AU632383B2 - Diagnostic probe for use in the detection of m.paratuberculosis - Google Patents

Description

2 2- This invention relates to diagnostic probes for use in the detection of Mycobacterium paratuberculosis, to methods of using such probes and to kits containing the same.

Paratuberculosis (Johne's disease) is one of the most serious I enteric infectious diseases affecting domestic ruminants. The disease Sspreads insidiously and causes immense economic losses in the world cattle industry (Chiodini et al., The Cornell Veterinarian, 74, 218-262 (1984)).

Confirming a tentative diagnosis of Johne's disease in the live animal is difficult because there is no rapid, specific diagnostic test for the condition. Culture of the causative organism, Mycobacterium paratuberculosis (M.ptb) is the most reliable diagnostic aid but can take several weeks or months while examination of faeces for acid-fast bacteria i gives both false positive and false negative results (Ris et al., New Zealand Veterinary Journal, 36, 112-114 (1988)). Furthermore, diagnosis by serological methods is unreliable (Sherman, Veterinary Medicine, 77-84 (1985)).

It is therefore an object of the present invention to provide a V method of diagnosis of Johne's disease in animals which is both rapid and reliable or which at least provides the public with a useful choice.

Accordingly, in a first aspect the present invention consists in a DNA molecule consisting of a nucleotide sequence characteristic of and ~ST\e\vC \ecst tuc-\4ot.hes \«VNL& st\~ specific for M.ptb, said sequence including at least nucleotides 406-414 of the sequence set out in Figure 2.

In still a further aspect, the invention provides a DNA molecule comprising a nucleotide sequence characteristic of M.ptb, said Scharacteristic sequence consisting of at least nucleotides 4-1456 of the sequence set out in Figure 2.

"T r- 3 In still a further aspect, the invention provides a DNA molecule comprising a nucleotide sequence characteristic of M.ptb, said characteristic sequence including the entire sequence set out in Figure 2.

It will also be appreciated that the invention includes DNA molecules comprising a nucleotide sequence characteristic of M.ptb which hybridise at high stringency with the characteristic sequences defined above.

In yet a further aspect, the invention provides a recombinant plasmid which includes a DNA molecule as defined above.

In still a further aspect the invention there is provided a diagnostic probe consisting of a DNA molecule as defined above optionally otr carrying a revealing label. Conveniently, the probe comprises a part of a o 4 recombinant plasmid.

In a further aspect, the invention consists in a method for idetecting the presence or absence of M.ptb in a sample which comprises the step of assaying said sample for M.otb DNA with a probe as defined above.

In yet a further aspect, the invention provides a diagnostic kit for use in detecting the presence or absence of M.ptb in a sample, which kit :tit comprises as one component an optionally labelled probe as defined above.

4 i Although the present invention is broadly as defined above, it will be appreciated by those persons skilled in the art that it is not limited thereto and that it also includes embodiments of which the following Sdescription provides examples. Furthermore, the present invention will be 'more fully understood by reference to the accompanying drawings in which: Figure 1 shows the approximate location of pAM-3 within the 3.8 Kb insert of the Xgtll recombinant; Figure 2 is the putative DNA sequence of pAM-3; L ~L~~~iB6 iX~1~T--liFI~.LXiirrXP~ 4 -4- Figure 3A is a Southern Blot showing the results obtained where selected microorganisms were digested with EcoRI, electrophoresed, blotted onto nitrocellulose and probed with pAM-3.

Figure 3B is a diagramatic representation of the aligned pAM-3 hybridisation bands seen with ovine and bovine M.ptb DNA.

Figure 4 is a dot blot of dilutions of ovine faeces mixed variously with M.ptb, M.phlei, no additions.

Figure 5 is a blot onto nylon membrane of various strains and isolates of M.ptb following digestion with Hinfl.

Figure 6A and 6B are dot blots showing the specificity of pAM-3 as a probe for M.ptb.

As defined above, the present invention generally relates to the detection of M.ptb using DNA probes. The key to developing a nucleic-acid probe is to identify and isolate a nucleic acid sequence which is unique to the organism to be detected. The identification and isolation of such a unique sequence characteristic of and specific for M.ptb will now be described.

