Title of Invention | "PRIMERS AND METHOD FOR IDENTIFICATION OF PATHOGENIC MYCOBACTERIA" |
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Abstract | The present invention relates to the rapid identification of the Pathogenic mycobacteria. More particularly, it relates to the development of new primers and a rapid method to identify the mycobacterial isolates at species level by gene amplification restriction analysis. This study was undertaken to develop a new rapid method to identify the mycobacterial isolates at species level by gene amplification restriction analysis using primers encoding 16S-23S rDNA spacer region and flanking parts of the 16S as well as 23S rDNA. This system accurately identifies clinically relevant Mycobacteria. This assay allows better and easier differentiation on gels as it targets a larger region than ITS sequences particularly between the 16S 23S rRNA genes, as the fragments generated from amplicons by this assay are bigger which can be easily separated and analyzed. |
Full Text | Title Novel Primers for a PCR-RFLP assay for identification of pathogenic Mycobacteria Field of the invention: The present invention relates to the identification of the pathogenic mycobateria. More particularly, it relates to the development of new primers and a rapid method to identify the mycobacterial isolates at species level by gene amplification restriction analysis. Background and prior art: Identification of a clinical isolate is primarily based on culture characteristics and biochemical tests. These conventional tests need several weeks and often inconclusive. Later several important PCR-RFLP assays targetting different gene regions are available for identification of mycobacteria. Telenti et al in 1993, (J Clin Microbiol 31 : 175-178) relates to hsp 65 gene regions of amplicon size of 439bp, but there was no clear differentiation between M.tuberculosis and M.fortuitum. Kim et al 2001 . (J Clin Microbiol 39 : 2102-2109) reveals rpo B gene region with the amplicon size 342 bp, but it differentiates a limited number of species by use of different cn/ymes (Mae III enzyme differentiates slow and rapid growing Mycobacteria, enzyme Hind 11 can differentiate M. tuberculosis complex from other mycobacteria, subspecies of M.kansasii and M.gastri can be differentiated by different pair of enzymes Mva I, Ace 11). Prabhakar et al 2004, (J Clin Microbiol 42 : 2724-2732) teaches gene region hup B of amplicon size 318 and 291bp, can only distinguish members of MTB complex from other mycobacterial species as well as for differentiating between members of MTB complex namely M. tuberculosis and M.bovis. but it can not identify other mycobacteria. Kasai et al 2000. (J Clin Microbiol 38 : 301 - 308) relates to gyr B gene region of 1257bp amplicon size, which differentiates only slowly growing mycobacteria. Dobner et al 1996, (J Clin Microbiol 34 : 866 - 869) reveals analysis of promoter of rRNA genome of amplicon size 0.3 to 0.4kb, with which only limited number of species can be differentiated (M.tuberculosis, M.gordonae, M.xenopi and M.terrae, M.avium M. intraceilulcire, M.srnegmalis). Vannechoutte et al 1993 (J Clin Microbiol 31 : 2061 - 2065) has used bacterial specific primers with amplicon size of 1500 bp and is not mycobacterial specific. Sansila et al 1998, (J din Microbiol 36 : 2399-2403) reveals (Internal transcribed spacer) ITS with amplicon size of 380bp, restricted to identify limited number of species, and it also needs hybridization. The present invention is related to mycobacterial specific primers targeting 3' of 16S rDNA (Position 468 to 492 of the M. tuberculosis 16S rRNA), the 16S - 23S rDNA spacer regions and 5' of the 23S rDNA (Position 477 to 457 of the M.tuberculosis 23S rRNA). Thus the target sequences are different. This assay allows better and easier differentiation on gels as it targets a larger region than targeting ITS sequences. ITS present in prokaryotic genomes has variable sequence. Roth et al 2000 (J Clin Microbiol 38 : 1094-1104) discloses the 200bp to 330 bp size amplicons, but