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EP0550531A1 - Identification d'organismes - Google Patents

Identification d'organismes

Info

Publication number
EP0550531A1
EP0550531A1 EP91916841A EP91916841A EP0550531A1 EP 0550531 A1 EP0550531 A1 EP 0550531A1 EP 91916841 A EP91916841 A EP 91916841A EP 91916841 A EP91916841 A EP 91916841A EP 0550531 A1 EP0550531 A1 EP 0550531A1
Authority
EP
European Patent Office
Prior art keywords
probe
nucleic acid
probes
organisms
hybridisation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91916841A
Other languages
German (de)
English (en)
Inventor
Gregory Glenn Lennon
Hans Rudolph Lehrach
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cancer Research Horizons Ltd
Original Assignee
Imperial Cancer Research Technology Ltd
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 Imperial Cancer Research Technology Ltd filed Critical Imperial Cancer Research Technology Ltd
Publication of EP0550531A1 publication Critical patent/EP0550531A1/fr
Withdrawn legal-status Critical Current

Links

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
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the identification of organisms, particularly microorganisms, and especially eubacteria.
  • one aspect of the invention provides a method of identifying an organism, the method comprising (1) obtaining nucleic acid derived from the organism, and (2) determining, for each of at least 5 nucleotide probes, whether the probe hybridises to the said nucleic acid.
  • the number of probes used is at least 10, 15, 20, 25 or more when one wishes to distinguish between large numbers of samples (for example hundreds) .
  • 10-20 would typically be enough in such a situation to establish a meaningful signature, provided that each probe does not hybridise so seldom or so frequently to the sample nucleic acid as to give the same highly negative or highly positive hybridisation signature to all the samples which one is testing and between which one wishes to differentiate.
  • fewer probes will usually be needed.
  • probes bind to more than one site on the sample nucleic acid, since each probe is then capable of yielding more than a binary "yes/no" result and nucleic acid which binds the probe three times can be differentiated from nucleic acid which binds it only once or twice.
  • the key feature of the methods of the present invention is that a plurality of short probes are used in concert to generate an informative "hybridisation signature".
  • a second aspect provides a method of assigning a "hybridisation signature" to a given organism's nucleic acid based on its pattern of hybridisations to more than one oligonucleotide probe.
  • each probe is such that, under appropriate hybridisation conditions, it will bind to about 25-75% of all nucleic acid of the types being assayed, preferably 30-70%, more preferably 40-60% and most preferably 40-50%.
  • Each probe whilst being specific (subject to the hybridisation conditions) for a given sequence corresponding to the length of the probe, is non-specific in the sense that, on its own, it cannot be used with any precision to distinguish between different lengths of nucleic acid which are all significantly longer than the probe.
  • the specificity of the assay method thus derives from the fact that a relatively high number of probes are used.
  • the probes are typically 4-15 nucleotides long, preferably 5-10 and often 8 or 9 nucleotides long.
  • a mixture of two or more similar probes can be treated as a single probe (ie used in a single hybridisation step) to increase the frequency with which the composite probe binds to sample nucleic acid.
  • probe is used in this specification to include such composite probes.
  • analogues and fragments thereof are those which provide for specific amplification by the PCR of DNA adjoining that to which the said four specific primers hybridise.
  • Probes and primers can be made by known means, for example by simple chemical synthesis using apparatus supplied by Applied Biosystems Inc.
  • nucleic acid in each sample can be arranged to be the same in each case, or can be measured by some other means, it is convenient to normalise the amount by determining the extent of binding of a probe which is known to be common to all of the samples under consideration, for example one in a highly conserved region. Such probes are hereinafter termed “conserved probes", as opposed to the previously discussed “variable probes” . The binding of the variable probes is then expressed as a ratio to the binding of the conserved probes. conserved probes are not subject to the same size constraints as variable probes.
  • Probes may comprise all or part of the sequence of a PCR primer.
  • the identification of the organism which is achieved by methods in accordance with the invention may be more or less precise according to how many probes are used and may be more or less informative according to what reference data one has for the same and other organisms.