A DNA probe for the specific identification of Mycobacterium paratuberculosis (M.ptb) 1. Construction of p'ne library of M.ptb and initial identification of DNA characteristic of M.ptb To identify DNA characteristic of and specific for M.ptb a gene library of M.ptb was formed. The library was constructed in Xgtll by a method described by Young et al. Proc Natl. Acad. Sci. USA, 82, 2583-2587 (1985) with some modifications.

DNA extracted from M.ptb (isolate 16620) which had been cultured in the Department of Veterinary Pathology and Public Health, Massey University, from a cow with clinical Johne's disease, was mechanically sheared and size-fractionated on an agarose gel. After electroelution, 4-7 Kb DNA fragments were methylated and end-filled to facilitate the addition of 32 P-labelled EcoRI linkers. Following EcoRI digestion to generate cohesive ends, excessive linkers were removed by centrifugation through a Centricon 30 microcentrator (Amicon Corporation, Danvers, MA, USA). The concentrated DNA was ligated with X gtll and packaged in vitro using the Protoclone Xgtll and Packaging System Protocol (Promega, Madison, WI, USA).

Approximately 2.2 x 105 individual recombinants were obtained.

Insert sizes of 20 randomly picked recombinants ranged from 1.5 to 6.5 Kb.

Library phage plaques were "lifted" onto nitrocellulose (Benton and 0 0 .0 0 o00 Davis, Science, 196, 180-182 (1977)) and screened by differential o o 0

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a oo hybridisation with chromosomal DNA from M.ptb (Massey University field 00 isolate) and M.phlei (Massey University field isolate) radiolabelled by o nick translation (Rigby et al. Mol. Biol, 113, 237-251 (1977)). Phage 00 which hybridised only to M.ptb were then identified by autoradiography. A 0 0 single recombinant containing an insert of approximately 3.8 Kb was selected for further analysis. The insert from this clone was digested oao with EcoRI and BamHI and the four resulting fragments separated on an 0 0 agarose gel. One fragment of approximately 1.6 Kb was electroeluted from 0 0 the gel and ligated to EcoRI- and BamHI-digested pGEM-2 (Promega). Max S* o Efficiency DH5(X competent cells (Bethesda Research Laboratories, A ,o Gaithersburg, MD USA) were transforimd with recombinant pGEM-2 according o°"o0 to the suppliers instructions and plated onto LB plates containing 100 pg/ml ampicillin. A single colony was selected and used to prepare a bulk growth of the recombinant plasmid (designated pAM-3) using standard techniques. (Auubel et al., CLurtent Protocols in Molecular Biology (1987)). The approximate location of the M.ptb cDNA insert included in pAM-3 within the 3.8 Kb insert is shown in Fig. 1.

I 1- -6 2. Deposition of Samples Samples of both the organism from which the characteristic sequence was derived (M.ptb isolate 16620) and E.coli DH5a carrying pAM-3 have been deposited at the American Type Culture Collection (ATCC) Rockvale, MD20852 USA on 18 October 1989. These samples have been assigned accession Nos. 53950 and 68128 respectively.

3. Nucleotide Sequencing of pAM-3 The M.ptb DNA insert in pAM-3 has been sequenced to determine the peptide sequence of the product it encodes.

The approach taken to generate the complete nucleotide sequence of the DNA insert was to randomly shear the DNA by sonication (Deininger, Anal Biochem 129 216 (1983)), shotgun clone the fragments into the M13 sequencing vector (Messing et al., Proc. Natl. Acad. Sci. USA 74 3642 (1977)) and sequence the clones by the dideoxy chain termination method (Sanger et al. Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977)).

Efficient overlapping and ordering of the sequence data from the random fragments was achieved using computer programs (Staden, Nucleic Acids Res. 14 217 (1986)).

The clones were sequenced and the complete sequence of the M.ptb DNA insert included in the pAM-3. DNA was shown to be 1608 bp in size.

Details of the putative sequence are shown in Figure 2.

Accordingly, in a first aspect the invention provides a DNA molecule consisting of a nucleotide sequence characteristic of and specific for Mptb. This DNA molecule may be isolated from an appropriate natural source or from Mptb isolate 16620 (ATCC 53950) or can be produced synthetically using any of those techniques known in the art.

L nan~~ 7 In one aspect, the DNA molecule of the invention is in the form of an oligonucleotide which includes at least nucleotides 406-414 of the sequence set out in Figure 2. In this form, the oligonucleotide must include sufficient flanking nucleotides from the Figure 2 sequence to enable a hybridisation reaction to occur which is specific for the presence of Mptb DNA. Conventionally, such a specific hybridisation reaction requires an oligonucleotide of at least 14 base pairs in length, with a 20 base pair length being preferred.

The synthetic oligonucleotides of this aspect of the invention can be prepared using conventional techniques, for example by the triester method of Matteucci et al (J Am. Chem. Soc. 103, 3185-3191(1981)), or by employing an appropriate DNA synthesizer (eg. the Applied Biosystems DNA synthesizer).

However, while oligonucleotides have the advantages of rapid hybridisation and simplicity of preparation, it is preferred that the DNA molecule of the invention be substantially greater in size than 20 base pairs. This preference is due to both the increased sensitivity and specificity of the hybridisation reaction with longer molecules.

In a preferred embodiment, the DNA molecule is approximately 1.45 kb in length and consists of nucleotides 4-1456 of the sequence set out in Figure 2. More preferably, the DNA molecule is approximately 1.6 kb in length and consists of the entire nucleotide sequence of Figure 2.

While the DNA molecule of the invention has been defined above in relation to the Figure 2 sequence, it will be appreciated by those persons skilled in the art that the Figure 2 sequence is not to be construed as strictly limiting. More particularly, it will be understood that a DNA molecule consisting of a nucleotide sequence which hybridises at high stringency with any of the DNA molecules fined above is also within the scope of this invention.

i 8- It will further be appreciated that the DNA molecule of the invention may comprise a part of a recombinant plasmid. Indeed, such an embodiment is preferred where the DNA molecule consists of all or a major portion of the nucleotide sequence of Figure 2. Any suitable recombinant plasmid can be employed in this regard with commercially available plasmids such as pGEM-2 being presently preferred.

In a second aspect the present invention relates to a method of diagnosing the presence or absence of M.ptb in a sample which employs a probe comprising a DNA molecule characteristic of and specific for M.ptb as described above. Such a method has application in the diagnosis of Johne's disease in animals or any disease associated with M.ptb in mammals, birds, reptiles, amphibians and fish. In these diagnostic applications the sample can be of tissue, tissue extract, body fluids, excreta and also M.ptb isolated following in vitro culture.

Alternatively, the method can also be applied in the detection of M.ptb in environmental samples such as soi:., water or animal and human foodstuffs.

As will be readily appreciated by the worker having ordinary skill in the art, the probe for use in the method may take a variety of forms.

In this regard, reference can usefully be made to Tenover, F.C., "Diagnostic Deoxyribonucleic Acid Probes for Infectious Diseases, Clinical Microbiology Reviews Volume 1 82-101 (1988) and Tenover, "DNA Probes for Infectious Diseases", CRC Press Inc, Boca Raton, Florida (1989) which review this area of technology.

While by no means essential, it is preferred that the probe comprise the DNA molecules cons-sting of at least a major portion of the nucleotide sequence of Figure 2. In particular, the probe should be based upon all or part of the nucleotides sequenced 4-1456 in Figure 2.

_1 1 9 o of 0010.

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*1i It is however presently most preferred that the probe comprise the entire nucleotide sequence of Figure 2 and form a part of a recombinant plasmid. pAM-3 is an example of such a probe.

While there are detection systems which do not require the probe to be labelled, the probe for use in the method preferably carries a detectable marker or label. This label is commonly a radioactive, fluorescent, antigenic or enzymic reporter molecule although any of those labels known in the art could be used. Suitable examples are the PhotoGene (Gibco BRL), Chemiprobe (Orgenics) and Enhanced Chemiluminescence (Amershan) labels.

Where, as is preferred, the probe comprises pAM-3 the entire 4.5 Kb plasmid can be labelled. This labelling of the entire plasmid increases the ease of detection during use.

Once prepared, the probes can be employed in any conventional reaction system which allows hybridisation to occur with the target DNA and the presence or absence of hybrid DNA to be detected. For example, where the probe is unlabelled it can be bound to a solid matrix formed from materials such as nitrocellulose, cellulose or plastic and have sample DNA which has been released by mechanical, chemical, heat, sonication or enzymic treatment applied to it. Target DNA complementary to the probe sequence becomes bound to the matrix via the probe. The matrix is washed to remove unbound DNA and then the target DNA is released by heating or transfer to a denaturing solution. Once released the target DNA can be detected by any known techniques.

An alternative reaction system involves the use of hydroxyapatite (HAP) which selectively binds double-stranded DNA. Sample DNA obtained as above can be mixed with the probe in single-stranded form in a reaction solution designed to allow hybridisation and prevent nuclease action. Any hybrid DNA in the reaction solution is then exposed to and bound by HAP

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10 and any unhybridised probe DNA removed by washing or by digestion by single- strand specific nuclease followed by precipitation with a suitable reagent such as ethanol or trichloroacetic acid.

Once again, hybrid DNA can be detected by any of those techniques known in the art.

Where the probe is labelled, the reaction system selected to detect hybrid DNA will of course depend upon the type of label employed. For example, where the label is selected from amongst the PhotoGene (Gibco BRL), Chemiprobe (Orgenics) and Enhanced Chemiluminescence (Amersham) labels, the reaction system adopted to detect hybrid nucleic acid molecules will vary in accordance with the manufacturer's instructions.

Alternatively, where desirable, the target DNA is first amplified by polymerase chain reaction (PCR) Mullis and Fallona, Methods in Enzymology, 155, 335-350 (1987)). Described generally, PCR amplification involves two oligonucleotide primers that flank the characteristic DNA sequence to be amplified and repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and the extension of the annealed primers with DNA polymerase. These primers hybridise to opposite strands of the target sequence and are oriented so DNA synthesis by the polymerase proceeds across the region between the primers, effectively doubling the amount of the DNA. Moreover, since the extension products are also complementary to and capable of binding primers, each successive cycle essentially doubles the amount of DNA synthesized in the previous cycle. This results in the exponential accumulation of the specific target segments, approximately 2 n where n is the number of cycles.

Where PCR is employed, the specific oligonucleotide primers used will depend upon the nucleotide sequence characteristic of and specific for M.ptb selected for use as the probe. These primers are generated by synthesis as described above in relation to the oligonucleotide probes.

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I C 11 In most cases, the primers selected will result in the amplification of a characteristic Mptb nucleotide sequence including nucleotides 406-414 of the sequence set out in Figure 2. However, it will be appreciated that where the selected diagnostic probe is either labelled pAM-3 or a labelled probe consisting of nucleotides 4-1456 of the Figure 2 sequence, the primers can be selected to define a DNA molecule consisting of any part or all of the sequence from nucleotides 4-1456 in Figure 2.

Preferably the PCR amplification procedure employed involves the formation of a reaction mixture which includes the sample, the S oligonucleotide primers defining the DNA sequence selected for amplification, deoxynucleotide triphosphates and a DNA polymerase. The DNA polyierase is included in the reaction mixture to enable polymerisation of complementary strands to occur. The DNA polymerase used is preferably also thermostable and capable of withstanding the temperature variations to which the reaction mixture is subjected. An example of a suitable DNA polymerase is the "Amplitaq" DNA polymerase marketed by Cetus Corporation.

Following formation of the polymerase chain reaction mixture, the next step in the method comprises heating the reaction mixture to a temperature at which the DNA in the mixture is dissociated into single strands. Generally, this dissociation occurs at temperatures in excess of 0 C with a temperature of 94 0 C being commonly employed.

