WO1990001560A1 - Sonde d'adn bacterienne - Google Patents

Sonde d'adn bacterienne Download PDF

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Publication number
WO1990001560A1
WO1990001560A1 PCT/GB1989/000850 GB8900850W WO9001560A1 WO 1990001560 A1 WO1990001560 A1 WO 1990001560A1 GB 8900850 W GB8900850 W GB 8900850W WO 9001560 A1 WO9001560 A1 WO 9001560A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna probe
oligonucleotide
probe
sample
iii
Prior art date
Application number
PCT/GB1989/000850
Other languages
English (en)
Inventor
David John Evans
Francis Ian Lamb
Original Assignee
David John Evans
Francis Ian Lamb
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
Priority to KR1019900700729A priority Critical patent/KR900701791A/ko
Application filed by David John Evans, Francis Ian Lamb filed Critical David John Evans
Publication of WO1990001560A1 publication Critical patent/WO1990001560A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • testing is carried out to detect the presence of bacteria. This is important, for example, in medical diagnostics, in the food, beverage and pharmaceutical industries, in the supply of water, and in public health work in general. It may be necessary or desirable to detect not just live bacteria but also or alternatively non-viable bacteria.
  • the testing may be carried out routinely, for example, for quality control purposes, or in the context of a specific investigation of bacterial contamination.
  • the methods generally used include microscopy after staining, and culture and colony counting. All these methods have disadvantages, for example, there is no stain that will reliably identify all bacteria. Furthermore, non-viable organisms may lose their normal staining properties, for example, non-viable Gram positive organisms may lose their positivity. It is, of course, 'not possible to culture organisms that are not viable, and even with viable organisms success is critically dependant on the choice of medium. Moreover, with certain organisms in vitro culture is difficult, and for some organisms, for example, T ⁇ pallidum, in vitro culture has not yet been achieved.
  • a particular problem is to detect bacteria in the presence of cells of higher organisms, for example, mammalian, avian, fish or plant cells. This problem is encountered especially in medical diagnostics, in the food industry and in public health work.
  • An example of a particular problem for conventional methods is the detection of prior bacterial contamination in food that has been treated by X -irradiation.
  • a further problem area is in the diagnosis of diseases in which bacteria are believed to be implicated but the aetiology of which is unknown.
  • the present invention relates to a method for detecting and/or determining the presence of bacteria, either viable or non-viable.
  • the present invention provides a DNA probe comprising an oligonucleotide of formula I, II, III . TV or V
  • V 5'-TTACTCACCCGTTCGCCGCT-3 » (V) or an oligonucleotide complementary to an oligonucleotide of formulaI, II, III, IV or V, (ie the corresponding cDNAs), or an oligonucleotide sub-sequence of 7 or more contiguous bases of an oligonucleotide of formula I, II,
  • a DNA probe comprising an oligonucleotide of formula I or an oligonucleotide sub-sequence thereof should especially be mentioned! and also the corresponding cDNAs.
  • a DNA probe of the present invention is capable of hybridising with bacterial nucleic acid material substantially exclusively, that is to say, it hybridises with prokaryotic nucleic acid material but does not hybridise substantially detectably with viral or eukaryotic nucleic acid material. Accordingly, an oligonucleotide or an oligonucleotide sub-sequence thereof of the present invention can function as a DNA probe specific for the detection of bacteria. It is particularly useful to use a DNA probe of the invention to hybridise with riboso al RNA (rRNA) . .
  • rRNA riboso al RNA
  • the present invention also provides a DNA probe that comprises any sub-sequence of an oligonucleotide of formula I, II, III, IV or V, or of a corresponding complementary oligonucleotide, that retains the ability to hybridise exclusively with bacterial nucleic acid material.
  • An oligonucleotide sub-sequence of the invention generally comprises at least 7 contiguous bases, preferably more than 7, more preferably 10 or more, and especially 16 or more contiguous bases.
  • An oligonucleotide sub-sequence of the invention may have less than 7 contiguous bases provided that it retains the ability to hybridise exclusively with bacterial nucleic acid material.
  • the present invention further provides a DNA probe which comprises an oligonucleotide of formula I, ⁇ , m, iv V, or a corresponding complementary oligonucleotide, or an oligonucleotide sub-sequence thereof, the oligonucleotide or sub-sequence thereof having one or more base changes, rearrangements, additions or deletions with the proviso that the resulting DNA probe is capable of hybridising with bacterial nucleic acid material exclusively.
  • a base change is especially one that result from the degeneracy of the genetic code.
