioned bacteria were isolated and digested with EcoRI restriction enzyme. The digest was electrophoresed in 0.8% agarose gel, blotted onto nitrocellulose paper and hybridized with DIG-labeled denatured 1291 bp DNA fragment as a probe. The Southern hybridization of 1291 bp fragment with genomic DNA of

Mycobacterium tuberculosis

revealed several bands confirming the presence of repetitive sequences with 1291 bp DNA fragment whereas no hybridization was found with the other mycobacteria listed above (FIG.

6

).

The hybridization of 1291 bp DNA fragment was done with genomic DNA from other pathogenic organisms listed below. The result of dot blot hybridization is shown in FIG.

7

.

Salmonella typhimurium

Staphylococcus aureus

Proteus vulgaris

Pseudomonas aeruginosa

Klebsiella pneumoniae

Enterobacter alginii

Vibrio cholerae

Bacillus subtilis

Edwardsiella

Salmon sperm DNA

Human placental DNA

E. coli

The 1291 bp DNA fragment did not hybridize to the genomic DNA of these pathogens (FIG.

7

).

VII. Hybridization of 1291 bp StuI-StuI DNA fragment with clinical isolates of

Mycobacterium tuberculosis

The Southern hybridization of 1291 bp DNA fragment with genomic DNA of clinical isolates of

Mycobacterium tuberculosis

should permit the detection of this 1291 bp DNA fragment in

Mycobacterium tuberculosis

and its use in epidemiology.

The genomic DNA from confirmed clinical isolates of

Mycobacterium tuberculosis

were isolated and digested with StuI restriction enzyme. The digests were electrophoresed, transferred onto nitrocellulose paper and hybridized with DIG-labeled denatured 1291 bp DNA fragment. The results with more than two hundred clinical isolates of

Mycobacterium tuberculosis

showed the presence of this fragment. The hybridization revealed several bands which confirmed the repetitive nature of the fragment. Several isotopes were identified, however all the isolates showed a strong band of 1291 bp (FIG.

8

).

VIII. Nucleotide sequence of 1291 bp StuI-StuI DNA fragment

The nucleotide sequence of 1291 bp DNA fragment was determined by dideoxy chain termination sequencing technique (Biggin, M. D. et al. 1983. Proceedings of the National Academy of Sciences, USA. 80:3963-3965). The products of the sequencing reactions were electrophoresed on 6% acrylamide/ 7M urea/ 0.5-2.5 X TBE gradient sequencing gels. The gels were dried under vacuum and exposed to Kodak XRP-1 film. The nucleotide sequences were determined independently for both strands of 1291 bp DNA fragment. Computer-aided analysis of the nucleotide sequence and deduced protein sequence was performed using databases and programs provided by the National Institute of Health, as well as the programs of Chou and Fasman and Hopp and Woods. (Chou, P. Y. and G. D. Fasman, Advances in Enzymol., 47:45-148, 1978; Hopp, T. P. and K. P. Woods, Proceedings of the National Academy of Sciences, USA, 78:3824-3828, 1981). The nucleotide sequence of the 1291 bp StuI-StuI region of 4.2 kb DNA fragment is represented in FIG.

9

. The nucleotide sequence of the DNA fragment shows several interesting features including the presence of repeat sequences and an IS like sequence with open reading frame. The nucleotide sequence of the DNA fragment of this invention along with IS and repeat sequences present within the sequence can be seen in FIG.

9

. In Table II, the presence of direct repeats in 1291 bp StuI-StuI DNA fragment is shown. Only 10 repeats of more than 9 nucleotide bases along with the sequence and their position have been shown. In Table III, the presence of hairpin loop site in IS like element present within 1291 bp StuI-StuI DNA fragment has been shown. The two inverted repeats are flanked with direct repeat GTT on either side.

TABLE II

The presence of Direct repeats in 1291 bp StuI-StuI DNA fragment

S.N.

Length(bp)

Sequence

Position

SEQ ID NO:

1.

26

CCG ATG TTG TTG TTG

588, 618

SEQ ID NO:3:

CCG GTG TTG GC

2.

10

TGA AGA AGC C

205, 1015, 1261

SEQ ID NO:4:

3.

10

GCC CGT GTT G

317, 332, 362

SEQ ID NO:5:

4.

10

TTG AGG ATG C

354, 384

SEQ ID NO:6:

5.

9

CCG GTG TTG

213, 273, 303, 603

6.

9

CCG ATG TTG

588, 618, 708, 738, 912

7.

9

GTT GAA GTC

158, 338, 653, 869

8.

9

GAA GAA GCC

206, 1016, 1166, 1262

9.

9

GTT GCC GGT

269, 518, 599, 629

10.

9

GCC GAC GTT

452, 677, 692, 812

TABLE III

Presence of hairpin loop site in IS like element present within 1291 bp Stul-StuI fragment

No.

Stem length (bp)

%

Loop length

Hairpin loop sequence (SEQ ID NO:7:)

1.

9

100

714

461 469 475

: : :

5′ TCCGGTGCCCGCGTT.......

:::::::::: :

3′ AGGCCACGGTTGTTA.......

: : :

1192 1184 1178

The inverted repeats are located at 461 to 469 (TCCGGTGCC) and at 1184 to 1192 (GGCACCGGA).

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 7

<210> SEQ ID NO: 1

<211> LENGTH: 1291

<212> TYPE: DNA

<213> ORGANISM: Mycobacterium tuberculosis

<400> SEQUENCE: 1

aggcctcggt gaccgtgatc atgttgccgc cgaaggtcat tacgttgtgt acgtcaatga 60

ccatctgctc gttgtttatg gggatgaatc gggagtggtg accgagagat cgatggcgaa 120

tctggccctg gttatcgccc gccaccaaga agccattgtt gaagtcgccc gtgtcgaaag 180

cgccggtatt gacgttgccg ggattgaaga agccggtgtt ggtgtcaccc gggttatagc 240

tgccggtatt ggtgtcaccc acgttgaagt tgccggtgtt ggtgttaccg acgttgaagc 300

cgccggtgtt gtagctgccc gtgttgtaga agcccgtgtt gaagtcgccg gcgttgagga 360

tgcccgtgtt gtagctgcca gcattgagga tgccggtatt gtcggtaccc gggttcccga 420

taccccagtt cccggtgccc gagtttgcga tgccgacgtt tccggtgccc gcgttgaaga 480

tgccaacgtt attggtgccc gaattgaaca ggccgctgtt gccggtgccc gagttccagc 540

cgctagcaat attgaagccc tgctggttgt cgccggacag cccgatgccg atgttgttgt 600

tgccggtgtt ggcgaacccg atgttgttgt tgccggtgtt ggcaaagcct tggttgaagt 660

cgcccgcgtt cccgaagccg acgttgtagt cgccgacgtt tccaaaaccg atgttgtaga 720

tcccgaggtt tccggatccg atgttgtagt ttcccaggct tccggaaccg acattgaata 780

ctccgatgtt tccactgccg atattgaagc tgccgacgtt gccgctgccc aagatgtttt 840

ggctgccgag gttgccgctg ccaaggatgt tgaagtcacc gacgtttccg ctgccgagaa 900

tgttgtaatt gccgatgttg gcgttgccga gaatgttcac gacgccccgg tttgccaggc 960

cgagattgaa gaccggtggg ccaccgaaaa atcccgacat gttgcttccg gtgttgaaga 1020

agcccgagat caaggccggc gttgtgatgg ccaccaggct catgttgaac aaacccgata 1080

cggtgttgcc cgagttgatc acgcccgata ccagcacgcc cgcgtttgcc aggccggagt 1140

taccgatggc ccccgacgaa gagtggaaga agccagaatt gttggcaccg gagttcagga 1200

agccggacgc gctaccggca ccgctgttga agaatcccga cgacggcgca ctggtcgagt 1260

tgaagaagcc gggctcccga aaatcaggcc t 1291

<210> SEQ ID NO: 2

<211> LENGTH: 430

<212> TYPE: PRT

<213> ORGANISM: Mycobacterium tuberculosis

<220> FEATURE:

<221> NAME/KEY: UNSURE

<222> LOCATION: (4)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (6)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (20)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (29)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (54)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (64)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (69)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (89)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (99)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (114)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (119)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (129)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (159)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (169)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (182)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (185)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (219)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (259)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (269)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (291)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (323)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (339)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (349)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (356)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (366)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (410)

<223> OTHER INFORMATION: amino acid has not been identified

<221> NAME/KEY: UNSURE

<222> LOCATION: (421)