all restriction fragment sizes of patterns shown for slowly growing mycobacteria and M.gordoncie and M.kansasii exhibits minor differences of 3 to 9 bp by using Hae 111 restriction enzyme. Khan et al 2005 (J Clin Microbiol 43 : 4466-4472) also reports observation about the primers of amplicon si/,e 215 to 296bp, where only for the differentiation of M.chelonae complex. Deepa et al 2005 (Indian J Med Res 121: 694-700) relates to 370bp to 450bp size amplicons. with the potential approach for identification and differentiation of especially slow growing mycobacterial species. Accurate identification of mycobacteria at species level is very important for patient management. The development of DNA probes for the identification of mycobacteria has provided rapid and specific approach, but it suffers from its high cost and probes are available for limited number of spaces or complex. All of these assays have been found to be easy and rapid in generating of RFLP patterns but their success in accurately identifying species is yet to be achieved. The patterns also become difficult to discriminate in case of small size of amplicons generated in most of these assays. In view of the above, there is a need to develop cost effective method, which can identify & differentiate all mycobacterial species with accuracy. An effort is made in present invention, to overcome all the above said drawbacks of existing method. The method of the present invention discloses specific primers targeting 16S-23S rDNA spacer region and flanking parts of the 16S as well as 23S rDNA that amplified a larger fragment (1.8 kb) compared to other references described above thereby allowing better differentiation oi'all mycobacterial species. Objective of the invention: Our main objective is to develop a cost effective, rapid method to differentiate mycobacterial isolates at species level with accuracy. Another objective of the present invention is to develop primers useful in amplifying fragments encoding 16S-23S r DNA spacer region and the flanking parts of 16S and 23S rDNA as well. Yet another objective of the present invention is amplification of fragment encoding 16S- 23S rDNA spacer region and Hanking parts of the 16S as well as 23S rDNA. Another objective of the present invention is to obtain a distinct gene amplification restriction analysis patterns by restriction of amplicons with three distinct restriction endonucleases (Hha I, Hinf 1 and Rsa I) to differentiate various mycobacterial species. Statement of the invention: The present relates to primers for identification of pathogenic Mycobacterium comprising the sequences SEQ ID NO 1 and SEQ ID NO 2 and a rapid RFLP assay method for identification of pathogenic Mycobacterium at species level by using novel primers of SEQ ID NO 1 and SHQ ID NO 2 comprises of: (a) obtaining isolated DNA from clinical isolates of mycobacterium species; (b) obtaining isolated DNA from known cultures of mycobacteria; (c) amplifying the DNA molecules of step (a) and (b) with forward and reverse primers represented by SEQ ID NO 1 and SEQ ID NO 2; (d) treating the resulting amplified nucleic acid molecule with restriction enzymes such as herein described to produce nucleic acid restriction fragments; and (e) analyzing the restriction fragments so produced using restriction fragment length polymorphism analysis so as to detect the species. Brief description of the drawings: Fig:l Diagrammatic representation of M. tuberculosis oligonucleotide primers targeting mycobacterial specific regions present at the 3' of 16S rDNA, the 16S - 23S rDNA spacer regions and 5" ol'the 23S rDNA of mycobacteria Fig: 2 Gene amplification restriction analysis of amplicons from different mycobacterial isolates digested with Hha 1 Fig 3. Gene amplification restriction analysis of amplicons from different mycobacterial isolates digested with Rsa 1 Fig 4 . Gene amplification restriction analysis of amplicons from different mycobacterial isolates digested with Hinf I Fig 5a. Nucleotide sequence of the 16S rRNA gene of isolate identified as M.lerrae