Following dissociation of the DNA into single strands, the reaction mixture is cooled to a second temperature at which annealing of the oligonucleotide primers to the complementary sequences of the single strands can occur. While this temperature will vary depending on the DNA sequence selected to be amplified, it is common for the temperature to be in the range of 45 0 C-65 0

C.

i 12 Upon annealing of the primers to the single strand DNA, the reaction mixture is maintained at a third temperature to allow polymerisation of complementary strands to occur. The temperature at which the reaction mixture is maintained in this step is of course dependent upon the DNA polymerase used and in particular on the activation temperature of the polymerase. For some DNA polymerases, this temperature may be the same as the temperature at which the annealing of the primers to the dissociated strands occurs. However, it is common that the third temperature is greater than that at which the annealing occurs. By way of example, where the preferred Amplitaq polymerase is employed, the polymerisation reaction takes place with the reaction mixture maintained at a temperature of 72 0

C.

Upon completion of the polymerisation of complementary strands, the steps of dissociation, annealing and polymerisation are repeated. The number of times these steps are repeated will depend on the degree of amplification desired for the selected DNA sequence. Conveniently, the dissociation, annealing and polymerisation steps are repeated at least times.

The above amplification procedures are suitable for automation. For example, there are various thermocycling devices commercially available which would be suitable for use in the present method. One such suitable apparatus is the Perkin-Elmer Cetus DNA Thermocycler.

The final step of this aspect of the diagnostic method of this invention is performed following the completion of the amplification step.

This final step involves the detection of the presence or absence in the reaction mixture of the selected nucleotide sequence. If the selected sequence is present in amplified quantities in the reaction mixture, the sample from which the DNA amplified was obtained is positive, 13 The procedure adopted to detect the presence or absence of the selected sequence in the reaction mixture involves assaying the reaction mixture with the diagnostic probe of the invention, which is preferably labelled. Any of those procedures known in the art cae i cmployed in the detection step. Conveniently, the procedure adopted is Southern blotting (Maniatis et. al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour (1982)). By this procedure, an electrophoresis gel is run and the DNA transferred to nitrocellulose filters and incubated. The labelled diagnostic probe is then added to the filters to allow hybridisation between the probe and a region of the DNA within the selected sequence.

The filters are then autoradiographed and any hybridisation which has occurred detected.

The method of the invention is illustrated by the following non-limiting examples.

The Use of M.ptb specific probe Differentiation between mycobacterial strains The precise identification and differentiation between mycobacterial strains was achieved by isolation of genomic DNA from target bacteria, digestion with restriction endonuclease EcoRI, electrophoresis, blotting (Southern, J. Mol. Biol., 98, 503-517 (1975)) and probing with pAM-3 labelled with a reporter molecule.

DNA from bovine M.ptb (Massey University field isolate), ovine M.ptb (Australian field isolate), M. avium serovar 2 (TMC 714), M. avium serovar 3 (TMC 725), M. bovis (TMC 410), M. smegmatis (Massey University field isolate), M. phlei (Massey University field isolate), Actinomyces bovis (NCTC 9341), Streptomyces griseus (NCTC 7807) and E.coli (Massey I i 14 University field isolate) was treated as described and probed with pAM-3 labelled by a Chemiprobe (Promega) non-radioactive labelling protocol (Fig. 3A).

pAM-3 hybridised strongly to approximately 15 bands in bovine M.ptb. In contras-, ovine M.ptb revealed approximately 16 strongly hybridising bands and two weakly hybridising bands. pAM-3 did not hybridise with any of the other microorganisms tested. This result indicates that pAM-3 hybridises to a sequence that is repeated in the M.ptb genome which is absent in the closely related M. avium serovars 2 t, ,10 and 3. Also, the complex banding pattern found with the two M.ptb 4 isolates distinguishes between them (Fig. 3B).

(ii) Demonstration of M. paratuberculosis in ovine faeces Freshly voided ovine faeces was mixed with either M.ptb (Massey University fifld isolate), or M. phlei (Massey University field isolate). A third sample had no additions, The samples were mixed at 225 rpm for 1 hour at 250C, sonicated for 5 minutes on ice and allowed to 4 4 settle for 1 hour at 74 0 C. The supernatant was removed and incubated with 04 lysosome for 2.5 hours at 37 0 C. Sodium dodecyl sulphate and proteinase K was then added and the samples incubated at 50 0 C for 16 hours. Following ml the addition of 0.4 volumes 5M potassium acetate the supernatant was extracted with phenol-chloroform and the DNA precipitated with ethanol. Dilutions of the resuspended DNA samples were dot blotted onto nitrocellulose, denatured with alkali, neutralized, and baked at 80 0 C for Shours.

The dot blot was hybridised with pAM-3 labelled by a Chemiprobe non-radioactive labelling protocol.

u;l~r~ 15 pAM-3 gave a positive result in the dilutions of faecal sample which had M.ptb added (Sample A positive result was not obtained with samples B and C (Fig. 4).

(iii) Differentiation between mycobacterial strains upon digest with Hinfl In a further example genomic DNA from M. paratuberculosis (ATCC 19698) and field isolates of M.ptb of ovine and bovine origin was digested with restriction endonuclease Hinfl, electrophoresed, blotted onto nylon membrane and probed with pAM-3 labelled with the Chemiprobe (Promega) non radioactive labelling protocol.

The results are shown in Figure 5. As can be clearly seen, in each case at least six hybridising bands were obtained. Also Isolate 29 gave a different banding pattern to the other isolates including the ATCC 19698 type strain, (iv) Specificity of pAM-3 Type strains and field isolates of mycobacteria from around the world were treated with lysozyme, sodium dteqcyl sulphate and proteinase K to extract DNA. Following treatment with phenol-chloroform lul aliquots of the extracted material were dot blotted onto nylon membrane.

The dot blots were hybridised with pAM-3 labelled with the Chemiprobe (Promega) non-radioactive labelling protocol.

The results are shown in Figures 6A and 6B. As can be clearly seen, pAM-3 gave a positive result with the M.ptb ATCC 19698 type strain and the 23 M.ptb field isolates plus an isolate from a human patient with Crohn's disease. pAM-3 did not hybridise with M.avium, M. intracellulare, M. scrofulaceum (MAIS) complex serovars 2, 4, 8, 9, 14, 16, 18 and 41, 16 M. kansasii ATCC 12478, M. tuberculosis strain H37Rv, M. bovis, woodpigeon mycobacteria M21, M.ptb vaccine strain 316 and the BCG vaccine strain.

In a further aspect, the present invention provides a diagnostic kit for use in the detection of M.ptb. Such a kit includes as one component a diagnostic probe comprising a characteristic sequence for M.ptb which is optionally labelled. Conveniently, the probe is an oligonucleotide probe as described above labelled with a reporter molecule. Alternatively, the probe is pAM-3 optionally carrying a revealing label.

While in no way intended to be limiting, 3 2 P can be used as a suitable label, Other examples of suitable but non-radioactive labels are the PhotoGene (Gibco BRL), Chemiprobe (Orgenics) and Enhanced Chemiluminescence (Amersham) labels.

In addition to the optionally labelled probe, the kit may also include a lysing agent capable of lysing the cells contained in the sample to be tested to release the DNA they contain. Any conventional lysing agent can be provided for this purpose.

Other optional components the kit may include are a denaturing solution and an alkaline fixation solution. These can be selected from amongst those available in the art and provided in appropriate amounts.

As still a further optional component, the kit may contain a separation suspension. While again not critical, hydroxyapatite in buffered solution containing 0.02Z sodium azide can be used.

Yet a further optional component of the kit is a wash solution which is suitably a buffered solution containing 0.02% sodium azide.

While the kit of the invention can usefully include those components set out above, it will be appreciated by those persons skilled in the art that other conventional diagnostic components can also be provided if desired.

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i i r i larr;;a~a~ L-1 17 Thus, in accordance with the present invention there is provided a DNA probe which is capable of determining whether or not M.ptb is present in a sample. The probe is specific for M.ptb in that it does not hybridise to other closely related mycobacteria which allows a rapid and conclusive diagnosis of paratuberculosis infection to be made. As such, the method of the invention employing the probe represents a considerable advance over those diagnostic procedures presently available.

It wi.ll be appreciated by those persons skilled in the art that the above description is provided by way of example only and that the present invention is not limited in scope thereto.

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Claims (21)

1. A DNA molecule consisting of a nucleotide sequence characteristic of and specific for M.ptb, said sequence being at least 14 nucleotides in length and including at least nucleotides 406-414 of the sequence set out in Figure 2.

2. A DNA molecule as claimed in claim 1 which has a nucleotide sequence consisting of 20 consecutive nucleotides from Figure 2. o 3. A DNA molecule as claimed in claim 1 which is approximately 1.45 kb in length.

4. A )NA molecule as claimed in claim 3 wherein the nucleotide sequence e 9 consists of nucleotides 4-1456 of the sequence set out in Figure 2.

5. A DNA molecule as claimed in claim 1 which is approximately 1.6 kb in length. a

6. A DNA molecule as claimed in claim 5 consisting of the entire lnucleotide sequence of Figure 2.

7. A DNA molecule as claimed in any one of claims 1-6 consisting of part of all of the M.ptb-sourced DNA contained within E.coli (ATCC 68128).

8. A DNA molecule consisting of a nucleotide sequence characteristic of and specific for M.ptb which sequence hybridises at high stringency with a DNA molecule as defined in any one of claims 1-7.

9. A recombinant plasmid which includes a DNA molecule as claimed in any one of claims 1-6. -19- Recombinant plasmid pAM-3 (as herein defined).

11. A diagnostic probe consisting of a DNA molecule as claimed in any one of claims 1-6 optionally carrying a revealing label.

12. A diagnostic probe consisting of a DNA molecule as claimed in claim 7 or claim 8 optionally carrying a revealing label.

13. A diagnostic probe based upon all or part of nucleotide sequence of the DNA molecule of claim 4, optionally carrying a revealing label.

14. A diagnostic probe consisting of a plasmid as claimed in claim 9 or claim 10 optionally carrying a revealing label. A method for detecting the presence or absence of M.ptb in a sample ***which comprises the step of assaying said sample for M-ptb with a diagnostic probe as defined in any one of claims 11-14.

16. A method as claimed in claim 15 including the preliminary step of 9amplifying any M.ptb DNA present in said sample having a selected nucleotide sequence, said sequence being hybridisable to said probe.

17. A method as claimed in claim 16 which comprises the steps: Mt(i forming a reaction mixture including DNA from said sample, deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP, oligonucleotide primers defining each end of said selected nucleotide sequence and a DNA polmerase; (ii) heating said reaction mixture to a first temperature to dissociate said DNA into single strands; (iii) cooling said reaction mixture to a second temperature to allow annealing of the primers to the dissociated strands; (iv) maintaining the reaction mixture at a third temperature to allow polymerisation of the complementary strand to occur; I repeating steps for a predetermined number of times; and (vi) detecting the said probe.

18. A method as defined diagnostic probe is

19. A method as defined diagnostic probe is

20. A method as defined diagnostic probe is presence/absence of the amplified sequence with in any one of claims 15-17 wherein the defined in claim 11. in any one of claims 15-17 wherein the defined in claim 13. in any one of claims 15-17 wherein the pAM-3 carrying a revealing label. 9* 0q 9 9, 9 5 9 .9 a (t9

21. A diagnostic kit suitable for use in detecting the presence or absence of M.ptb in a sample which kit includes an optionally labelled diagnostic probe as defined in any one of claims 11-14.

22. A DNA molecule as defined in claim 1 substantially as herein described with reference to any example thereof.

23. A diagnostic probe as defined in any one of claims 11-14 substantially as herein described with reference to any example thereof.

24. A method for detecting the presence or absence of M.ptb in a sample substantially as herein described with reference to any example thereof or to the accompanying drawings. Dated this 10th day of September, 1992 MASSEY UNIVERSITY By its Patent Attorneys DAVIES COLLISON CAVE