WO1996008579A1

WO1996008579A1 - Methods for the detection of pathogenic mycobacteria

Info

Publication number: WO1996008579A1
Application number: PCT/NZ1995/000089
Authority: WO
Inventors: Alan Murray; Eamonn Patrick Gormley
Original assignee: Alan Murray; Eamonn Patrick Gormley
Priority date: 1994-09-16
Filing date: 1995-09-18
Publication date: 1996-03-21
Also published as: AU3621095A

Abstract

This invention relates to methods of detecting pathogenic mycobacteria with reference to a characteristic 152 bp DNA sequence. Identification of the specific species of pathogenic mycobacteria is also possible through reference to both the characteristic DNA sequence above and the genomic DNA which flanks it in that particular species.

Description

METHODS FOR THE DETECTION OF PATHOGENIC MYCOBACTERIA

This invention relates to methods for the detection of pathogenic mycobacterial organisms, to nucleic acid probes and amplification primers suitable for use in such methods and to diagnostic kits containing such probes and/or primers.

BACKGROUND

Members of the genus Mycobacterium are acid fast organisms which can be subdivided into either fast or slow growing species. Among the slow growing species, there are a number which cause significant morbidity and mortality in both man and animals. In the developed world, the most important species in this respect belong to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex and Mycobacterium paratuberculosis. These organisms are die causative agents of tuberculosis in man and animals (particularly cattle), tuberculosis in birds and a chronic enteritis (Johne's disease) in ruminant animals. In addition, strains of the M. avium complex have become important opportunistic colonisers of patients with acquired immunodeficiency syndrome (AIDS).

Traditional diagnostic methods for the identification and differentiation of mycobacteria include acid-fast staining, the growth characteristics of the organism and a complex series of biochemical tests. However, these tests are expensive, time-consuming and sometimes inconclusive.

It is accordingly an object of the present invention to provide methods for the detection of pathogenic mycobacterial organisms which are specific, sensitive and rapid, or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect the invention consists in a method of detecting the presence or absence of one or more of a group of pathogenic mycobacterial organisms in a sample comprising the step of testing DNA contained in or from said sample to detect the presence or absence of part or all of a double-stranded DNA molecule characteristic of said organisms, one strand of said DNA molecule consisting of nucleotides 1 to 152 of the nucleotide sequence of Figure 1.

The term "pathogenic" as used herein includes all mycobacterial species conventionally regarded as causing disease or illness, as well as attenuated strains of such species which are now avirulent. Examples of "pathogenic" mycobacteria are therefore M. tuberculosis, M. paratuberculosis, M. avium and bovis (including M. bovis strain BCG). In a preferred embodiment, the testing step comprises:

(i) testing said sample with an optionally labelled nucleic acid probe capable of hybridising to part or all of one strand of said characteristic 152 bp DNA molecule, said testing being performed under conditions such that said probe will hybridise to said characteristic 152 bp DNA molecule if present to form hybrid DNA;

(ii) detecting the presence or absence of said hybrid DNA in the sample.

In this embodiment it is further preferred that the testing be performed under high stringency hybridisation conditions.

Alternatively, said testing step includes the sub-steps of:

(i) amplifying part or all of said characteristic 152 bp DNA molecule if present in said sample; (ii) detecting the presence or absence or said amplified DNA in said sample.

Suitably, the amplification procedure employed is the polymerase chain reaction, the ligase detection reaction or the ligase chain reaction.

In a further aspect, the invention can be said to consist in a method of detecting the presence or absence of a specific species of pathogenic mycobacterial organism in a sample comprising the steps of:

(a) testing DNA contained in or from said sample to detect the presence or absence of part or all of a double-stranded DNA molecule characteristic of pathogenic mycobacteria, one strand of said DNA molecule consisting of nucleotides 1 to 152 of the nucleotide sequence of Figure 1;

(b) where said DNA molecule is present in said sample, identifying the specific species of pathogenic mycobacterial organism with reference to the genomic DNA flanking said DNA molecule.

Preferably, said identification step (b) involves analysis of the flanking genomic DNA by restriction fragment length polymorphism (RFLP).

In still a further aspect, the invention consists of a method of detecting and identifying one or more of a group of pathogenic mycobacterial organisms in a sample which may contain said organisms comprising the steps of:

(i) testing DNA contained in or from said sample:

(a) with a primary labelled nucleic acid probe capable of hybridising to part or all of one strand of a double-stranded DNA molecule characteristic of pathogenic mycobacteria, one strand of said DNA molecule consisting of nucleotides 1-152 of the nucleotide sequence of Figure 1, said testing being performed under conditions such that said probe will hybridise to said characteristic 152 bp DNA molecule if present in the sample to form labelled hybrid DNA; and (b) with one or more secondary labelled nucleic acid probe, each secondary probe carrying a different label and being capable of hybridising to part or all of one strand of a double-stranded DNA molecule characteristic of and specific for a different pathogenic mycobacterial species, said species-specific DNA molecule having the nucleotide sequence of the part of the genomic DNA of that mycobacterial species which flanks one end of said characteristic 152 bp

DNA molecule, said testing being performed under conditions such that each probe will hybridise to any DNA having the nucleotide sequence of said species-specific DNA molecule present in the sample to form iabeiied hybrid DNA; and (ii) detecting and identifying the specific mycobacterial species present with reference to the differential label(s) of said hybrid DNA(s) in the sample.

