Title of Invention

"OLIGONUCLEOTIDE PRIMER USEFUL FOR DETECTION OF MYCOBACTERIUM SPECIES"

Abstract Present invention deals with a set of oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEID NO 7 and SEQ ID NO 8 useful for the detection of Mycobacterium tuberculosis.
Full Text Field of invention;
The present invention relates to a set of oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 useful for the synthesis of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 1 and its parts thereof having SEQ ID 2, SEQ ID NO 3, SEQ ID 4.
More particularly, it relates to a Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 useful for rapid diagnosis of Mycobacterium tuberculosis infection in clinical samples and to identifing clinical isolates of Mycobacterium tuberculosis.
Background and Prior art of the invention;
Present invention provides a Mycobacterium tuberculosis specific set of oligonucleotide primers, the DNA fragments contained within the primers, the nucleotide sequence of the DNA fragments and primers, 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 fragments 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. Resurgence of tuberculosis (TB) is a major cause of infectious mortality. According to a WHO report, the number of people dying of TB in 1995 was larger than in any other year in the history. Reference may be made to Moran, N.
1996. WHO Issues Another Gloomy Report. Nature Medicine 4:377 and Joint International Union Against Tuberculosis and World Health Organization Study Group. Tubercle 63:157-169, 1982, wherein 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.
The causative agent of tuberculosis is a gram positive acid fast bacterium Mycobacterium tuberculosis or Mycobacterium bovis, which are the tubercle bacilli of the family of Mycobacteriaceae. Mycobacterium 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. Infection with Mycobacterium tuberculosis is always transmitted from host to host.
Hence the essential components of any tuberculosis control program include diagnosis, chemotherapy and immunoprophylaxis. 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; a test which can diagnose presence of a few bacilli of Mycobacterium tuberculosis in a relatively short time. A number of methods have been suggested for identification of Mycobacterium tuberculosis present in the clinical samples such as sputum, CSF, blood, pleural fluid etc. Reference may be made to 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 DC, pp. 85-110 wherein microscopic examination of acid fast bacilli present in the clinical samples is the most
commonly used method in the diagnostic labs. However there are several drawbacks and the most important being unless the number of bacilli in the sample is sufficiently high (more than 10 4/ ml), this method is not reliable, moreover it does not identify Mycobacterium tuberculosis specifically. In the other method, the presence of Mycobacterium tuberculosis in clinical samples is established by growth of bacilli in specific media followed by 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 time is required in biochemical testing before identification can be made. It has been reported that not all Mycobacterium tuberculosis positive samples become culture positive. The other drawback is that virtually very few bacilli are present in many clinical sample such as CSF, therefore culture positivity in CSF samples is very rare.
Methods based on antigen and antibody detection in body fluids have been developed as reagents for diagnosis. Reference may be made to Young, D. B. and Mehlert, A. 1989, Serology of Mycobacteria: Characterization of Antigens Recognized by Monoclonal Antibodies. Rev. Infec. Dis. 12: S431-S435. Although these methods are convenient but are not reliable because most of the antibody/ antigen reagents used are not specific to tuberculosis and they cross react with other mycobacteria,. Hence specific diagnosis of tuberculosis by antigen and antibody reagents will only be reliable if they are specific to Mycobacterium tuberculosis.
More recently nucleic acid probes (sequence specific DNA fragments) have been developed as reagents for rapid diagnosis and monitoring of the epidemiology of tuberculosis. Reference may be made to 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. Reference may be made to 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 . Reference may be made to 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. However IS6110 has some drawbacks. Several Mycobacterium tuberculosis strains with one copy or no copy of IS6110 have been reported. Reference may be made to 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.
In this quest for sequence specific DNA probe for specific and unambiguous rapid detection of Mycobacterium tuberculosis, a unique 1291 bp DNA fragment of Mycobacterium tuberculosis of SEQ ID NO 1 is identified. This DNA fragment was cloned and characterized by determination of nucleotide sequence of 1291 bp identification of IS like sequence and various repeat sequences (Table I and Table II). Reference may be made to US Patent " A Mycobacterium tuberculosis specific DNA fragment, Ranjana Srivastava, Deepak Kumar and Brahm Shanker Srivastava. US 6,114,514 " where we have described the identification of these fragments, method of producing such fragments, use of this fragment as DNA probe by DNA:DNA hybridization in southern hybridization assay. This is a very useful probe for identification of the presence of Mycobacterium tuberculosis in clinical samples, clinical isolates of Mycobacterium tuberculosis and epidemiology to detect point source outbreaks, transmission and as a marker for strain typing. It relates to production and identification of a DNA fragment which contains 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.
TABLE I
The presence of Direct repeats in 1291 bp Stul-Stul DNA fragment
S.N. Length Sequence Position
(Table Removed)
TABLE II
Presence of hairpin loop site in IS like element present within 1291 bp Stul-Stul fragment (SEQ ID NO 1)
No. Stem length Loop length Hairpin loop sequence
(Table Removed)
The inverted repeats are located at 461 to 469 (TCCGGTGCC) and at 1184 to 1192 (GGCACCGGA) of 1291 bp fragment of SEQ ID NO 1. The two inverted repeats are flanked by GTT on either side.
US 6114514 Present invention
It deals with a 1291 bp • It deals with a 4 set of
Mycobacterium tuberculosis oligonucleotide primers to amplify
specific DNA fragment which is the DNA fragment of 1291bp, and
useful for rapid diagnosis of other DNA fragment of (777bp,

Mycobacterium tuberculosis 181bp and 131bp which are a part
infection in clinical samples and of the said 1291 bp fragment
useful for the identification of disclosed in US 6114514)
clinical isolates of Mycobacterium
tuberculosis.
• 1291bp fragment is produced by the • 1291 bp fragment is generated by
screening of genomic library which PCR amplification using set of
is a time consuming and complex oligonucleotide primers. It is a
method simple way to produce such
fragment
Objects of the invention:
The main object of the present invention is to provide a set of oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 useful for the synthesis of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 1 and its parts thereof having SEQ ID 2, SEQ ID NO 3, SEQ ID 4.
Another object of the present invention is to provide a Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 useful for rapid diagnosis of Mycobacterium tuberculosis infection in clinical samples and to identifying clinical isolates of Mycobacterium tuberculosis.
Further another object of the present invention is to provide a set of oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 for the detection of polymorphism in clinical isolates of Mycobacterium tuberculosis.
Summary of the invention:
Present invention deals with a set of oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8 for synthesis of Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 1 and its parts thereof It also relates the Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4.
Brief description of figures:
Figure 1 represents the nucleotide sequence of 1291 bp DNA fragment of Mycobacterium tuberculosis of SEQ ID NO 1 amplified by oligonucleotide primers of SEQ ID NO 5
Figure 2 represents the nucleotide sequence of the oligonucleotide primers of SEQ ID NO 5
Figure 3 represents the nucleotide sequence of 777 bp DNA fragment of Mycobacterium tuberculosis of SEQ ID NO 2 amplified by oligonucleotide primers of SEQ ID NO. 6.
Figure 4 represents nucleotide sequence of the oligonucleotide primers of SEQ ID NO 6.
Figure 5 represents nucleotide sequence of the 181 bp DNA fragment of Mycobacterium tuberculosis of SEQ ID NO. 3 amplified by oligonucleotide primers of SEQ ID NO 7.
Figure 6 represents nucleotide sequence of the oligonucleotide primers of SEQ ID NO. 7
Figure 7 represents nucleotide sequence of the 131 bp DNA fragment of Mycobacterium tuberculosis of SEQ ID NO. 4 amplified by oligonucleotide primersofSEQIDN0 8.
Figure 8 represents nucleotide sequence of the oligonucleotide primers of SEQ ID NO. 8.
Figure 9 agrose gel electrophoresis of PCR amplified product wherein Lane 1 represents 777bp DNA fragment; Lane 2 represents 100 bp ladder plus DNA marker (MBI Fermentas); Lane 3 represents 181 bp DNA fragment and Lane 4 represents 131 bp DNA fragment.
Detailed description of the invention;
Accordingly, the present invention provides a set of oligonucleotide primers useful for the synthesis of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 1 and its parts thereof having SEQ ID 2, SEQ ID NO 3, SEQ ID 4; wherein the said primers comprising:
a) SEQ ID NO 5
Forward Primer: 5'-aggcctcggtgaccgtg-3' Reverse Primer: 5'-aggcctgattttcgggagc-3'
b) SEQ ID NO 6
Forward Primer: 5'tgttgccgccgaaggtcatta-3' Reverse Primer: 5'-gcagtggaaacatcggagtat-3'
c) SEQ ID NO 7
Forward Primer: 5'-aaggccggcgttgtgatgg-3' Reverse Primer: 5'-gcgcgtccggcttcctga-3'
d) SEQ ID NO 8
Forward Primer: 5'- tgttgccgccgaaggtcatta-3'
Reverse Primer: 5'-cttcttggtggcgggcgataa-3'
In an embodiment of the present invention the said primers are derived from 1291
bp sequence of Mycobacterium tuberculosis.
In another embodiment of the present invention, the said primers of SEQ ID NO 5 is useful for the amplification of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 1.
In further another embodiment of the present invention, the said primers of SEQ ID NO 6 is useful for the amplification of 777 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 2 from 22nd -798th nucleotide base pairs of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 1.