by rRNA sequencing Fig 6a. Nucleotide sequence of the 16S rRNA gene of isolate identified as M.avium by rRNA sequencing Fig 7a. Nucleotide sequence of the 16S rRNA gene of isolate identified as M.fortuitum 5b* Confirmation of mycobacterial isolate as M.terrae by BLAST analysis 6b* Confirmation of mycobacterial isolate as M.avium by BLAST analysis 7b* Confirmation of mycobacterial isolate as M.fortuitum by BLAST analysis *B1.AST ANALYSIS Detailed description of the invention: Designing of primers: Oligonucleotide primers were designed targeting mycobacterial specific regions present at the 3' of 16S rDNA, the 16S - 23S rDNA spacer regions and 5" oi'the 23S rDNA oi mycobacteria. The sequences of the primers designed are SKQ ID NO 1: 5' - GAT, TGA, CGG, TAG, GTG, GAG, AAG, AAG > 3' (Position 468 to 492 of the M tuberculosis I6S rRNA) and SI-Q ID NO 2: 5' >CAC, GGG, C'CC, GCT, GCT, ACT, CG (Position 477 to 457 oi'the M.tuberculosis 23S rRNA). Mycobacterial reference strains and isolates: Reference strains and biochemically characterized clinical isolates of M.tuberculosis, M.bovis. M.avium, M.intracellulare, M.scrofulaceum, M.flavescens, M.gordonae, M.kansasii, M.marinum, M.terrae, M.fortuitum, M.chelonae and M.smegmatis obtained from Mycobacterial Repository maintained at National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Indian Council of Medical Research, Dr. M. Miyazaki Marg, Taj Ganj, Agra-1. U.P. , India were investigated. Sample preparation: DNAs were isolated by a physiochemical procedure of freeze boiling and treatment with lyso/yme and proteinase K by a method as described earlier (van Embden et al 1993), Enzymatic amplification of the rDNA: The gene amplification was performed in reaction mixtures of 50 uJ containing 50 mM KCI, 16 mM Iris HC1 (pll-9.0 at 25°C), 1.5 mM MgChand 0.1% Triton X-100, 200 uM of each of the four deoxynucleoside triphosphate (dNTPs) ,0.6 uM of each of primer and 2.5 units of Taq DNA polymerase. The reaction mixture was divided in 48 ul / reaction tubes, subsequently 2ul of isolated DNA samples were added to the each reaction tubes. Amplification was performed in a thermal cycler (M.J. Research Inc., U.S.A./ Applied Biosystems, U.S.A.) 35 cycles of amplification were carried out after initial denaturation for 2 min at 94°C, denaturalion at 94°C for 1 min, annealing at 59°C for 1 min, extension at 72°C for 2 min. Finally a 10 min extension period at 72°C was carried out. A reaction tube containing 2ul of distilled water was used to control for possible contamination and we have also tested the PCR condition by adding various concentrations of Tetramethylammonium Chloride (TMAC), Dimethlyl Sulfoxide (DMSO) and glycerol to reduce nonspecific amplification. The presence of specific PCR products were analysed by 1.5% agarose gel containing 50ng / ml ethidium bromide in TBE running buffer (89 mM Tris, 89 mM boric acid, 2 mM KDTA, pH- 8.0) and electrophoresis for 60 min at 2.5 V/cm. The length of the amplicons generated was about 1.8 kilo base pairs. Restriction analysis: Three restriction en/ymes (5 U each), Hha I, Hinf I, Rsa 1 were used and restriction was carried out for 2 hours at 37 °C in 30 ul volumes in shaking waterwath. Reaction was stopped by the addition of 6 ul of 6X loading dye containing bromophenol blue (0.09% wt./voU xylene cyanol (0.09% wt./vol.). 60% Glycerol and 60 mM KDTA. The restriction fragment patterns were separated by gel electrophoresis of 20 fil of each restriction mixture at 1.2 V/cm for 5 hours in 2.0 % molecular screening agarose gel (Roche Molecular Biochemicals, Germany) in IX TBE buffer containing 50 ng / ml. of ethidium bromide. Band patterns were analysed by Gel Documentation system using Quantity One Software. 1) Processing of samples for isolation of template DNA for PCR: 1. Cell suspension : Two loopfuls of mycobacterial growth was taken in 400u.l T.E. buffer. 2. Freezing thawing : Suspension was heated for 5 to 7 minutes in boiling water, then immediately cooled at -20°C for 15-20 minutes. 3. Lysozyme treatment : 50ul (20 mg/ml) of lysozyme was added and incubated at 37°C for 2 hours. 