  • Any oligonucleotide or oligonucleotide sub-sequence of the present invention may be part of a larger nucleotide with the proviso that the resulting larger nucleotide is capable of hybridising exclusively with bacterial nucleic acid material.
  • a DNA probe of the present invention may be produced by chemically linking the appropriate deoxyribonucleotides, as individual base(s) and/or as subsequence(s) of the desired probe, using any chemical method of DNA synthesis, see for example, Caruthers al ⁇ (1982) New Methods for Synthesising Deoxynucleotides, in "Genetic Engineering” eds Setlow J. K. and Hollaender A. pp 119-45, Plenum Press and Narang S. A. (1983) DNA Synthesis Tetrahedron 39.3-22.
  • Solid phase synthesis is generally preferred, for example, there may be used a solid phase synthesiser, for example, Cyclone synthesiser made by Biosearch Inc. (New Brunswick Scientific Co.), the precursors used being phosphoramidites (L. J. McBride and M. H. Caruthers, Tetrahedron Lett., 2 : 245, 1983).
  • a double stranded oligonucleotide may be produced initially by chemical synthesis and then cloned in a suitable bacterial host. Such methods have been described, for example in T. Maniatis et al "Molecular Cloning - A
  • the present invention also provides a
  • DNA probe of the invention when produced by recombinant DNA technology.
  • chemical synthesis is generally the method of choice.
  • the present invention provides a method of detecting and/or determining bacteria, which comprises contacting a solid or liquid sample under investigation with a DNA probe of the invention under hybridising conditions and detecting and/or determining hybridised DNA.
  • the present invention further provides a DNA probe of the present invention for use as in the detection and/or determination of bacteria using a hybridisation technique.
  • the DNA probe of the present invention hybridises exclusively with bacterial nucleic acid material, that is to say, with DNA and/or RNA from bacteria but not with nucleic acids from other life forms, for example, viruses, fungi or higher life forms.
  • Detection of bacterial nucleic acid material may be carried out using any qualitative or quantitative method of nucleic acid hybridisation, for example. Southern blotting. Northern blotting, dot blotting or hybridisation in solution. Such methods are all well known.
  • Northern blotting may be carried out in the present invention, for example, as follows:
  • the nucleic acid material is extracted by lysis, generally by physical or chemical means, for example, chemical lysis with guanidinium chloride or physical lysis by sonication, followed by extraction, generally with chloroform, and precipitation, generally with ethanol.
  • the extracted nucleic acid material is bound to a solid support, for example, diazotised aminobenzyloxymethyl paper or nitrocellulose or charge-modified Nylon, for example, a nitrocellulose filter.
  • the bound nucleic acid material is hybridised, optionally in the presence of formamide, with a probe of the invention that has previously been labelled.
  • Specifity of hybridisation is controlled by subsequent washing of the hybridised material, for example, by the number of washes, the temperature at which the washing is carried out, and the nature and strength of the solutions used for washing. Detection of hybridisation is carried out using a method appropriate for the label used.
  • Labels suitable for use in detecting nucleic acid in hybridisation are known and include labels that can be detected directly, in particular radioisotopes, for example, *-- ⁇ * ⁇ ?, ⁇ -->S , **-*H, 125j anc g labels that can be detected indirectly, for example, using enzymic or im unolo ical techniques.
  • Biotin is an example of the latter type of label, and is generally detected by methods which rely primarily on the affinity of biotin for avidin or streptavidin which may themselves by labelled or which may be reacted with a further specific reactant that is itself labelled, for example, a labelled antibody. Methods for labelling DNA are well known.
  • the oligonucleotide of formula I is highly specific for bacterial nucleic acid. It hybridises with all major classes of bacteria and shows no detectable hybridisation with mammalian PNA on Northern blotting. Sub-sequences of this oligonucleotide generally have decreasing specificity with decreasing size. Furthermore, the specificity of the oligonucleotide of formula I generally decreases when the oligonucleotide is incorporated in a larger molecule, the specificity in this case decreasing with increasing molecular size.
  • the lesser specificity of an oligonucleotide smaller or larger than that of formula I may be acceptable, particularly if the oligonucleotide is larger or smaller than formula I by only a few bases, for example oligonucleotide sub-sequences having from 18 to 25 bases have >95% of the specificity of the oligonucleotide of formula I, which has 26 bases, and oligonucleotide sub ⁇ sequences having 16 bases still have good specificity. Below 16 bases the specificity tends to decrease, but sub ⁇ sequences of only 7 bases may be adequate for certain purposes. Generally, however, it is preferable to use a DNA probe having more than 16 bases, and for the best specificity the oligonucleotide of formula I itself is particularly preferred.
  • the sensitivity of the test method used can be altered by the choice of nucleic acid material to be determined and by the choice of label and detection method. There is considerably more rRNA than DNA per average bacterial cell. Accordingly, detection of rRNA is generally preferable.
  • Radioisotope labels are generally determined directly, and can afford qualitative or quantitative results.
  • Other labels for example, biotin can be detected using secondary markers. Sensitivity can be increased in some cases by using detection techniques that involve amplification of the original label.
  • the bacterial detection test of the present invention may be carried out on any sample for which evidence of bacterial contamination is required.
  • the samples may be already liquid or may be solid. In the latter case any bacteria or bacterial fragments present may be washed off or otherwise extracted from the sample.
  • Many suitable methods are known, that is to say, described in the literature of the art or in actual use in the art.
  • the present method may also be used to determine by an in situ hybridisation method the presence of bacteria in a sample that has previously been sectioned histologically.
  • a sample to be analysed may be any for which the determination of bacteria, for example, in the presence of eukaryotic cells, is desired.
  • the sample may be of a body fluid, or tissue, or any other sample of a medical nature. It may be a sample of food, beverage or a pharmaceutical undergoing analysis during manufacture for quality control purposes or it may be a sample of food or beverage undergoing analysis for suspected bacterial contamination. It will be appreciated that the present test will find application in public health work in general as well as in quality control and also research laboratories. - O -
  • the present invention also provides RNA sequences that are complementary to the DNA sequences of formulae I, II, III, IV and V, and also to the corresponding cDNA sequences, and to oligonucleotide sub-sequences thereof.
  • RNA sequences may themselves be useful in the detection and/or determination of bacteria, and the present invention provides such a method, which comprises contacting a solid or liquid sample under investigation with such an RNA probe under hybridising conditions and detecting and/or determining hybridised R NA probe.
  • the invention further provides kits analogous to the DNA probe-containing kits described above, but comprising an RNA probe of the invention.
  • a cDNA probe may be used to detect and/or determine DNA in a sample.
  • Samples of bacteria were grown at 37°C on a rotary platform shaker (200rpm) to mid-log phase (optical density at 540 nm of 0.5-1.0) and 2.0 M glycine was added to a final concentration of 150 mM. After a further 16 hours o growth bacteria were harvested by centrif gation at 5000 rpm for 5 minutes in a Sorvall GSA rotor, to obtain 0.5-1.0 g wet weight of bacterial pellet. This was resuspended by gentle trituration in 15 ml of 10 mM tris- HC1 pH8, 1 mM EDTA, and placed on wet ice.
  • RNA was obtained from Staphylococcus aureus, Streptococcus species, Pseudomonas species, Escherichia coli, diphtheroid, Proteus species and Mycobacterium smegmatis ATCC 607.
  • the oligonucleotide probe used has the sequence
  • oligonucleotide was separated from unincorporated isotope by gel filtration on a Pharmacia PD-10 prepacked column previously equilibrated with, and eluted with 10 M tris- HCl pH8, 1 M NaCl. Peak excluded fractions were pooled, 1 mg of E. coli tRNA was added, NaCl was added to 0.3 M and the oligonucleotide was precipitated by addition of 2 volumes of ethanol with incubation at -20°C for at least 16 hours.
  • the suspension was centrifuged in an Eppendorf microfuge for 5 minutes, resuspended in 1 ml of 70% ethanol, recentrifuged, dried in vacuo for 5 minutes, and dissolved in 50 ul of 10 M trisHCI pH8, 1 mM EDTA.
  • RNA was then transferred overnight to nitrocellulose (Schleicher and Schuell type BA85) by capillary suction as described in Maniatis e_t a ⁇ _ (loc. cit.) using 1.5 M NaCl, 0.15 M sodium citrate as transfer fluid.
  • the nitrocellulose sheet was then removed and dried at 37°C, after which it was baked at 80°C for 2 hours. (Gel electrophoresis was use to determine the size of the RNA used, but hybridisation can alternatively be carried out by directly blotting the samples onto nitrocellulose.)
  • the filter was prehybridised in a plastic bag containing 10 ml of 900 mM NaCl, 90mM tris-HCl pH7.6, 6 mM EDTA, 0.04% bovine serum albumin, 0.04% Ficoll 400, 0.04% polyvinylpyrrolidone, 100 ug/ml sonicated (to 500-1000 bp) salmon sperm DNA (heated immediately before use to 95°C for 10 minutes), and 70 ug/ml yeast tRNA, at 55°C for 3 hours. This solution was then removed and replaced with 1 ml of the same solution containing one million cpm per ml of the labelled oligonucleotide.
  • the filter was incubated at 37°C for 2 days, after which it was washed at room temperature in a Hybaid apparatus using 1000 ml of 0.3 M NaCl, 0.03 M sodium citrate, followed by 500 ml of 0.03 M NaCl, 0.003 M sodium citrate, followed by 500 ml of 0.15 M NaCl', 0.015 mM sodium citrate. After drying at 37°C the filter was exposed to Fuji X-ray film for 16-60 hours at room temperature.

Abstract

Un oligonucléotide selon les formules (I), (II), (III), (IV) ou (V) ou une sous-séquence de celui-ci, est une sonde d'ADN spécifique pour la détection de bactéries.
PCT/GB1989/000850 1988-07-29 1989-07-31 Sonde d'adn bacterienne WO1990001560A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1019900700729A KR900701791A (ko) 1988-08-09 1989-07-28 치료제

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB888818066A GB8818066D0 (en) 1988-07-29 1988-07-29 Bacterial dna probe
GB8818066.6 1988-07-29

Publications (1)

Publication Number Publication Date
WO1990001560A1 true WO1990001560A1 (fr) 1990-02-22

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Application Number Title Priority Date Filing Date
PCT/GB1989/000850 WO1990001560A1 (fr) 1988-07-29 1989-07-31 Sonde d'adn bacterienne

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AU (1) AU4042889A (fr)
GB (1) GB8818066D0 (fr)
WO (1) WO1990001560A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0601606A1 (fr) * 1992-12-11 1994-06-15 Canon Kabushiki Kaisha Méthode de détection de bio-individus en utilisant une sonde d'acide nucléique du type non naturel et sonde pour utilisation en cela
WO1995013396A2 (fr) * 1993-11-11 1995-05-18 U-Gene Research B.V. Procede d'identification de micro-organismes, et dispositifs pour sa mise en oeuvre
EP0692540A2 (fr) 1994-06-17 1996-01-17 Becton, Dickinson and Company Amorces d'oligonucléotides et sondes pour la détection de bactéries
WO1999014317A1 (fr) * 1997-09-19 1999-03-25 Synbiotics Corporation Procedes servant a diagnostiquer l'anemie feline infectieuse
US6280946B2 (en) 1998-08-07 2001-08-28 Boston Probes, Inc. PNA probes, probe sets, methods and kits pertaining to the universal detection of bacteria and eucarya
EP0479117B1 (fr) * 1990-10-05 2001-11-28 F. Hoffmann-La Roche Ag Méthodes et réactifs pour l'identification bactérienne
US6664045B1 (en) 1998-06-18 2003-12-16 Boston Probes, Inc. PNA probes, probe sets, methods and kits pertaining to the detection of microorganisms
US7108980B1 (en) 1998-08-07 2006-09-19 Boston Probes, Inc. Methods for the analysis of microcolonies of bacteria and yeast

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987005907A1 (fr) * 1986-04-04 1987-10-08 Institut Pasteur Sondes oligonucleotidiques et procedes pour la detection par hybridation des acides nucleiques de bacteries et autres etres vivants
EP0250662A1 (fr) * 1986-06-25 1988-01-07 The Regents Of The University Of California Détection de mycoplasma par hybridation d'ADN
EP0277237A1 (fr) * 1986-07-10 1988-08-10 Toray Industries, Inc. Procede et sonde de depistage de bacteries dans des echantillons

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987005907A1 (fr) * 1986-04-04 1987-10-08 Institut Pasteur Sondes oligonucleotidiques et procedes pour la detection par hybridation des acides nucleiques de bacteries et autres etres vivants
EP0250662A1 (fr) * 1986-06-25 1988-01-07 The Regents Of The University Of California Détection de mycoplasma par hybridation d'ADN
EP0277237A1 (fr) * 1986-07-10 1988-08-10 Toray Industries, Inc. Procede et sonde de depistage de bacteries dans des echantillons

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0479117B1 (fr) * 1990-10-05 2001-11-28 F. Hoffmann-La Roche Ag Méthodes et réactifs pour l'identification bactérienne
EP0601606A1 (fr) * 1992-12-11 1994-06-15 Canon Kabushiki Kaisha Méthode de détection de bio-individus en utilisant une sonde d'acide nucléique du type non naturel et sonde pour utilisation en cela
US5545521A (en) * 1992-12-11 1996-08-13 Canon Kabushiki Kaisha Method for detecting bioindividuals by use of nonnatural type nucleic acid probe
WO1995013396A2 (fr) * 1993-11-11 1995-05-18 U-Gene Research B.V. Procede d'identification de micro-organismes, et dispositifs pour sa mise en oeuvre
NL9301957A (nl) * 1993-11-11 1995-06-01 U Gene Research Bv Werkwijze voor het identificeren van micro-organismen, en daarvoor bruikbare hulpmiddelen.
WO1995013396A3 (fr) * 1993-11-11 1995-06-08 U Gene Research Bv Procede d'identification de micro-organismes, et dispositifs pour sa mise en oeuvre
EP0692540A2 (fr) 1994-06-17 1996-01-17 Becton, Dickinson and Company Amorces d'oligonucléotides et sondes pour la détection de bactéries
WO1999014317A1 (fr) * 1997-09-19 1999-03-25 Synbiotics Corporation Procedes servant a diagnostiquer l'anemie feline infectieuse
US6664045B1 (en) 1998-06-18 2003-12-16 Boston Probes, Inc. PNA probes, probe sets, methods and kits pertaining to the detection of microorganisms
US6280946B2 (en) 1998-08-07 2001-08-28 Boston Probes, Inc. PNA probes, probe sets, methods and kits pertaining to the universal detection of bacteria and eucarya
US6656687B1 (en) 1998-08-07 2003-12-02 Boston Probes, Inc. Multiplex PNA-ISH assay
US7108980B1 (en) 1998-08-07 2006-09-19 Boston Probes, Inc. Methods for the analysis of microcolonies of bacteria and yeast

Also Published As

Publication number Publication date
AU4042889A (en) 1990-03-05
GB8818066D0 (en) 1988-09-01

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