<223> OTHER INFORMATION: amino acid has not been identified

<400> SEQUENCE: 2

Arg Pro Arg Xaa Pro Xaa Ser Cys Cys Arg Arg Arg Ser Leu Arg Cys

1 5 10 15

Val Arg Gln Xaa Pro Ser Ala Arg Cys Leu Trp Gly Xaa Ile Gly Ser

20 25 30

Gly Asp Arg Glu Ile Asp Gly Glu Ser Gly Pro Gly Tyr Arg Pro Pro

35 40 45

Pro Arg Ser His Cys Xaa Ser Arg Pro Cys Arg Lys Arg Arg Tyr Xaa

50 55 60

Arg Cys Arg Asp Xaa Arg Ser Arg Cys Trp Cys His Pro Gly Tyr Ser

65 70 75 80

Cys Arg Tyr Trp Cys His Pro Arg Xaa Ser Cys Arg Cys Trp Cys Tyr

85 90 95

Arg Arg Xaa Ser Arg Arg Cys Cys Ser Cys Pro Cys Cys Arg Ser Pro

100 105 110

Cys Xaa Ser Arg Arg Arg Xaa Gly Cys Pro Cys Cys Ser Cys Gln His

115 120 125

Xaa Gly Cys Arg Tyr Cys Arg Tyr Pro Gly Ser Arg Tyr Pro Ser Ser

130 135 140

Arg Cys Pro Ser Leu Arg Cys Arg Arg Phe Arg Cys Pro Arg Xaa Arg

145 150 155 160

Cys Gln Arg Tyr Trp Cys Pro Asn Xaa Thr Gly Arg Cys Cys Arg Cys

165 170 175

Pro Ser Ser Ser Arg Xaa Gln Tyr Xaa Ser Pro Ala Gly Cys Arg Arg

180 185 190

Thr Ala Arg Cys Arg Cys Cys Cys Cys Arg Cys Trp Arg Thr Arg Cys

195 200 205

Cys Cys Cys Arg Cys Trp Gln Ser Leu Gly Xaa Ser Arg Pro Arg Ser

210 215 220

Arg Ser Arg Arg Cys Ser Arg Arg Arg Phe Gln Asn Arg Cys Cys Arg

225 230 235 240

Ser Arg Gly Phe Arg Ile Arg Cys Cys Ser Phe Pro Gly Phe Arg Asn

245 250 255

Arg His Xaa Ile Leu Arg Cys Phe His Cys Arg Tyr Xaa Ser Cys Arg

260 265 270

Arg Cys Arg Cys Pro Arg Cys Phe Gly Cys Arg Gly Cys Arg Cys Gln

275 280 285

Gly Cys Xaa Ser His Arg Arg Phe Arg Cys Arg Glu Cys Cys Asn Cys

290 295 300

Arg Cys Trp Arg Cys Arg Glu Cys Ser Arg Arg Pro Gly Leu Pro Gly

305 310 315 320

Arg Asp Xaa Arg Pro Val Gly His Arg Lys Ile Pro Thr Cys Cys Phe

325 330 335

Arg Cys Xaa Arg Ser Pro Arg Ser Arg Pro Ala Leu Xaa Trp Pro Pro

340 345 350

Gly Ser Cys Xaa Thr Asn Pro Ile Arg Cys Cys Pro Ser Xaa Ser Arg

355 360 365

Pro Ile Pro Ala Arg Pro Arg Leu Pro Gly Arg Ser Tyr Arg Trp Pro

370 375 380

Pro Thr Lys Ser Gly Arg Ser Gln Asn Cys Trp His Arg Ser Ser Gly

385 390 395 400

Ser Arg Thr Arg Tyr Arg His Arg Cys Xaa Arg Ile Pro Thr Thr Ala

405 410 415

His Trp Ser Ser Xaa Arg Ser Arg Ala Pro Glu Asn Gln Ala

420 425 430

<210> SEQ ID NO: 3

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Mycobacterium tuberculosis

<400> SEQUENCE: 3

ccgatgttgt tgttgccggt gttggc 26

<210> SEQ ID NO: 4

<211> LENGTH: 10

<212> TYPE: DNA

<213> ORGANISM: Mycobacterium tuberculosis

<400> SEQUENCE: 4

tgaagaagcc 10

<210> SEQ ID NO: 5

<211> LENGTH: 10

<212> TYPE: DNA

<213> ORGANISM: Mycobacterium tuberculosis

<400> SEQUENCE: 5

gcccgtgttg 10

<210> SEQ ID NO: 6

<211> LENGTH: 10

<212> TYPE: DNA

<213> ORGANISM: Mycobacterium tuberculosis

<400> SEQUENCE: 6

ttgaggatgc 10

<210> SEQ ID NO: 7

<211> LENGTH: 714

<212> TYPE: DNA

<213> ORGANISM: Mycobacterium tuberculosis

<400> SEQUENCE: 7

cgcgttgaag atgccaacgt tattggtgcc cgaattgaac aggccgctgt tgccggtgcc 60

cgagttccag ccgctagcaa tattgaagcc ctgctggttg tcgccggaca gcccgatgcc 120

gatgttgttg ttgccggtgt tggcgaaccc gatgttgttg ttgccggtgt tggcaaagcc 180

ttggttgaag tcgcccgcgt tcccgaagcc gacgttgtag tcgccgacgt ttccaaaacc 240

gatgttgtag atcccgaggt ttccggatcc gatgttgtag tttcccaggc ttccggaacc 300

gacattgaat actccgatgt ttccactgcc gatattgaag ctgccgacgt tgccgctgcc 360

caagatgttt tggctgccga ggttgccgct gccaaggatg ttgaagtcac cgacgtttcc 420

gctgccgaga atgttgtaat tgccgatgtt ggcgttgccg agaatgttca cgacgccccg 480

gtttgccagg ccgagattga agaccggtgg gccaccgaaa aatcccgaca tgttgcttcc 540

ggtgttgaag aagcccgaga tcaaggccgg cgttgtgatg gccaccaggc tcatgttgaa 600

caaacccgat acggtgttgc ccgagttgat cacgcccgat accagcacgc ccgcgtttgc 660

caggccggag ttaccgatgc cccccgacga agagtggaag aagccagaat tgtt 714

Mycobacterium tuberculosis specific DNA fragment

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