In yet another embodiment of the present invention, the said primers of SEQ ID NO 7 is useful for the amplification of 181 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 3 from 1032nd - 1213th nucleotide base pairs of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 1.
In still another embodiment of the present invention, the said primers of SEQ ID NO 8 is useful for the amplification of 131 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 4 from 22nd -152nd nucleotide base pairs of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 1 and 1st -131st nucleotide base pairs of 777 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 2. Further, the present invention also provides a Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 2 comprising 777 from 22nd - 798th nucleotide base pairs of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 1.
Present invention also provides a Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 3 comprising 181 nucleotide base pairs 3 from 1032nd -1213th nucleotide base pairs of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 1.
Further, the present invention also provides a Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 4 comprising 131 nucleotide base pairs from 22nd -152nd nucleotide base pairs of 1291 nucleotide base pairs Mycobacterium tuberculosis specific DNA fragment of SEQ ID 1 and 1st -131st nucleotide base pairs of 777 nucleotide base pairs* Mycobacterium tuberculosis specific DNA fragment of SEQ ID2.
In an embodiment of the present invention, the Mycobacterium tuberculosis specific DNA fragment of SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 having repetitive sequences to detect Mycobacterium tuberculosis.
Further, the present invention also provides a kit useful for the rapid diagnosis of
Mycobacterium tuberculosis infection in clinical samples wherein the sai kit
comprises of:
i) a set of oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO
7, and SEQ ID NO 8 or Mycobacterium tuberculosis specific DNA fragments
of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 and; ii) other additives selected from restriction enzymes, reverse transcriptases,
polymerases, ligases, linkers, nucleoside triphosphates as substrate, suitable
buffers, labels, and/or other accessories Mycobacterium tuberculosis specific four sets of oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 & SEQ ID NO 8 which respectively amplify DNA fragments of 1291, 777, 181 and 131bp, use of such oligonucleotide primers and DNA fragments, 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 fragments and the set of oligonucleotide primers to diagnose tuberculosis and strains of Mycobacterium tuberculosis, the set of oligonucleotide primers are derived from 1291 bp sequence of Mycobacterium tuberculosis and these four sets of primers amplify DNA of 1291, 777, 181 and 131 bp DNA fragments which contain several repeat DNA sequences and can be used as DNA probe for identification of Mycobacterium tuberculosis. To produce a sequence specific DNA fragment of Mycobacterium tuberculosis comprising of 1291 bp DNA fragment described above, a genomic library of Mycobacterium tuberculosis DNA was constructed in phage lambda gtl 1 vector. The library of genomic fragments was screened with genomic DNA of Mycobacterium tuberculosis as a probe to screen and select DNA fragments which rapidly hybridize to Mycobacterium tuberculosis genomic DNA, thus providing 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. It is a very complex and time consuming method for synthesis of 1291bp Mycobacterium tuberculosis specific DNA fragment. To simplify it concerned set of oligonucleotide primers has been designed. These oligonucleotide primers of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 & SEQ ID NO 8 derived from 1291 bp sequence of Mycobacterium tuberculosis and these four sets of primers amplify DNA of 1291, 777, 181 and 131 bp DNA fragments from Mycobacterium tuberculosis infection in clinical samples which may include sputum, cerebrospinal fluid, pleural fluid, urine, ascitic fluid, gastric samples, bronchial lavage, pericardial fluid, pus, blood or lymph node aspirate .
The nucleotide sequence of the specific DNA fragment has been determined and DNA primers have been 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.
