Title of Invention

"A PROCESS FOR THE DESIGNING OF PRIMERS USEFUL FOR THE DETECTOPN OF MYCOBACTERIUM TUBERCULOSIS"

Abstract

This invention relates to a process for the designing of primers for the detection of mycobacterium tuberculosis comprising, Constructing a genomic library of M.tuberculosis using novel vector PGEM4Z cut with enzyme Hinc II and ligating at a temperature of 15-37°C to a number of Mycobacterium tuberculosis fragments cut with the restriction enzyme RSal, transferring the ligation mixture in E-coli HB 101, Screening the whole library by hybridization onto nitrocellulose membrane with P-32 labelled M. tuberculosis at a temperature of 50-75C, Further Characterization of the selected clones for specificity and sensitivity using slot blot hybridization with non Mycobacterial species DNA and atypical Mycobacterium DNA, Sequencing the cloned repeat element which leads to the designing of several primers for use in polymerase chain reaction.

Full Text

F1ELD OF INVENTION This invention relates to a process for the preparation of primers useful for the detection of Mycobacterium tuberculosis. PRIOR ART Tuberculosis is currently an important health problem throughout the world There seems to be an alarming resurgence in the number of tuberculosis cases due to the emergence of AIDS epideimic. The laboratory diagnosis of this condition has been hampered by the slow growth of the causative organism. Mycobacterium tuberculosis. At present microscopic examination is the most rapid diagnostic method available. More than 10,000 bacilli per milli litre of sputum are necessary to secure microscopic positivity. The limitation of this method is low sensitivity. Conventional culture takes 4 weeks to detect the bacilli and further 4 weeks to confirm sensitivity to drugs. Altogether it takes 8 weeks to start treatment for the patient. Culture through radiometric systems and biphasic culture may require two weeks to confirm diagnosis. Besides research in serology and structural components such as tuberculostearic acid is not widely used in laboratories in some cases due to high costs involved and in others due to lack of sensitivity. Detection of M.tuberculosis by the polymerase chain reaction (PCR) has been found useful in pulmonary as well as extra-pulmonary tuberculosis cases like tuberculosis meningitis and tuberculosis pleurisy. Eventhough PCR appeared to be a solution for rapid diagnosis of tuberculosis, the best primers and the protocol are yet to be determined. Several procedures have been described to detect M.tuberculosis genome-the target sequence being the main difference. Notable primers designed so far are constructed from sequences of mycobacterial 65 kD antigen, 38 kD antigen, DNA J gene and insertion sequence IS6110. Among these primers the primer pair which amplify 123 basepair fragment from the insertion element IS6110 has been most widely used. But earlier observation has shown that 40% of the strains from Madras have only a single copy of 1S6110 and 4-5% have no copy of IS6110. OBJECTS OF THE INVENTION An object of this invention is to propose a novel set of primers for M.tuberculosis DNA amplification and to thereby improve the efficacy of PCR. Another object of this invention is to provide a process for the preparation of primers useful for the detection of M.tuberculosis DNA amplification for improving the efficacy of PCR by designing a novel set of primers. Still another object of this invention is to evaluate the potential of these primers in amplifying M.tuberculosis DNA from clinical specimens in comparison with IS6110. According to this invention there is provided a process for the preparation of primers useful for the detection of Mycobacterium tubeculosis comprising constructing a genomic library of M.tuberculosis, selecting specific clones being sensitive for detecting M.tuberculosis and then subjecting said clone to the step of sequencing and characterisation so as to obtain the primers. In accordance with this invention a genomic library of M.tuberculosis has been first constructed by using a novel vector and enzymes. After screening the whole library, 10 clones have been selected for checking its specificity and sensitivity in detecting M.tuberculosis by hybridisation. One clone has been found to be specific for M.tuberculosis complex. The mycobacterial fragment of this clone, pTRC4 is fully sequenced and characterized. For this purpose 4 sets of primers have been designed and tested by polymerise chain reaction. Among