4. SDS and proteinase K treatment : 56f.il of 10% SDS was added and after that 8f.il (10 mg/ml) of proteinase K was added and incubated at 65°C in water bath for 1 hour. 5. Addition of CTAB/NaCl and 5 M NaCl : 80ul 5 M NaCl and 64f.il CTAB/NaCl was added and incubated for 30 minutes at 65°C. 6. Chlorotbrm/Isoamyl alcohol treatment : 645u.l of chloroform and isoamyl alcohol (24:1) was added. After mixing this was centrifuged at 8000Xg for 5 minutes. Phases are separated and middle layer was observed. 300f.il supernatant was taken into new microcentrifuge tube without disturbing middle layer. 7. Addition of Isopropanol : 180f.il (0.6% of volume) isopropanol was added and incubated at -20°C for 30 minutes. This was centrifuged for 15 minutes at 8000Xg and supernatant was discarded. 8. Washing with 70% ethanol (chilled) : ISOjil of cold 80% ethanol was added gently side by side and centrifuged for 5 minutes at 8000Xg and discard the supernatant. 9. Drying and rcdissolution: DNA pellet was dried at room temperature until ethanol evaporate and redissolved in 30f.il T.E. buffer and kept the samples in - 20°C for further process. Examples Example 1 : Mycobacterial isolates were identified on the basis of growth rate, pigmentation and several biochemical test viz, catalase test at room temperature & at 68oC, nitrate reduction, tween 80 hydrolysis, aryl sulphatase, growth on 5% NaCl and "MacConkcy Agar test etc. as per the CDC manual (Vestal 1977). Details of these biochemical tests are shown in table -1. These tests were performed for confirmation of identity of mycobacterial isolates in the experiments conducted. Examples 2: All the isolates showing biochemical variable features which were identified by in- house developed gene-amplification restriction analysis and is sequenced for final confirmation. These were confirmed as same species as identified by gene amplification restriction analysis. Some of the examples are shown in figs. 4 to 6. Table 1. Growth characteristics and identification of mycobacteria (Table Remove) Table 2. Fragments sixes of rl)NA generated by restriction of 1.8 kb segment with Hha I, Hint' 1 and Rsa I restriction enzymes from different species of mycobacteria Species Restriction fragment sizes (bp) 5. M.foriuitum 420.380,230,220,180,160,140 568,470,425,277 6. M.cheloncw 450.400,350,320,258 811,710,674,251 7. M.smegmaiis 517,425,387,341 805,670,281,253 8. M.marinum 650,480,420,220 700,673,377,255 Hha I 1. M. tuberculosis 800, 400, 320, 220 2. M.bovis 800,400,320,220 3. M.avium 500,450,400,250 4. M.intracellurae 670.480.410,220 9. M.flavescens 500,450,400,350 10. M.terrae 850,450,420,220 11. M.scrofulaceum 888,584,526 12. M.gordonae 876,607,526,330 13. M.kansasii 785,503,445,260 Hinfl 799,659,316 799,659,316 715,673,259 715,673,259 848,677,255 682,470,364,255 702, 548, 300 810,640,340 714,677,255 Rsa I " '1000.40(U06,50 1000.400,300,50 551,379.273,240,208 551,379,273,240,208 733,442,296,266 622,367,282,231,197 622,369.233,205,189,170 540.374,235,200,189,170 639,650,454,377 643,379,238,202,193,168 1000,400,300,50 700,450,240,208 536,372,235,197,182,172,160 This system is based on the amplification of approximately 1.8 kb fragment encoding 16S-23S rDNA spacer region and flanking parts of the 16S as well as 23S rDNA. This assay was applied on reference strains and biochemically characterized clinical isolates of mycobacterial species i.e. M. tuberculosis, M.bovis, M.avium, M,intracellulare,M.scrofulaceum, M.flavescens, M.gordonae, M.kansasii, M.marmum, M. terras, M.fortuitum, M.chdonae and M.smegmalis to validate the technique. Distinct gene amplification restriction analysis patterns were obtained by restriction of amplicons with three distinct restriction endonucleases (Hha I, Hinf [ and Rsa I) to differentiate various mycobacterial species. Restriction patterns generated by any of the enzyme used distinguished M.tuberculosis from M.avium, M.intracellularae and other important nonchromogenie slow growers. M.scrofulaceum showed pattern resembling with