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention.
EXAMPLE 1
From the nucleotide sequence of 1291 bp DNA fragment of Mycobacterium tuberculosis of SEQ ID NO 1, four sets of oligonucleotide primers SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO. 7 and SEQ ID NO.8 were selected and custom synthesized. These four sets of primers may be used to amplify DNA fragments from Mycobacterium tuberculosis genome. This amplification may be done by Polymerase chain reaction which may consist of specified primers, four deoxyribonucleotides, buffer, target DNA and Taq DNA polymerase. The target DNA may consist of DNA from the culture of Mycobacterium tuberculosis or clinical specimens containing Mycobacterium tuberculosis. For amplification any DNA polymerase used for PCR may be used. The amplification may involve denaturation of DNA at 95° C followed by annealing and extension at 72°C. The annealing temperature may vary from 50 - 65°C. (Persing, D.H., Smith, T.F. , Tenover, F.C. White, T.J.)1993, American Soc. Microbiology, Washington DC. The amplified product may be checked by agarose gel electrophoresis subsequently by DNA hybridization with DIG-labeled 1291 bp DNA fragment. (Sambrook, H., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning, a laboratory manual, 2nd ed. CSH, NY.) The amplification of M. tuberculosis genomic DNA with oligonucleotide primer of SEQ ID NO 5 resulted in amplification of 1291 bp fragment of SEQ ID NO. 1, oligonucleotide primer of SEQ ID NO 8 resulted in amplification of 131 bp DNA fragment Of SEQ ID NO. 4, the third set of oligonucleotide primer of SEQ ID NO 6, amplified 777 bp DNA fragment of SEQ ID NO 2 while the fourth set of oligonucleotide primer SEQ ID NO 7, amplified 181 bp DNA fragment of SEQ ID NO 3. The nucleotide sequence was determined by Dideoxy Chain Termination sequencing technique. ( Beggin, M. D. et al. 1983. Proceeding of National academy of Sciences, USA, 80: 3963-65.) The genomic DNA from different mycobacteria and other pathogens were isolated and the three sets of primers were used to amplify their genomic DNA by setting a Polymerase chain reaction. The four sets of primers amplified
genomic DNA of specific sizes of fragments of 1291, 131, 777 and 181 bp from Mycobacterium tuberculosis and not from other mycobacterial strains including M. smegmatis, M. phlei, M.fortuitum, M. chelonae, M.flavescens, M. triviale, M. duvali, M. marinum, M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, M. aurum, M. microti, M. szulgai and several other non mycobacterial strains listed as 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.
EXAMPLE 2
The four amplified M. tuberculosis DNA fragments of 1291 bp of SEQ ID NO. 1, 131 bp of SEQ ID NO. 4, 777 bp of SEQ ID NO. 2, 181 bp of SEQ ID NO 3 hybridized to M. tuberculosis DNA but not to the genomic DNA from other mycobacterial and non mycobacterial strains including M. smegmatis, M. phlei, M. fortuitum, M. smegmatis, M. chelonae , M. flavescens, M. chelonae, M. triviale, M. duvali, M. marinum, M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, M. aurum, M. microti, M. szulgai and several other non mycobacterial strains listed as 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 . Genomic DNA from 300 clinical isolates of Mycobacterium tuberculosis were isolated . The four sets of primers amplified genomic DNA of specific sizes of fragments of 1291, 131, 777 and 181 bp as from M. tuberculosis H37Rv. Hence the DNA fragments of 1291, 131, 777 and 181 bp are present in all clinical isolates of Mycobacterium tuberculosis and can be reliably amplified using three sets of primers described in invention. Thirty two out of 300 clinical isolates of Mycobacterium tuberculosis lacked IS6110.
Advantages;
The main advantages of the present invention are:
1. It provides an easy and simple method for the production of 1291 bp Mycobacterium tuberculosis specific fragment which is useful for rapid diagnosis of Mycobacterium tuberculosis infection in clinical isolates.
2. It provides an easy, simple and time consuming method for the production of 1291 bp Mycobacterium tuberculosis specific fragment which is useful for identification of clinical isolates of Mycobacterium tuberculosis.








WE CLAIM:
1. A set of oligonucleotide primer useful for detection of Mycobacterium species wherein
the said primers are selected from the group comprising:
a) SEQ ID NO 5
Forward Primer: 5'-aggcctcggtgaccgtg-3' Reverse Primer: 5'-aggcctgattttcgggagc-3'
b) SEQ ID NO 6
Forward Primer: 5'tgttgccgccgaaggtcatta-3' Reverse Primer: 5'-gcagtggaaacatcggagtat-3'
c) SEQ ID NO 7
Forward Primer: 5'-aaggccggcgttgtgatgg-3' Reverse Primer: 5'-gcgcgtccggcttcctga-3'
d) SEQ ID NO 8
Forward Primer: 5'- tgttgccgccgaaggtcatta-3' Reverse Primer: 5'-cttcttggtggcgggcgataa-3'
2. The oligonucleotide primer useful for detection of Mycobacterium species as claimed
in claim 1 wherein the fragment amplified is selected from the group consisting of Seq ID
no. 1, SEQ ID 2, SEQ ID NO 3, and SEQ ID 4.
3. A set of oligonucleotide primers, useful for identifying Mycobacterium tuberculosis substantially as herein described with reference to the examples and figures accompanying this specification.

Documents:

1760-DEL-2004-Abstract-(20-10-2010).pdf

1760-del-2004-abstract.pdf

1760-DEL-2004-Claims-(20-10-2010).pdf

1760-del-2004-claims.pdf

1760-DEL-2004-Correspondence-Others-(20-10-2010).pdf

1760-del-2004-correspondence-others.pdf

1760-del-2004-description (complete).pdf

1760-del-2004-description (provisional).pdf

1760-DEL-2004-Drawings-(20-10-2010).pdf

1760-del-2004-drawings.pdf

1760-DEL-2004-Form-1-(20-10-2010).pdf

1760-del-2004-form-1.pdf

1760-del-2004-form-18.pdf

1760-DEL-2004-Form-2-(20-10-2010).pdf

1760-del-2004-form-2.pdf

1760-del-2004-form-3.pdf

1760-del-2004-form-5.pdf


Patent Number 244289
Indian Patent Application Number 1760/DEL/2004
PG Journal Number 49/2010
Publication Date 03-Dec-2010
Grant Date 29-Nov-2010
Date of Filing 17-Sep-2004
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 RANJANA SRIVASTAVA CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, LUCKNOW-226 001, U.P. INDIA.
2 DEEPAK KUMAR CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, LUCKNOW-226 001, U.P. INDIA.
3 BRAHM SHANKER SRIVASTAVA CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, LUCKNOW-226 001, U.P. INDIA.
PCT International Classification Number C12N 15/11
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA