GB2228485A - DNA probes for tuberculosis - Google Patents
DNA probes for tuberculosis Download PDFInfo
- Publication number
- GB2228485A GB2228485A GB8903968A GB8903968A GB2228485A GB 2228485 A GB2228485 A GB 2228485A GB 8903968 A GB8903968 A GB 8903968A GB 8903968 A GB8903968 A GB 8903968A GB 2228485 A GB2228485 A GB 2228485A
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- GB
- United Kingdom
- Prior art keywords
- tuberculosis
- dna
- hybridizing
- probe
- mycobacterium tuberculosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 201000008827 tuberculosis Diseases 0.000 title claims abstract description 36
- 239000003298 DNA probe Substances 0.000 title claims abstract description 14
- 239000013612 plasmid Substances 0.000 claims abstract description 18
- 239000012634 fragment Substances 0.000 claims abstract description 17
- 241000186366 Mycobacterium bovis Species 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000002105 Southern blotting Methods 0.000 claims abstract description 7
- 238000003745 diagnosis Methods 0.000 claims abstract description 6
- 238000009396 hybridization Methods 0.000 claims abstract description 6
- 241000186365 Mycobacterium fortuitum Species 0.000 claims abstract description 5
- 238000001514 detection method Methods 0.000 claims abstract description 5
- 206010036790 Productive cough Diseases 0.000 claims abstract description 4
- 239000005547 deoxyribonucleotide Substances 0.000 claims abstract description 4
- 125000002637 deoxyribonucleotide group Chemical group 0.000 claims abstract description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 4
- 210000003802 sputum Anatomy 0.000 claims abstract description 4
- 208000024794 sputum Diseases 0.000 claims abstract description 4
- 238000004458 analytical method Methods 0.000 claims abstract description 3
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 claims abstract description 3
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims abstract description 3
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 3
- 210000002700 urine Anatomy 0.000 claims abstract description 3
- 108020003215 DNA Probes Proteins 0.000 claims description 5
- 239000013611 chromosomal DNA Substances 0.000 claims description 5
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims 5
- 239000000523 sample Substances 0.000 description 21
- 108020004414 DNA Proteins 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 5
- 241001302239 Mycobacterium tuberculosis complex Species 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 3
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000186359 Mycobacterium Species 0.000 description 2
- 241000187478 Mycobacterium chelonae Species 0.000 description 2
- 241000186364 Mycobacterium intracellulare Species 0.000 description 2
- 241000187481 Mycobacterium phlei Species 0.000 description 2
- 241000187490 Mycobacterium scrofulaceum Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000588622 Moraxella bovis Species 0.000 description 1
- 241000187482 Mycobacterium avium subsp. paratuberculosis Species 0.000 description 1
- 208000026681 Paratuberculosis Diseases 0.000 description 1
- 101000892431 Proteus hauseri Type II restriction enzyme PvuII Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000012568 clinical material Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 108091035233 repetitive DNA sequence Proteins 0.000 description 1
- 102000053632 repetitive DNA sequence Human genes 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 229920006298 saran Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
DNA probes having potential applications to the diagnosis of tuberculosis can be derived from deoxyribonucleotide sequences capable of hybridizing with those present in a naturally occuring plasmid known to exist in Mycobacterium fortuitum. The hybridizing sequence is preferably one occurring in a DNA fragment of approximately 5000 base pairs which is present in DNA of M. tuberculosis cut with restriction endonuclease EcoRI and which detects banding patterns on Southern blotting distinguishing different strains of M. tuberculosis as well as distinguishing between M. tuberculosis, M. bovis and BCG vaccine organism. The DNA probes can be used for the detection of M. tuberculosis in clinical samples such as sputum, urine or cerebrospinal fluid by the techniques of dot blot analysis, solution hybridization or polymerase chain reaction.
Description
DEOXYRIBONUCLEIC ACID PROBES FOR DIAGNOSIS OF TUBERCULOSIS
This invention relates to deoxyribonucleic acid probes ("DNA probes") for diagnosis of tuberculosis.
Laboratory diagnosis of tuberculosis is unsatisfactory because several weeks may be required to obtain a definite result and the detection of small numbers of Mycobacterium tuberculosss bacteria in heavily contaminated samples is often difficult. The specific identification of mycobacteria is also difficult, and especially the differentiation between the members of the M. t ubercul osis complex: M. tuberculosis itself, the bovine strain M. bovis and the vaccine strain BCG (which may cause disease in immunologically suppressed individuals). The use of DNA probes can provide a sensitive method for the rapid detection of small numbers of specific bacteria in the presence of other organisms.
DNA probes for dentifying strains of the M. tuberculosis complex are commercially available but depend on detecting ribosomal R.NA and require the bacteria to be cultivated first.
Although they are capable of identifying the M. tuberculosis complex they are not suitable for detecting bacteria in a specimen of sputum.
DNA probes derived from chromosomal DNA of M tuberculosis have been described (K.D. Eisenach, J. T. Crawford a J.H. Bates.
1988. Repetitive DNA sequences as probes for Mycobecterf um tuberculosis. Journal of Clinical Microbiology 26 : 2240-2245).
The existing probes are not capable of distinguishing between closely related members of the M. tuberculosis complex.
It has now been found that DNA probes having potential applications to the diagnosis of tuberculosis can be derived from deoxyribonucleotide sequences capable of hybridizing with those present in a naturally occurring plasmid.
As part of an investigation into antibiotic resistance, the presence of plasmids in M. tuberculosis was sought by hybridizing the total DNA from three clinical isolates with DNA of a naturally occurring plasmid known to exist in M. fortuftum (A. Labidi, C. Dauguet, K. S. Goh & H. L. David. 1984. Flasmid profiles of Mycobacterium fortultum complex isolates. Current
Microbiology 11 : 235-240). Plasmids have not hitherto been found in M tuberculosis, and it was hoped that they would be revealed by the use of the plasmid DNA as a probe.
Surprisingly, while this did not reveal the presence of any plasmids in M. tuberculosis, it did show that there were chromosomal DNA fragments capable of hybridizing with the plasmid DNA. Moreover, in total DNA from the three clinical isolates digested with restriction endonuclease BamHI or PvuII, the size of the hybridizing fragments was not the same for each strain.
According to the invention there is provided a DNA probe for diagnosis of tuberculosis which comprises a deoxyribonucleotide sequence capable of hybridizing with DNA of a plasmid occurring in M. fortuitum and with chromosomal DNA of M. tuberculosis.
The hybridizing sequence is preferably one occurring in a
DNA fragment of approximately 5000 base pairs which is present in DNA of M. tuberculosis cut with restriction endonuclease
EcoRI and which detects banding patterns on Southern blotting distinguishing different strains of M. tuberculosis as well as distinguishing between M. tuberculosis, M. bovis and BCG vaccine organism.
DNA probes according to the invention can be used for the detection of M. tuberculosis in clinical samples such as sputum, urine or cerebrospinal fluid by the techniques of dot blot analysis, solution hybridization or polymerase chain reaction.
A probe having a hybridizing sequence occurring in the DNA fragment of approximately 5000 base pairs, when used in combination with Southern blotting of restriction-endonucleasedigested DNA from such clinical material or from bacteria isolated from it, can distinguish M. tuberculosis from M. bovis and BCG, and is of value for epidemiological purposes because it is also able to differentiate between clinical isolates of H. .t ubercul osf s.
Although the fragment of approximately 5000 base pairs can itself be used as a probe, the effective nucleotide sequence is believed to comprise only part of this fragment. This effective sequence may be identified and isolated, and if not too large may be synthesized, by methods known per se.
The specific descr ption which follows is given by way of illustration.
METHOD OF ISOLATION OF THE PROBES
As part of an investigation into the possible presence of plasmids in clinical isolates of M. tuberculosis, total DNA from three such isolates was subjected to Southern blotting and probed with a naturally occurring plasmid from M. fortuf tum This plasmid, referred to as pSU300, was obtained from
M. fortuitum strain CIPT 14-041-0003 in the Collection de 1' Institut Pasteur, Tuberculose, Paris, France. The results showed that there were chromosomal DNA fragments in the strains of H. tuberculosis capable of hybridizing to this plasmid, and also that in material digested with BamHI or PvuII the size of the hybridizing fragments was not the same for each strain.
In order to isolate these hybridizing fragments, a total DNA library from a clinical isolate of M. tuberculosis (strain 50401) was constructed in the lambda phage vector EMBL4 by ligation of a partial Sau3AI digest of the H. tuberculosis DNA with BdHI-digested EMBL4. The library was screened with a DNA probe derived by labelling a recombinant plasmid pSU3Oi. This plasmid was constructed by ligating an EcoRI digest of plasmid pSU300 with an EcoRI digest of the Escherichia coli plasmid vector pUCi9. Positive plaques were purified through further rounds of plaque screening. The probes described below were obtained from the recombinant phage, referred to as EMBL4/A-3, of one of the positive plaques obtained by this procedure.
The DNA from this recombinant phage was extracted and digested with EcoRI. Agarose gel electrophoresis and Southern blotting demonstrated that EcoRI-digested EMBL4/A-3 contained two fragments of approximate sizes 9000 base pairs (9 kb) and 5000 base pairs (5 kb) which hybridized with the plasmid pSU300.
These fragments were each excised from the gel and are referred to as probe A3/1 (the 9 kb fragment) and probe A3/2 (the 5 kb fragment) respectively.
PROPERTIES OF PROBE A3/1
Probe A3/1 was radioactively labelled with 32P using the Multiprime random primer extension method (Amersham) and hybridized to Southern blots of PvuII-digested total DNA from eight clinical isolates of M. uberculosis (isolate numbers 50410, 60925, 61066, 61104, 61125, 61267, 61377, 61513) as well as M. bovis (field strain, Central Veterinary Laboratory) and
BCG (NCTC 5692). After agarose gel electrophoresis, the DNA fragments were transferred to a Hybond-N filter and fixed by baking at 80-C for 1 hour. The filter was prehybridized at 68 C in hybridization buffer. Hybridization with the probe was carried out in the same buffer at 68-C overnight.
The hybridization buffer consisted of 5x Denhardt1s solution, 5x SSPE buffer, 0.2% sodium dodecyl sulphate (SDS) and 100 Fg./ml. sonicated salmon sperm DNA. The Denhardt's solution and the SSPE buffer were made up as stock solutions as follows.
50x Denhardt's solution: 0.5 g. Ficoll (mw 400,000); 0.5 g. polyvinylpyrrolidone (mw 40,000); 0.5 g. bovine serum albumin. The ingredients were dissolved in sterile deionized distilled water to a final volume of one litre which was dispensed into aliquots and stored at -20iC.
20x SSPE buffer: 3. 6M Nazi; 20mM ethylenediaminetetraacetic acid (EDTA); O.2M NaH2P0/Na2HP04, pH 7.7. The ingredients were dissolved in one litre of deionized distilled water and autoclaved.
The filter was then washed twice with 2x SSC, once with 2x SSC containing 0. 1% SDS and once with 0. 1X SSC containing O. 1X SDS. All washes were done at 68-C. The SSC was made up as a stock solution as follows.
20x SSC: 3M Nail; O.3M sodium citrate. The ingredients were dissolved in distilled water to a final volume of one litre and autoclaved after the pH had been adjusted to 7.0.
The filter was covered with Saran wrap and exposed tb X-ray film (RX, FuJi) for 16 hours at room temperature.
Each strain of M. tuberculosis hybridized to probe A3/1 exhibited several hybridizing bands; some elements of this pattern varied from strain to strain while others remained constant. M. bovis and BCG also hybridized to probe A3/1 with a pattern which retained the conserved features of the
M. tuberculosis pattern.
The following species of mycobacteria (one strain each except where indicated) did not hybridize with probe A3/1 to any significant extent: M. paratuberculosis, M. intracellulare, M. scrofulaceum, M. phlei, M. fortuitum (three strains) and
M. chelonei (two strains).
Probe A3/1 was therefore specific for the M. tubercu2oss complex (which includes M. bovis and BCG > , with some ability to differentiate between strains.
PROPERTIES OF PROBE A3/2
Probe A3/2 was tested in a similar way, with nine strains of
M. tuberculosis as well as M. bovis and BCG. The conditions were the same as described above for probe A3/1, except that autoradiography was for 6. 5 hours at room temperature.
Each M. tuberculosis strain showed between five and fifteen strongly hybridizing fragments, as well as a number of weaker bands. The pattern of bands was markedly different for each strain. The number of bands and the strength of the signal, as well as the variation between strains, indicated the presence of a randomly inserted repetitive DNA element in the chromosome of these strains.
M, bowls and BCG showed a simpler pattern of two and three major bands respectively. These organisms could therefore be easily distinguished from M. tuberculosis and from each other.
The following species of mycobacteria (one strain each except where indicated) did not hybridize with probe A3/2:
H. paratuberculosis, M. intracellulare, M. scrofulaceum, M. phlei, M fortuitum (three strains) and M. chelonei (two strains > .
Probe A3/2 was therefore specific for the M. tuberculosis complex and was in addition able to distinguish between
M. tuberculosis, H. bovis and BCG, and to distinguish between strains of M. t tuberculosis.
Claims (3)
1. DNA probe for diagnosis of tuberculosis which comprises a deoxyribonucleotide sequence capable of hybridizing with DNA of a plasmid of Mycobacterium fortuitum and with chromosomal DNA of Mycobacterium tuberculosis.
2. DNA probe as claimed in claim 1 in which the sequence is one occurring in a DNA fragment of approximately 5000 base pairs which is present in DNA of Mycobacterium tuberculosis cut with restriction endonuclease EcoRI and which detects banding patterns on Southern blotting distinguishing different strains of Mycobacterium tuberculosis as well as distinguishing between
Mycobacterium tuberculosis, Mycobacterium bovis and BCG vaccine organism.
3. Process for the detection of Mycobacterium tuberculosis in clinical samples such as sputum, urine or cerebrospinal fluid comprising the use of a DNA probe as claimed in claim 1 or claim 2 by the techniques of dot blot analysis, solution hybridization or polymerase chain reaction.
Priority Applications (12)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8903968A GB2228485A (en) | 1989-02-22 | 1989-02-22 | DNA probes for tuberculosis |
| GB909000411A GB9000411D0 (en) | 1989-02-22 | 1990-01-09 | Probes and kits for the direction of mycobacteria |
| DE69009744T DE69009744T3 (en) | 1989-02-22 | 1990-02-22 | Probes, equipment and methods for detection and differentiation of mycobacteria |
| EP90903862A EP0460041B2 (en) | 1989-02-22 | 1990-02-22 | Probes, kits and methods for the detection and differentiation of mycobacteria |
| ES90903862T ES2057539T5 (en) | 1989-02-22 | 1990-02-22 | PROBES, KITS AND PROCEDURES FOR THE DETECTION AND DIFFERENTIATION OF MICOBACTERIES. |
| JP50393490A JP3247371B2 (en) | 1989-02-22 | 1990-02-22 | Probes, kits and methods for detection and differentiation of Mycobacterium tuberculosis |
| AU51627/90A AU623236B2 (en) | 1989-02-22 | 1990-02-22 | Probes, kits and methods for the detection and differentiation of mycobacteria |
| PCT/GB1990/000276 WO1990010085A1 (en) | 1989-02-22 | 1990-02-22 | Probes, kits and methods for the detection and differentiation of mycobacteria |
| GB9118275A GB2248298B (en) | 1989-02-22 | 1991-08-20 | Probes,kits and methods for the detection and differentiation of mycobacteria |
| IN781MA1991 IN173129B (en) | 1989-02-22 | 1991-10-16 | |
| US08/160,524 US5851761A (en) | 1989-02-22 | 1993-12-01 | Probes, kits and methods for the detection and differentiation of mycobacteria |
| JP2000295176A JP2001136989A (en) | 1989-02-22 | 2000-08-23 | Probe, kit and method for detection and differentiation of mycobacterium tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8903968A GB2228485A (en) | 1989-02-22 | 1989-02-22 | DNA probes for tuberculosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB8903968D0 GB8903968D0 (en) | 1989-04-05 |
| GB2228485A true GB2228485A (en) | 1990-08-29 |
Family
ID=10652080
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8903968A Withdrawn GB2228485A (en) | 1989-02-22 | 1989-02-22 | DNA probes for tuberculosis |
| GB909000411A Pending GB9000411D0 (en) | 1989-02-22 | 1990-01-09 | Probes and kits for the direction of mycobacteria |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB909000411A Pending GB9000411D0 (en) | 1989-02-22 | 1990-01-09 | Probes and kits for the direction of mycobacteria |
Country Status (1)
| Country | Link |
|---|---|
| GB (2) | GB2228485A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851761A (en) * | 1989-02-22 | 1998-12-22 | Cogent Limited | Probes, kits and methods for the detection and differentiation of mycobacteria |
-
1989
- 1989-02-22 GB GB8903968A patent/GB2228485A/en not_active Withdrawn
-
1990
- 1990-01-09 GB GB909000411A patent/GB9000411D0/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851761A (en) * | 1989-02-22 | 1998-12-22 | Cogent Limited | Probes, kits and methods for the detection and differentiation of mycobacteria |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9000411D0 (en) | 1990-03-07 |
| GB8903968D0 (en) | 1989-04-05 |
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| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |