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WO1996008579A1 - Procedes de detection de mycobacteries pathogenes - Google Patents

Procedes de detection de mycobacteries pathogenes Download PDF

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Publication number
WO1996008579A1
WO1996008579A1 PCT/NZ1995/000089 NZ9500089W WO9608579A1 WO 1996008579 A1 WO1996008579 A1 WO 1996008579A1 NZ 9500089 W NZ9500089 W NZ 9500089W WO 9608579 A1 WO9608579 A1 WO 9608579A1
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WIPO (PCT)
Prior art keywords
dna molecule
dna
sample
strand
probe
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PCT/NZ1995/000089
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English (en)
Inventor
Alan Murray
Eamonn Patrick Gormley
Original Assignee
Alan Murray
Eamonn Patrick Gormley
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Alan Murray, Eamonn Patrick Gormley filed Critical Alan Murray
Priority to AU36210/95A priority Critical patent/AU3621095A/en
Publication of WO1996008579A1 publication Critical patent/WO1996008579A1/fr

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    • 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

Definitions

  • 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.
  • 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).
  • AIDS acquired immunodeficiency syndrome
  • 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 testing step comprises:
  • testing be performed under high stringency hybridisation conditions.
  • said testing step includes the sub-steps of:
  • the amplification procedure employed is the polymerase chain reaction, the ligase detection reaction or the ligase chain reaction.
  • 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:
  • said identification step (b) involves analysis of the flanking genomic DNA by restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • 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:
  • 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.
  • 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.
  • 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).
  • the testing step employs a DNA amplification procedure.
  • 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).
  • 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.
  • the probe is an oligonucleotide of at least 14 nucleotides in length.
  • the probe comprises at least 20 consecutive nucleotides from the nucleotide sequence between nucleotide 1 and nucleotide 152 of the sequence of Figure 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 sets out the nucleotide sequence of the 165 bp BamHl/Aspl fragment from pAM-3.
  • FIGS 2 to 5 show various EcoRl digests of mycobacterial DNA.
  • Figure 6 shows -Scgl digests of M.bovis BCG and M.tuberculosis DNA.
  • 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 - ⁇ mHl. Following size fractionation on an agarose gel, a 1.6 Kb fragment was isolated and cloned into the plasmid vector pGEM-2 (Promega).
  • pAM-3 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).
  • P ⁇ 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.
  • the present invention provides a method of detecting the presence ⁇ r 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.
  • 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).
  • 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 fo ⁇ namide 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.
  • 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.
  • the reaction system employed will depend upon the identity of the label used and will follow the manufacturer's instructions for that labelling system.
  • 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.
  • 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.
  • nucleic acid probe for use in this embodiment of the invention can take a variety of forms.
  • 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.
  • a probe specific for the sense strand (believed to be SEQ ID No.1) will be employed.
  • the probe can vary in terms of size.
  • the probe can comprise DNA hybridisable to one strand of the entire 152 bp nucleotide sequence of the characteristic DNA molecule.
  • 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.
  • 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.
  • 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.
  • 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 DH5 ⁇ , 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
  • 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 PCR amplification.
  • PCR polymerase chain reactio
  • 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.
  • the primers defining the sequence are generated.
  • 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.
  • This detection step can involve any suitable procedure with gel electrophoresis, Southern blotting and restriction enzyme digestion being examples.
  • LDR ligase detection reaction
  • LCR ligase chain reaction
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • RFLP restriction fragment length polymorphism
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DNA present in the sample will carry both labels.
  • the method operates as both a general screening method and a species-specific mycobacterial identification method.
  • 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.
  • a DNA 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.
  • 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.
  • 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.
  • 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.
  • the Multiprime rapid hybridisation system (Amersham International, UK) was used to label the 165 bp probe. Briefly, approximately 25 ng DNA in 28 ⁇ l water was denatured at 100°C for 3 minutes then rapidly cooled on ice and 10 ⁇ l labelling buffer, 5 ⁇ l primer solution, 50 ⁇ Ci ⁇ 2 ?) dCTP and 2 ⁇ l Klenow DNA polymerase added. The reaction was incubated at room temperature overnight. Unincorporated nucleotides were removed using a NICKTM column (Pharmacia LKB Biotechnology, Uppsala, Sweden).
  • 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.
  • 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).
  • 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).
  • M.paratuberculosis M.ptb
  • TMC 714 M.avium serovar 2
  • M.bovis field isolate KML
  • M.smegmatis field isolate K5
  • 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
  • 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.
  • 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:
  • M.tuberculosis and M.bovis BCG could not be differentiated when the DNA was digested with -EcoRl.
  • kits for use in the detection of pathogenic mycobacterial organisms in a sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • the kit may also include one or more of the following:
  • the kit can also be intended for use with an amplification procedure such as PCR
  • the kit will include paired amplification primers.
  • 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)).
  • 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.
  • the kit will desirably also include other conventional components supplied for use in amplification procedures.
  • the applicants have inter alia provided a method of quickly, accurately and reliably differentiating between pathogenic and non- pathogenic mycobacteria.
  • the applicants have provided methods of differentiating between different but closely related species of pathogenic mycobacteria.
  • ADDRESSEE A J PARK & SON
  • B STREET: HUDDART PARKER BUILDING, POST OFFICE SQUARE
  • GATCCCGTGA CAAGGCCGAA GAGCCCGCGA CCGTGCGGTC GTCGACGACC GAGTGTGAGC AGACCCCCTG GTGAAGGGTG AATCGACAGG TACACACAGC CGCCATACAC TTCGCTTCAT GCCCTTACGG GGGGCGGCCA ACCCAGAAGG AG
  • GATCCCGTGA CAAGGCCGAA GAGCCCGCGA CCGTGCGGTC GTCGACGACC GAGTGTGAGC AGACCCCCTG GTGAAGGGTG AATCGACAGG TACACACAGC CGCCATACAC TTCGCTTCAT GCCCTTACGG GGGGCGGCCA ACCCAGAAGG AGATTCTCAA TGACG

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Abstract

Procédés de détection de mycobactéries pathogènes par référence à une séquence d'ADN caractéristique 152 bp. On peut également identifier spécifiquement l'espèce de mycobactéries pathogènes par référence aussi bien à la séquence d'ADN caractéristique précitée qu'à l'ADN génomique qui l'encadre dans l'espèce en question.
PCT/NZ1995/000089 1994-09-16 1995-09-18 Procedes de detection de mycobacteries pathogenes WO1996008579A1 (fr)

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Application Number Priority Date Filing Date Title
AU36210/95A AU3621095A (en) 1994-09-16 1995-09-18 Methods for the detection of pathogenic mycobacteria

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NZ264473 1994-09-16
NZ26447394 1994-09-16

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6672790A (en) * 1989-11-17 1991-05-23 Massey University Diagnostic probe for use in the detection of m.paratuberculosis
AU4114793A (en) * 1992-04-28 1993-11-29 Gen-Probe Incorporated Nucleic acid process probes to (mycobacterium tuberculosis)
NZ244901A (en) * 1991-10-25 1995-07-26 Pasteur Institut Mycobacterium paratuberculosis nucleotide sequence and its use in vectors and host cells for cloning and expression of nucleotide sequences; vaccines containing the host cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6672790A (en) * 1989-11-17 1991-05-23 Massey University Diagnostic probe for use in the detection of m.paratuberculosis
NZ244901A (en) * 1991-10-25 1995-07-26 Pasteur Institut Mycobacterium paratuberculosis nucleotide sequence and its use in vectors and host cells for cloning and expression of nucleotide sequences; vaccines containing the host cells
AU4114793A (en) * 1992-04-28 1993-11-29 Gen-Probe Incorporated Nucleic acid process probes to (mycobacterium tuberculosis)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CLIN. MICRO. REV., Vol. 1, No. 1, 1988, TENOVER F.C., "Diagnostic Deoxyribonucleic Acid Probes for Infectious Diseases", pages 82-101. *

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