M.tuberculosis and M.bovis by using Rsa 1 en/yme, however, this could be well differentiated by en/ymes Hha I and Hinf I. M.avium-M.intracellularae complex could be differentiated to M.avium and M.intracellulare by using restriction enzyme Hha I. Restriction enzyme Rsa I and Hinf I were unable to discriminate the M.avium-M.intracellulare complex. Restriction pattern of rapid growers i.e. M.fortuitum and M.chelonae showed distinct band pattern by all three restriction enzymes. Results indicated that this system using novel primers is a simple, rapid and reproducible method to identify clinically relevant mycobacteria. Advantages: 1. This system accurately identifies clinically relevant Mycobacteria. 2. This assay allows better and easier differentiation on gels as it targets a larger region than ITS sequences particularly between the 16S - 23S rRNA genes, as the fragments generated from amplicons by this assay are bigger which can be easily separated and analy/ed. 3 Both slow growing and rapid growing mycobacteria investigated in our study could be well differentiated by using either of these enzymes. 3. Restriction patterns generated by any of the enzyme used distinguished M. tuberculosis from M.avium, M.intracellulurae and other important nonchromogenic slow growers. 5. M.scrofulaceum showed pattern resembling with M.tuberculosis and M.bovis by using Rsa I en/.ymc, however, this could be well differentiated by enzymes Hha I and Hinf 1. 6. M.avium-M.intracellularae complex could be differentiated to M.avium and M.inlracel/ulare by using restriction enzyme Hha I. 7. Restriction pattern of rapid growers i.e. M.fortuitum and M.chelonae showed distinct band pattern by all three restriction enzymes. 8. It is apparent that Hha 1 restriction enzyme will be sufficient for identification of most of species of mycobacteria. BLAST ANALYSIS 5b* Confirmation of mycobacterial isolate as M.terrae by BLAST analysis: BLASTN 2.2.8 |,Ian-()5-2004] Reference; Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402. RID: 1079004039-5343-162701455790.BLASTQ3 Query= (404 letters) Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences) 2,102,977 sequences; 10,130,642,339 total letters If you have any problems or questions with the results of this search please refer to the BLAST FAQs Taxonomy reports Distribution of 100 Blast Hits on the Query Sequence Score E Sequences producing significant alignments: (bits) Value g ; 282746] S [cfb|AY2l5 Hbi . ij Mycobac terium terrae isolate N17... 329 2e-87 g 282746bjgbjAY21SJ43.1j Mycobacterium sp. N288 16S ribos... 329 2e-87 cj. ,282Y4b79 jgbJ_AY:'lb ;2b . 1 [ Mycobacterium sp . N360CC 16S rib... 32J> 2e-87 c_j- [28274569 |gb|AY2 Lb US . 11 Mycobacterium smegmatis isolate ... 329 2e-87 g i:!8?74SOi gb|AY?Ib247.1| Mycobacterium Eortuitum isolate ... 329 2e-87 ^,[28274499 gb|AY21b245.1[ Mycobacterium fortuitum isolate ... 329 2e-87 g . j 20372930|emb|AJ4b8'JSb.1|MSP458955 Mycobacterium sp. LIV-. . . 329 2e-87 y;j i 20 r/2926 | emb j AJ4S6y SI. 1 [MSP458951 Mycobacterium sp . I,IV- . . . 329 2e-87 gi ]28274610jgb[AY215356.1j Mycolsacterium sp. N1740C 16S rib... 321 6e-85 g:i ! 28274600 j gb AY215346 . 1 j Mycobacterium sp . N582 16S ribos... 321 6e~8b 6b* Confirmation of mycobacterial isolate as M.avium by BLAST analysis: BLASTN 2.2.8 |Jan-05-20()4| RID: 1081404056-24416-174141481761.BLASTQ3 Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences) 2,224,734 sequences; 10,371,704,583 total letters If you have any problems or questions with the results of this search please refer to the BLAST FAQs Taxonomy reports Query= (343 letters) Distribution of 100 Blast Hits on the Query Sequence Score E Sequences producing significant alignments: (bits) Value gi | 27733724 j cjb [ AFb47 900 , 1 Mycobacterium avium subsp . silva... 356 2e 95 g :. I 27733723|g_b]AF547899[.1| Mycobacterium avium subsp. parat... 35J 2e-95 c;. [ 2773 V722 | gb| AF g, j28274459jgta|AY215205.1| Mycobacterium sp. N1873C 36S rib... 356 2e-95 g.. 28274457 [gb[AY21b203 . 1 | Mycobacterium sp . NlOb 16S ribos... 