Conveniently, the detection step includes the addition of an amount of a DNA ligase to said sample sufficient to ligate any primary and secondary probes hybridised to adjacent regions of a DNA strand.

In still a further aspect, the invention provides a method of detecting the presence or absence of a specific species of pathogenic mycobacteria in a sample comprising the step of testing DNA contained in or from said sample to detect the presence or absence of (i) at least part of a DNA molecule, one strand of which consists of nucleotides 1-152 of the nucleotide sequence of Figure 1; and (ii) at least part of the nucleotide sequence of the genomic DNA of said species immediately flanking DNA molecule (i).

Preferably, the testing step employs a DNA amplification procedure.

Most conveniently, the amplification procedure employed is the polymerase chain reaction, and the method uses a first amplification primer which binds to one strand of part of DNA molecule (i) and a second amplification primer which binds to the opposite strand of said genomic flanking DNA (ii). In a further aspect, the invention consists in an optionally labelled nucleic acid probe capable of hybridising to part or all of one strand of a double-stranded DNA molecule characteristic of a group of pathogenic mycobacterial species, one strand of said DNA molecule consisting of nucleotides 1 to 152 of the nucleotide sequence of Figure 1.

Conveniently, the probe is an oligonucleotide of at least 14 nucleotides in length.

Alternatively, the probe comprises at least 20 consecutive nucleotides from the nucleotide sequence between nucleotide 1 and nucleotide 152 of the sequence of Figure 1.

As still a further alternative, the probe is a Bam l/Aspl digestion fragment derived from pAM-3 (ATCC 68128), a sub-fragment thereof, or has a nucleotide sequence which hybridises at high stringency thereto.

Preferably, the probe carries an identifying label.

The invention still further provides a paired set of amplification primers, said primers defining part or all of a double-stranded DNA molecule characteristic of a group of pathogenic mycobacteria, one strand of said DNA molecule consisting of nucleotides 1 to 152 of the nucleotide sequence of Figure 1.

The invention also provides a paired set of amplification primers for use in detecting the presence or absence of a specific species of pathogenic mycobacteria, one primer of said set being capable of binding to one strand of part of a double-stranded DNA molecule, one strand of said DNA molecule having a nucleotide sequence consisting of nucleotides 1-152 of Figure 1; and the other primer of said set being capable of binding to the opposite strand of part of the genomic DNA of said species immediately flanking said 152bp double stranded DNA molecule.

In still a further aspect, the invention consists in a diagnostic kit for use in detecting the presence of a pathogenic mycobacterial organism in a sample which includes an optionally labelled nucleic acid probe as defined above or amplification primers as defined above.

In a final aspect, the invention consists in a diagnostic kit for use in detecting and identifying one or more of a group of pathogenic mycobacterial organisms in a sample, said kit including: (i) a primary labelled nucleic acid probe capable of hybridising to part or all of one strand of a double-stranded DNA molecule characteristic of said organisms, one strand of said DNA molecule consisting of nucleotides 1 to 152 of the nucleotide sequence of Figure 1; and (ii) at least one secondary labelled nucleic acid probe, each probe carrying a different label and being capable of hybridising to part or all of one strand of a double- stranded DNA molecule characteristic of and specific for a different pathogenic mycobacterial species, said species-specific DNA molecule having the nucleotide sequence of the part of the genomic DNA of that mycobacterial species which flanks one end of the characteristic 152 bp DNA molecule.

DESCRIPTION OF THE DRAWINGS

While 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 but that it also includes embodiments of which the following description provides examples. Moreover, a better understanding of the present invention will be gained from reference to the accompanying drawings in which

Figure 1 sets out the nucleotide sequence of the 165 bp BamHl/Aspl fragment from pAM-3.

Figures 2 to 5 show various EcoRl digests of mycobacterial DNA.

Figure 6 shows -Scgl digests of M.bovis BCG and M.tuberculosis DNA.

DETAILED DESCRIPTION OF THE INVENTION

In previous investigations, a gene library was constructed from bovine M.paratuberculosis field isolate (M.ptb isolate 16620, ATCC 53950). The library was used to search for species-specific sequences by differential screening of replicate plaque-lifts. Recombinant phage which hybridised strongly with M.ptb but not with M.phlei were selected for further study. One of these recombinants was chosen at random and the insert DNA digested with EcoRl and -δσmHl. Following size fractionation on an agarose gel, a 1.6 Kb fragment was isolated and cloned into the plasmid vector pGEM-2 (Promega). The resulting plasmid was designated pAM-3, which when used as a probe against a wide range of bacterial DNA, was shown to be specific for M.ptb (see NZ 231429). In additional investigations, part of the pAM-3 sequence, designated P^, has been shown to function as a promoter which can drive expression of heterologous genes in the vaccine strain M.bovis BCG (see PCT/EP92/02431). P^ has been characterised as a 152 bp fragment, the sequence of one strand of which is given in Figure 1 from nucleotides 1 to 152.

Following on from the above investigations, it has now surprisingly been found that this region P^ exists in pathogenic mycobacteria such as M. tuberculosis, M. paratuberculosis, M. avium andΛ/. bovis (including M. bovis strain BCG) but not in non-pathogenic mycobacteria such as M. smegmatis and M. phlei. It is this surprising finding upon which the first aspect of the present invention is broadly based.

In this first aspect, the present invention provides a method of detecting the presence υr absence of a pathogenic mycobacterial organism in a sample suspected to contain such an organism. The essential step of the method is the testing of DNA contained in or from the sample to detect the presence or absence of part or all of the double-stranded DNA molecule which the applicants have found to be characteristic of pathogenic mycobacterial organisms. As indicated above, one strand of this characteristic DNA molecule has a nucleotide sequence consisting of nucleotides 1 to 152 of the sequence set out in Figure 1 (herein SEQ ID No.1).

As those persons skilled in the art will readily appreciate, the testing of the sample can involve any conventional procedure. It is however presently preferred that the testing step be performed using an optionally labelled nucleic acid probe capable of hybridising to part or all of one strand of the characteristic DNA molecule. In this embodiment, the testing step will proceed under conditions which will allow the probe to hybridise to the DNA of any pathogenic mycobacterial organism containing the characteristic DNA molecule which is present in the sample such that hybrid DNA is formed.

The stringency of the hybridisation conditions under which the testing step is performed can be varied as desired through, for example, variations in temperature, salt concentration and foπnamide concentration. However, it is preferred that the testing step be performed under conditions of high stringency in order to maximise the specificity of the assay.

The reaction system which is employed for the testing step may be any of those systems known in the art. However, the system selected will to a great extent depend upon the labelling of the probe. For example, where the probe is not labelled, 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 somcation 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.

In contrast, where the probe is labelled, the reaction system employed will depend upon the identity of the label used and will follow the manufacturer's instructions for that labelling system.

Following on from the hybridisation step, the second step of the method involves the detection of the presence or absence of hybrid DNA in the sample. This detection step can again employ any of those detection procedures known in the art. The actual procedure employed will however generally depend upon whether or not the probe was labelled, and if so, upon the labelling system used. For example, where the label is selected from amongst the PhotoGene (Gibco BRL), Chemiprobe (Orgenics) and Enhanced Chemi-Luminescence (Amersham) labels, the procedure adopted to detect hybrid nucleic acid molecules will vary in accordance with the manufacturer's instructions.

Those persons skilled in the art will understand that the nucleic acid probe for use in this embodiment of the invention can 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 1 82-102 (1988) which reviews this area of technology.

As will also be appreciated, the nucleic acid which forms the probe and which is capable of hybridising to the characteristic DNA molecule can be DNA or RNA. It is however preferred that the probe be a DNA probe.

The nucleic acid probe can also be capable of hybridising to either the sense or anti-sense strand of DNA coding for the characteristic DNA molecule. Conventionally, a probe specific for the sense strand (believed to be SEQ ID No.1) will be employed.

It will still further be appreciated that the probe can vary in terms of size. For example, where the probe is a DNA probe, it can comprise DNA hybridisable to one strand of the entire 152 bp nucleotide sequence of the characteristic DNA molecule. Indeed, the probe can in many cases be longer than 152 nucleotides in length provided that it remains hybridisable to part or all of one strand of the characteristic 152 bp DNA molecule under high stringency hybridisation conditions. Such a probe, which consists of the entire 1 nucleotide sequence of Figure 1 (herein SEQ ID No.2), is exemplified herein.

Alternatively, the probe may be in a form of an oligonucleotide probe of from 14 to 4 nucleotides in length which is hybridisable to any part of the characteristic 152 bp DN molecule. Such oligonucleotide probes have the advantage of being stable over time a therefore suitable for use in diagnostic kits. Oligonucleotide probes also hybridise to the target DNA at very rapid rates and are simple to prepare by synthesis using an appropria DNA synthesiser. Example of such a DNA synthesiser is the Applied Bio-Systems DN synthesiser.

As indicated above, a probe which the applicants have found to be suitable for use in th present detection method is a 165 bp probe consisting of SEQ ID No.2, preferabl labelled. Such a probe can be obtained by digestion of pAM-3 (contained withi E.coli DH5α, deposited on 18 October 1989 at the American Type Culture Collectio (ATCC) at 12301 Park Lawn Drive, Rockville, Maryland, USA under ATCC Accessio No. 68128) as & BamRVAspl digestion fragment

In the alternative to detecting the presence or absence of the characteristic 152 bp DN molecule through use of a nucleic acid probe, it will also be appreciated that othe standard techniques can be used. Examples of such techniques are the DNA amplificatio procedures.

Conveniently, the amplification step involves amplification by polymerase chain reactio (PCR) (Mullis and Fallona, Methods Enzvmolog-v. 155 335-350 (1987)). Describe generally, PCR amplification involves two oligonucleotide primers that flank th characteristic DNA sequence to be amplified and repeated cycles of heat denaturation o the DNA, annealing of the primers to their complementary sequences, and the extensio of the annealed primers with DNA polymerase. These primers hybridise to opposit strands of the target sequence and are oriented so DNA synthesis by the polymeras proceeds across the region between the primers, effectively doubling the amount of th

DNA. Moreover, since the extension products are also complementary to and capable o binding primers, each successive cycle essentially doubles the amount of DN synthesized in the previous cycle. This results in the exponential accumulation of th specific target sequence, approximately 2" where n is the number of cycles. Where PCR is to be employed and the target DNA sequence is identified (in this case, part or all of the characteristic 152 bp DNA molecule), the primers defining the sequence are generated. Suitably, these primers are generated synthetically as described above in relation to the generation of oligonucleotide probes. These oligonucleotide probes can be prelabelled to enable the detection and/or capture of the accumulated product.

Following amplification by PCR, the presence or absence of the target DNA in amplified quantities is detected. This detection step can involve any suitable procedure with gel electrophoresis, Southern blotting and restriction enzyme digestion being examples.

Separate amplification procedures which can be employed are the ligase detection reaction (LDR) and the ligase chain reaction (LCR) (Landegran et al., Science 241 1047-1080 (1988)). Described generally, these reactions involve the hybridisation of two oligonucleotide probes to denatured DNA contained in a sample with the probes being selected such that the 3' end of one probe is immediately adjacent the 5' end of the other probe when bound to the target DNA (once again, part or all of the characteristic 152 bp DNA molecule). DNA ligase is then added to covalently link these two oligonucleotides, with the linking only occurring where the entire target DNA sequence complementary to both probes is present

The hybridisation/ligation procedures can be repeated cyclically using a thermostable DNA ligase to effect linear or exponential amplification of the target DNA depending on whether probes are provided which are hybridisable to one or both strands of the target

DNA.

Again, once the amplification is complete, any conventional procedure for detecting the presence or absence of amplified target DNA in the reaction mixture can be employed. It is however preferred that the detection step be effected through the use of differentially labelled oligonucleotide probes, with the presence of the amplified target DNA in the sample being shown through the detection of both labels on a particular DNA molecule.

In addition to the general screening method described above which allows the detection of the presence or absence of a pathogenic mycobacterial organism in a sample, in a second aspect the applicants also provide a method for distinguishing between species of pathogenic mycobacterial organisms. This second aspect of the invention is based upon the applicants' further surprising finding that genomic DNA of the pathogenic mycobacterial organisms flanking the characteristic 152 bp DNA molecule is different from one mycobacterial species to another. One method of this second aspect of the invention involves a first step of detecting the presence or absence of the characteristic 152 bp DNA molecule in a sample. This step can be performed as described above and will preferably involve hybridisation of a labelled DNA probe to part or all of the characteristic DNA molecule.

Where the sample is found to contain the characteristic 152 bp DNA molecule, the second step of the method is the identification of the species of pathogenic mycobacterial organism present in the sample with reference to the genomic DNA flanking the characteristic 152 bp DNA molecule. This step can be performed in a number of ways.

As one (presently preferred) example, the identification of the pathogenic mycobacterial organism present in the sample can be made on the basis of a restriction fragment length polymorphism (RFLP) analysis. Such a procedure is exemplified herein.

In the alternative, and as a further aspect of the present invention, the presence and identity of one or more specific pathogenic mycobacterial organisms which may be present in a sample can be detected, once again in two steps.

The first step of this aspect is that of testing the sample and proceeds in two sub-steps. The first sub-step involves testing DNA contained in or from the sample with a primary labelled nucleic acid probe hybridisable to part or all of one strand of the characteristic 152 bp DNA molecule. As described previously, the probe may take a variety of forms and carry one of a variety of labels.

The second sub-step of the testing procedure involves testing the DNA with at least one secondary labelled nucleic acid probe, with each secondary probe carrying a different label to each other secondary probe as well as to the primary probe. Moreover, each secondary probe must be capable of hybridising to part or all of one strand of a double- stranded DNA molecule characteristic of and specific for a different species of pathogenic mycobacterial organism. This species-specific DNA molecule will be the part of the genomic DNA of that species of mycobacteria which flanks one end or the other of the characteristic 152 bp DNA molecule.

The secondary labelled probe or probes may once again take a variety of forms and carry any conventional label(s). It must however be borne in mind that each probe must carry a different label to all other probes employed in the method. The identity, and sequence, of the secondary probe(s) will obviously be dependent upon the sequence of the flanking genomic DNA of each pathogen. This can be determined by isolating genomic DNA from each pathogen, identifying the region containing the characteristic 152bp DNA molecule, and sequencing the DNA immediately flanking the 152 bp sequence.

As an example of this, nucleotides 153-165 of the Figure 1 sequence represent the sequence of flanking DNA fromM paratuberculosis. A secondary labelled probe can be designed to bind to this region of the M. paratuberculosis DNA.

While in no way excluding the performance of the present method with a single secondary labelled probe, it will be appreciated that it will be usual for a number of such secondary probes to be used. This will be particularly so where the sample in question is suspected to contain any one or more of a number of different pathogenic mycobacterial species.

It will also be appreciated that the sub-steps of the testing procedure can be performed either sequentially or simultaneously. Furthermore, where the sub-steps are performed sequentially, either sub-step can precede the other.

Once the testing procedure has been completed and the probes (both primary and secondary) hybridised to the DNA in the sample, the second step of the method involves the detection and identification of the mycobacterial species present in the sample with reference to the labels carried by the probes. Thus, where the sample contains a single pathogenic mycobacterial species and the sample has been tested with both the primary probe and a secondaiy probe specific for that mycobacterial species, DNA present in the sample will carry both labels. However, where the sample contains a pathogenic mycobacterial species but has not been tested with a secondary probe specific for that mycobacterial species, DNA present in the sample will cany only a single label, this being the label carried by the primary probe. In this way, the method operates as both a general screening method and a species-specific mycobacterial identification method.

While the method can be performed as outlined above, an optional although preferred step involves the addition of a DNA ligase to the sample as part of the LDR or LCR to facilitate the detection of the DNA of a particular mycobacterial species. As described previously, where the primary and secondary probes for a particular mycobacterial species are selected to have adjacent 3' and 5' ends respectively, the ligase will link the probes together, thereby increasing the ease of detection. It will further be appreciated that the method can be adapted for use as part of a PC amplification protocol. The DNA sequence of each pathogen selected for amplificatio will be defined by paired primers, one of which binds to one strand (sense or anti-sense of the characteristic 152bp DNA molecule and the other of which binds to the opposite strand (sense or anti-sense) of the genomic DNA flanking the 152bp DNA molecule.

The sequence of the primer to bind to the genomic flanking DNA strand can be determined by extracting and sequencing the flanking DNA as described previously.

The invention will now be illustrated by the following non-limiting examples.

EXAMPLES

A. MATERIALS AND METHODS

1. Isolation of the 165 bp fragment pAM-3 (ATCC 68128) was digested with the restriction endonucleases Asp 1 and BamH 1 and the resulting two fragments separated by electrophoresis on a 2% agarose gel. The

165 bp fragment, which contained the characteristic 152 bp DNA molecule, was eluted from the gel using a Geneclean 11 kit (Bio 101 Inc La Jolla) according to the manufacturer's instructions.

The nucleotide sequence of the 165 bp fragment is shown in Figure 1.

Base pairs 1-152 lie outside of the M/?/Z>-specific IS900 sequence (Green et al., Nucl Acids Res 17 9063-9073 (1989)).

Base-pairs 153-165 are identical to bp 1439-1451 of the IS900.

Aliquots of the 165 bp fragment were stored frozen at -20°C until ready for use.

2. Labelling of DNA

The Multiprime rapid hybridisation system (Amersham International, UK) was used to label the 165 bp probe. Briefly, approximately 25 ng DNA in 28 μl water was denatured at 100°C for 3 minutes then rapidly cooled on ice and 10 μl labelling buffer, 5 μl primer solution, 50 μCi^²?) dCTP and 2 μl Klenow DNA polymerase added. The reaction was incubated at room temperature overnight. Unincorporated nucleotides were removed using a NICK™ column (Pharmacia LKB Biotechnology, Uppsala, Sweden).

3. Southern blotting Genomic DNAs from selected bacteria were digested with restriction endonucleases, size fractionated on 0.9% agarose gels (unless otherwise stated) by electrophoresis and blotted onto nylon membrane (Hybond-N, Amersham International, UK) by Southern transfer using standard protocols (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Laboratory Press ( 1989)).

4. Hybridisation

The Multiprime rapid hybridisation system was used for high stringency experiments. Briefly, the probe was denatured at 100°C for 3 minutes, cooled on ice and mixed with the Southem blot in hybridisation solution to give a probe concentration of approximately 8 ng/ml. Hybridisation was performed at 65°C with constant shaking for 2 hours.

After removal of the probe, the blots were washed twice in 2X SSC, 0.1% SDS for 10 minutes at room temperature followed by IX SSC, 0.1% SDS for 15 minutes at 65°C. Finally, two washes in 0.7% SSC, 0.1% SDS for 15 minutes at 65°C were done and the membranes exposed to Kodak X-Omat X-RAY film overnight at -70°C with an intensifying screen (Cronex, DuPont).

Low stringency hybridisation was carried out in hybridisation solution containing 35% formamide at 37°C for 16 hours (Sambrook et al. (1989)). Washes were done in 5X SSC, 0.1% SDS at room temperature for one hour.

To re-use Southern blots, the membranes were incubated at 45°C for 30 minutes in 0.4 M

NaOH and then transferred to a solution containing 0.1X SSC, 0.1% (w/v) SDS, 0.2 M Tris-HCl pH 7.5 for 15 minutes. This treatment removes the bound probe from the membrane. Autoradiography of washed blots confirmed the absence of detectable signal.

B. EXPERIMENTAL

The following experiments illustrate the use of the invention.

Experiment 1

Genomic DNA from the following bacteria was digested with -EcoRl and blotted onto nylon membrane by Southem transfer: M.paratuberculosis (M.ptb) (ATCC 53950), M.ptb (caprine field isolate Scand IP), M.ptb (bovine field isolate Vic 820/89), M.avium serovar 2 (TMC 714), M.bovis (field isolate KML) and M.smegmatis (field isolate K5). After hybridisation with the probe at high stringency, the autoradiograph was examined after overnight exposure with the results being shown in Figure 2. The autoradiograph revealed a single band in each of the mycobacteria except i M.smegmatis which gave no signal. The three positively reacting species gave a band a different position, i.e. they could be differentiated on the basis of an RFLP. Th approximate sizes of the bands are: Kb

M.bovis 9.5

M.ptb isolates 3.8

M.avium 2.2

In a separate experiment using the same conditions, other bacterial DNAs were tested M.bovis (field isolate KML), M.bovis (field isolate 82), M.bovis (field isolate 83), M.bovi BCG (Institut Pasteur), M.ptb (bovine field isolate 820/89), M.phlei (Massey Universit field isolate), E.coli (Massey University field isolate), with the results shown in Figur 3. A single band was seen with the M.bovis, M.bovis BCG and M.paratuberculosi isolates. No signal was obtained with M.phlei and E.coli.

The approximate sizes of the bands are:

Kb

M.bovis isolates 9.5 M. bovis BCG 6.5

M.ptb 3.8

In yet a further experiment, genomic DNA from a human isolate of M. tuberculosis was digested with EcoRl and included on a gel with M.bovis and M.bovis BCG. When the 165 bp probe was hybridised to the Southern blot of this gel, a single band was seen in each of the three species (Figure 4). The approximate sizes of the bands are:

Kb

M. tuberculosis 6.5

M.bovis BCG 6.5 M.bovis 9.5

Thus, M.tuberculosis and M.bovis BCG could not be differentiated when the DNA was digested with -EcoRl.

Experiment 2

Two of the Southern blots from Experiment 1 (Figures 2 and 3) were washed to remove the probe and re-probed under conditions of low stringency. Under these conditions a single strong signal was again seen ia M.bovis, M.ptb and M.avium in the same position as seen under high stringency. In addition, three minor bands appeared in the M.ptb isolates. However, no signal was observed with M.smegmatis, M.phlei and E.coli (Figure 5)-

Experiment 3

In an attempt to distinguish between M. tuberculosis and M.bovis BCG, DNA from these mycobacteria were digested with -spl, Aval 1, BamHl, BsfiLl 1, Hindi 1, Nrul, Pstl, Sail and Bcgl, electrophoresed, blotted and probed with the 165 bp fragment. A single band RFLP was demonstrated only with the Bcgl digest (Figure 6).

The approximate size of the fragments with the Bcgl digest:

Kb M.tuberculosis 1.0

M.bovis BCG 1.1

C. DISCUSSION

The above experiments, illustrating the use of the invention, show that the ³²P-labelled 165 bp fragment hybridises specifically to pathogenic mycobacteria under conditions of high or low stringency. In addition, M.avium serovar 2, M.bovis, M.bovis BCG, M.ptb and M. tuberculosis can be differentiated from each other on the basis of a RFLP.

A summary of fragment sizes for EcoRl and Bcgl digests respectively is as follows:

Kb Kb

M.avium serovar 2 2.2 ND M. bovis 9.5 ND

M. bovis BCG 6.5 1.1

M.ptb 3.8 ND

M.tuberculosis 6.5 1.0

(ND means Not Determined)

In still a further aspect, the invention provides diagnostic kits for use in the detection of pathogenic mycobacterial organisms in a sample. Such kits can include as components at least one nucleic acid probe with the identity of the probe being dependent on its intended method of use. For example, where the probe is to be used to generally screen a sample for the presence or absence of any pathogenic mycobacterial organism, the probe will normally be a labelled DNA probe hybridisable to part or all of one strand of the double-stranded DNA molecule having the nucleotide sequence of nucleotides 1-152 of the Figure 1 sequence. Further, this probe could also be used in the species-specific identification method of the invention, where the mycobacterial species present is identified on the basis of a RFLP.

In the alternative, where the method which is to be employed is simultaneously a general screen and a species-specific identification method, the kit can include a probe labelled as above as a primary probe together with one or more secondary labelled probes capable of identifying the species of mycobacteria in question with reference to the genomic DNA of that mycobacterial species which flanks the characteristic 152 bp DNA molecule.

In addition to the probe, the kit may also include one or more of the following:

(a) a lysing agent capable of lysing the cells in the sample of the medium to be tested;

(b) a denaturing solution;

(c) a neutralising solution;

(d) an alkaline fixation solution; and (e) Standard Saline Citrate (SSC).

Each of the above components can be selected from those known and available in the art and provided in appropriate amounts.

The kit can also be intended for use with an amplification procedure such as PCR In this case, the kit will include paired amplification primers. For use in a general screening method, these primers will be selected to define part or all of the characteristic double- stranded DNA molecule (nucleotides 1-152 of the Figure 1 sequence (SEQ ID No. l)). For use in species-specific identification, the primers will be selected for each pathogen so that one binds to part of one strand of the characteristic 152 bp DNA molecule, and the other binds to the opposite strand of the genomic DNA of the pathogen which immed ately flanks the characteristic 152 bp DNA molecule.

In this format, the kit will desirably also include other conventional components supplied for use in amplification procedures.

INDUSTRIAL APPLICATION

Thus, in accordance with the present invention the applicants have inter alia provided a method of quickly, accurately and reliably differentiating between pathogenic and non- pathogenic mycobacteria. In addition, the applicants have provided methods of differentiating between different but closely related species of pathogenic mycobacteria. These latter methods in particular offer substantial advantages over the mycobacterial identification methods presently employed. It will be appreciated by those persons skilled in the art that the description provided above is by way of example only and that the present invention is limited only by the lawful scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

( 1 ) APPLICANT: ALAN MURRAY, a joint British and New Zealand citizen o 6 Williams Terrace, Palmerston North, New Zealand; and EAMON PATRICK GORMLEY, a citizen of the Republic of Ireland of 80 Wikiriwh

Crescent, Palmerston North, New Zealand

(2) TITLE OF INVENTION: METHODS FOR THE DETECTION OF PATHOGENIC MYCOBACTERIA

(3) NUMBER OF SEQUENCES: 2

(4) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: A J PARK & SON (B) STREET: HUDDART PARKER BUILDING, POST OFFICE SQUARE

(C) CITY: P O BOX 949, WELLINGTON

(D) COUNTRY: NEW ZEALAND

(5) COMPUTER READABLE FORM: (A) MEDπJM TYPE: 3.5,DS,HD FLOPPY DISC

(B) COMPUTER: IBM PC COMPATIBLE

(C) OPERATION SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 6.1 FOR WINDOWS

(6) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 18 SEPTEMBER 1995

(C) CLASSIFICATION:

(7) ATTORNEY/AGENT INFORMATION:

(A) NAME: BENNETT, MICHAEL R.

(8) TELECOMMUNICATION INFORMATION (A) TELEPHONE: (644) 473 8278 (B) TELEFAX: (644) 4723358

472 3351 (2) INFORMATION FOR SEQUENCE ID NO. 1 :

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 152 BASE PAIRS

(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 1:

GATCCCGTGA CAAGGCCGAA GAGCCCGCGA CCGTGCGGTC GTCGACGACC GAGTGTGAGC AGACCCCCTG GTGAAGGGTG AATCGACAGG TACACACAGC CGCCATACAC TTCGCTTCAT GCCCTTACGG GGGGCGGCCA ACCCAGAAGG AG

(3) INFORMATION FOR SEQUENCE ID NO. 2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 165 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

GATCCCGTGA CAAGGCCGAA GAGCCCGCGA CCGTGCGGTC GTCGACGACC GAGTGTGAGC AGACCCCCTG GTGAAGGGTG AATCGACAGG TACACACAGC CGCCATACAC TTCGCTTCAT GCCCTTACGG GGGGCGGCCA ACCCAGAAGG AGATTCTCAA TGACG

Claims

CLAIMS:

1. A method of detecting the presence or absence of one or more of a group o pathogemc mycobacterial organisms in a sample comprising the step of testing DN contained in or from said sample to detect the presence or absence of part or all of double-stranded DNA molecule characteristic of said orgamsms, one strand of said DN molecule consisting of nucleotides 1 to 152 of the nucleotide sequence of Figure 1.

2. A method as claimed in claim 1 wherein the testing step comprises: (i) testing said sample with an optionally labelled nucleic acid probe capable of hybridising to part or all of one strand of said characteristic 152 bp DN molecule, said testing being performed under conditions such that said probe will hybridise to said characteristic 152 bp DNA molecule if present to form hybrid DNA; (ii) detecting the presence or absence of said hybrid DNA in the sample.

3. A method as claimed in claim 1 wherein said testing step includes the sub-steps of: (i) amplifying part or all of said characteristic 152 bp DNA molecule if present in said sample; (ii) detecting the presence or absence or said amplified DNA in said sample.

4. A method as claimed in claim 3 wherein the amplification procedure employed is the polymerase chain reaction, the ligase detection reaction or the ligase chain reaction.

5. A method of detecting the presence or absence of a specific species of pathogenic mycobacterial organism in a sample comprising the steps of:

(b) where said DNA molecule is present in said sample, identifying the specific species of pathogemc mycobacterial organism with reference to the genomic DNA flanking said DNA molecule.

6. A method as claimed in claim 5 wherein said identification step (b) involves analysis of the flanking genomic DNA by restriction fragment length polymorphism (RFLP).

7. A method of detecting and identifying one or more of a group of pathogenic mycobacterial organisms in a sample which may contain said organisms comprising the steps of:

(i) testing said sample: (a) with a primary labelled nucleic acid probe capable of hybridising to part or all of one strand of a double-stranded DNA molecule characteristic of pathogenic mycobacteria, one strand of said DNA molecule consisting of nucleotides 1-152 of the nucleotide sequence of Figure 1, said testing being performed under conditions such that said probe will hybridise to said characteristic 152 bp DNA molecule if present in the sample to form labelled hybrid DNA; and (b) with one or more secondary labelled nucleic acid probe, each secondary probe carrying a different label and being capable of hybridising to part or all of one strand of a double-stranded DNA molecule characteristic of and specific for a different pathogenic mycobacterial species, said species- specific DNA molecule having the nucleotide sequence of the part of the genomic DNA of that mycobacterial species which flanks one end of said characteristic 152 bp DNA molecule, said testing being performed under conditions such that each probe will hybridise to any DNA having the nucleotide sequence of said species-specific DNA molecule present in the sample to form labelled hybrid DNA; and (ii) detecting and identifying the specific mycobacterial species present with reference to the differential label(s) of said hybrid DNA(s) in the sample.

8. A method of detecting die presence or absence of a specific species of pathogenic mycobacteria in a sample comprising the step of testing DNA contained in or from said sample to detect the presence or absence of

(i) at least part of a DNA molecule, one strand of which consists of nucleotides 1-152 of the nucleotide sequence of Figure 1; and (ii) at least part of the nucleotide sequence of the genomic DNA of said species immediately flanking DNA molecule (i).

9. A method as claimed in claim 8 which employs a DNA amplification procedure.

10. A method as claimed in claim 9 wherein the amplification procedure employed is the polymerase chain reaction, and wherein the method uses a first amplification primer which binds to one strand of part of DNA molecule (i) and a second amplification primer which binds to the opposite strand of said genomic flanking DNA (ii).

11. An optionally labelled nucleic acid probe capable of hybridising to part or all o one strand of a double-stranded DNA molecule characteristic of a group of pathogeni mycobacterial species, one strand of said DNA molecule consisting of nucleotides 1 t 152 of the nucleotide sequence of Figure 1.

12. A probe as claimed in claim 11 which is an oligonucleotide of at least 1 nucleotides in length.

13. A probe as claimed in claim 11 which comprises at least 20 consecutive nucleotide from the nucleotide sequence between nucleotide 1 and nucleotide 152 of the sequenc of Figure 1.

14. A probe as claimed in claim 11 which is a Bair.W Aspl digestion fragment derive from pAM-3 (ATCC 68128), a sub-fragment thereof, or has a nucleotide sequence whic hybridises at high stringency thereto.

15. A probe as claimed in any one of claims 11 to 14 carrying an identifying label.

16. A paired set of amplification primers, said primers defining part or all of a double- stranded DNA molecule characteristic of a group of pathogenic mycobacteria, one strand of said DNA molecule consisting of nucleotides 1 to 152 of the nucleotide sequence o Figure 1.

17. A paired set of amplification primers for use in detecting the presence or absence of a specific species of pathogenic mycobacteria, one primer of said set being capable of binding to one strand of part of a double-stranded DNA molecule, one strand of said DNA molecule having a nucleotide sequence consisting of nucleotides 1-152 of Figure 1 and the other of said set being capable of binding to the opposite strand of part of the genomic DNA of said species immediately flanking said 152 bp double-stranded DNA molecule.

18. A d agnostic kit for use in detecting the presence of a pathogenic mycobacterial organism in a sample which includes a nucleic acid probe as claimed in any one of claims 11 to 15 or a set of primers as claimed in claim 16 or claim 17.

19. A diagnostic kit for use in detecting and identifying one or more of a group of pathogemc mycobacterial organisms in a sample, said kit including: (i) a primary labelled nucleic acid probe capable of hybridising to part or all of one strand of a double-stranded DNA molecule characteristic of said organisms, one strand of said DNA molecule consisting of nucleotides 1 to 152 of the nucleotide sequence of Figure 1; and (ii) at least one secondaiy labelled nucleic acid probe, each probe carrying a different label and being capable of hybridising to part or all of one strand of a double- stranded DNA molecule characteristic of and specific for a different pathogenic mycobacterial species, said species-specific DNA molecule having the nucleotide sequence of the part of the genomic DNA of that mycobacterial species which flanks one end of the characteristic 152 bp DNA molecule.