these only one set of primers, 844 and 1016 bp (base pet) in TRC4 sequence 2.123 bp is consistent in amplifying 173 bp product from clinical specimens infected with M.tuberculosis and a new pair of primers priTRCI and priTRC2 have been designed from this sequence. The process for the preparation of primers useful for the detection of Mico-bacterium tuberculosis according to a preferred embodiment is herein described and illustrated in the detail in the following description. According to the process of this invention a novel vector such as pGEM4Z is 'eut' with a novel enzyme such as Hinc II and is ligated at a temperature of 15-37°C to enumerable number of mico bacterial fragments cut with the restriction enzymes called R sa I. The ligation mixture is then transferred in E-coli HB 101. More that 300 colonies were obtained on the ampricilin containing vacto-agar plates. These clones/colonies were transferred onto nitrocellulose membrane and hybridised with P-32 labelled M.tuberculosis at a temperature of 50-75°C. The clones which give strong signal are aligned with the plate and clones are purified. All these strongli lighting clones are further characterized for specificity and sensitivity using slot-blot hybridization with non-micro bacterial species DNA and atyfrical mico-bacterial DNA. In general the polymerase chain reaction process is performed by using specimen that may contain the DNA sequence of interest is heated to denature double standard DNA in the presence of cocktail mix containing all the 4 Nucleotide triphosphates (NTP), PCR buffer, Tag polymerase enzyme and primers I and II. Then specific oligoneucleotides 'primes' bind to unique DNA sequence of interest. Heat stable DNA polymerase extends the primers to create a complete and complementary strand of DNA. These three steps are repeated sequentially 25 to 40 times thereby creating millions of copies of the target DNA sequence, and the amplified sequences can be detected easily by gel electrophoresis. In accordance with this invention, a DNA probe has been developed which is present as a repeat element in all of the 170 clinical isolates tested by restriction fragment length polymorphism. Hence this is a better target for polymerase chain reaction to detect M.tuberculosis from clinical samples. This cloned repeat element is then sequenced and several primers are designed for use in the polymerase chain reaction. A new pair of primers priTRCl and priTRC2 which will amplify M. tuberculosis DNA by the above process has been identified. We Claim 1. A process for the designing on of primers for the detection of mycobacterium tuberculosis comprising: - i) Constructing a genomic library of M.tuberculosis using novel vector PGEM4Z cut with enzyme Hinc II and ligating at a temperature of 15-37°C to a number of Mycobacterium tuberculosis fragments cut with the restriction enzyme RSal, transferring the ligation mixture in E-coli HB 101 ii) Screening the whole library by hybridization onto nitrocellulose membrane with P-32 labelled M. tuberculosis at a temperature of 50-75C iii) Further Characterization of the selected clones for specificity and sensitivity using slot blot hybridization with non Mycobacterial species DNA and atypical Mycobacterium DNA iv) Sequencing the cloned repeat element which leads to the designing of several primers for use in polymerase chain reaction. 2. A process for the preparation of primers as claimed in claim 1, wherein only one set of primers, 844 and 1016 bp (base pair) in the mycobacterial fragment of the clone TRC4 sequence 2.123 bp is consistent in amplifying 173,bp product from clinical specimens infected with M.tuberculosis. 3. A process for the preparation of primers as claimed in claim 1, wherein a new pair of primers pri TRC1 and pri TRC2 have been designed from this sequence. 4. A process for the preparation of primers useful for the detection of Mycobacterium tuberculosis a's illustrated and described herein.

Documents:

1172-DEL-2003-Abstract-(19-02-2009).pdf

1172-del-2003-abstract.pdf

1172-DEL-2003-Claims-(19-02-2009).pdf

1172-del-2003-claims.pdf

1172-DEL-2003-Correspondence-Others-(19-02-2009).pdf

1172-del-2003-correspondence-others.pdf

1172-del-2003-correspondence-po.pdf

1172-del-2003-description (complete).pdf

1172-DEL-2003-Form-1-(19-02-2009).pdf

1172-del-2003-form-1.pdf

1172-del-2003-form-18.pdf

1172-DEL-2003-Form-2-(19-02-2009).pdf

1172-del-2003-form-2.pdf

1172-DEL-2003-Form-3-(19-02-2009).pdf

1172-DEL-2003-Others-Document-(19-02-2009).pdf