356 2e-95 g ; [44477jembjX52934.I|MPA16SRN Mycobacterium paratuberculos.. . 356 2e~95 y:|2l427630 gb|AK41C479.1J Mycobacterium avium Myc373 16S r... 356 2e 95 g. 4 1 396888 j tjbj AEO'172 36 . 1 j Mycobacterium avium subsp. parat... 356 2e-95 10717166jgb|AF306455.1|AF306455 Mycobacterium avium 16S ... 356 2e-95 7b* Confirmation of mycobacterial isolate as M.fortuitum by BLAST analysis: BLASTN 2.2.8 |Jan-05-2004] Reference: Altschul, Stephen P., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402. RID: 1081413889-10600-171861811926.BLASTQ3 guery= (314 letters) Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences) 2,225,351 sequences; 10,374,915,205 total letters If you have any problems or questions with the results of this search please refer to the BLAST FAQs Taxonomy reports Distribution of 100 Blast Hits on the Query Sequence Score E Sequences producing significant alignments: (bits) Value 3 - j 2"'73374 8 | gb|AF':j47»24 . 1 [ Mycobacterium fortuitum subsp. f... 341 5e-91 g j2T/33747|gbJAFb47923.1j Mycobacterium fortuitum subsp. a... 341 5e-91 g .. 2752762.1jembJAJ536039 . .1|MF0536039 Mycobacterium fortuity... 341 5e-91 g.: i2827_45_Ol[g_bj_AY2_lB24_7. l[ Mycobacterium fortuitum isolate ... 341 5e-91 g: 28274499[gb|AY215245.1[ Mycobacterium fortuitum isolate ... 3_4_1 5e~91 g;|16304849|embjAJ416915.1[MF04169_15 Mycobacterium fortuitu... 341 5e-91 gi j 16304842: |embjAJ41_6908 . l|MF0416908 Mycobacterium fortuitu... 341 5e-91 g:j44^279[embJX52933 . 1 JMFOljISRN Mycobacterium fortuitum 16S ... 34JI 5e 91 g., j 32250956JgblAY3p62C7. 1 | Mycobacterium sp . HSC1158 16S ri . . . 339 2e-90 g: I 322509B1|gb[AY306202.1j Mycobacterium sp. HSC324 16S rib... 339 2e-90 We Claim: 1. Primers for identification of pathogenic Mycobacterium represented by SEQ ID NO 1 and SEQ ID NO 2. 2. A rapid RFLP assay method for identification of pathogenic Mycobacterium at species level, said assay comprising the steps: (a) obtaining isolated DNA from pathogenic mycobacterium species; (b) amplifying the DNA molecules of step (a) with forward and reverse primers represented by SEQ ID NO 1 and SEQ ID NO 2; (c) treating the resulting amplified nucleic acid molecule with restriction enzymes such as herein described to produce nucleic acid restriction fragments; and (d) analyzing the restriction fragments as obtained in step (c) for 1.8kb size amplicon and presence of said amplicon is an indicative of pathogenic Mycobacterium species. 3. A rapid RFLP assay method as claimed in claim 2, wherein, the restriction endonuclease is selected from a group comprising LIha I, Hinf I and Rsa I. 4. A rapid RFLP assay method as claimed in claim 2, wherein, the pathogenic mycobacterium is selected from Mycohacterium tuberculosis and Mycobacterium leprae species. 5. Primers and a rapid method substantially such as herein described with drawings, examples and foregoing description. |
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2418-del-2006- description (complete).pdf
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2418-DEL-2006-Correspondence-Others (09-02-2010).pdf
2418-DEL-2006-Correspondence-Others-(29-06-2010).pdf
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Patent Number | 242073 | |||||||||||||||||||||
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Indian Patent Application Number | 2418/DEL/2006 | |||||||||||||||||||||
PG Journal Number | 33/2010 | |||||||||||||||||||||
Publication Date | 13-Aug-2010 | |||||||||||||||||||||
Grant Date | 09-Aug-2010 | |||||||||||||||||||||
Date of Filing | 07-Nov-2006 | |||||||||||||||||||||
Name of Patentee | DEPARTMENT OF BIOTECHNOLOGY | |||||||||||||||||||||
Applicant Address | BLOCK-2, (7TH FLOOR), C.G.O. COMPLEX, LODI ROAD, NEW DELHI, INDIA. | |||||||||||||||||||||
Inventors:
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PCT International Classification Number | C12Q1/68; C12Q1/68 | |||||||||||||||||||||
PCT International Application Number | N/A | |||||||||||||||||||||
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PCT Conventions:
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