  • the methods may be used to place the organism in any of the taxonomic groups (Kingdom, phylum, class, sub-class, order, sub-order, family, genus, species or sub-species), to identify similarities with organisms having desirable or undesirable characteristics or even to identify the particular strain of organism.
  • the methods are particularly useful in screening microorganisms, especially bacteria, which are collected more or less randomly from the wild to determine whether the sample microorganism has been studied previously or whether it falls into a group of microorganisms which are believed to be of particular interest.
  • Another use for the methods of the invention is to identify, at least to an extent, the nature of a pathogen.
  • papillomaviruses which are found must be typed, especially to determine whether they are type 16 or type 18, the two types thought to be responsible for cervical cancers and other abnormalities.
  • probes which generate only binary hybridisation signatures (binding or not binding) then six probes may be necessary to distinguish between 35-60 types (as 2 6 is 64) . However, fewer probes may be employed if the probes bind multiply or if one wishes only to know whether the virus is type 16 or 18 or another type.
  • probes may be used, with consequent savings of time and materials. It should be noted that the probes are not individually capable of specifically binding to a sequence motif unique to the virus type in question; it is still the hybridisation signature obtained by the use of a plurality of probes which yields the desired information.
  • Further uses include the typing of other pathogens, for example HIV, rhinoviruses and Streptococcus species, and the typing of desirable or undesirable bacterial and viral strains in cheese-making, for example the lysin-producing bacteriophages and the desirable bacteria Lactococcus lactis subsp. lactis and subs, cremori ⁇ .
  • pathogens for example HIV, rhinoviruses and Streptococcus species
  • desirable or undesirable bacterial and viral strains in cheese-making for example the lysin-producing bacteriophages and the desirable bacteria Lactococcus lactis subsp. lactis and subs, cremori ⁇ .
  • the methods of the invention are generally applicable to identifying or typing viruses, bacteria, mycoplasmas
  • Plants including trees
  • Plants may be typed as an initial screening process to identify those most likely to be useful in some context (for example desirable bread-making strains of wheat, oil- or pharmaceutical- bearing plants or nutritionally valuable or disease- resistant strains of crop plants) and this may be particularly valuable as part of a plant breeding program where one may otherwise have to wait until the hybrid is mature before being able to identify its characteristics.
  • the nucleic acid which is probed may be DNA, RNA or cDNA.
  • the method may involve any suitable combination of nucleic acid and probe.
  • This includes, but is not limited to, chromosomal nucleic acid isolated in toto (DNA) , ribonucleic acid, or specific nucleic acid regions amplified in order to increase the sensitivity of the method.
  • specifically amplified nucleic acid the important concept is that regions of nucleic acid variability will be located between highly conserved nucleic acid sequences, such that oligonucleotide primers can be designed that will complement the conserved regions and will allow the amplification of the variable regions located in between them.
  • suitable loci include the ribosomal RNA genes, such as the bacterial 16S and 23S genes, and regions between them.
  • Primers are likely to consist of oligonucleotides of length appropriate to their use in the polymerase chain reaction.
  • Probes are likely to consist of oligonucleotide nucleic acids between six and ten bases in length, tagged either radioactively or non- radioactively.
  • the nucleic acids to be probed may be positioned on a support such as a nylon filter membrane.
  • Hybridisation of the probes to the nucleic acids must allow sufficient specific hybrid molecules to form and to be detected. Accurate quantitation of the amounts of probe bound to each nucleic acid is important, and may be achieved through the use of storage phosphor screen autoradiography or by other means.
  • Figure 1 shows partial DNA sequences from the 16S ribosomal RNA genes of four bacteria
  • Figure 2 illustrates the generation of distinct hybridisation signatures for four bacteria, using only two probes
  • Figure 3 illustrates diagrammaticc.lly the use of six probes to generate respective hybridisation signatures for six organisms
  • Figure 4 shows partial DNA sequences from several human papillomaviruses
  • FIG. 5 shows partial sequences (as they might appear following reverse transcription) from four human rhinoviruses.
  • Figure 1 shows nucleotide sequence alignments of the beginning of several 16S ribosomal RNA genes from different bacterial organisms.
  • the sequence of the E. coli 16S rRNA gene is the reference sequence (GenEMBL Accession No J01695) .
  • the next three sequences are from distantly related bacteria Streptomyces coelicolor, GenEMBL Accession No Y00411; Pseudoj ⁇ ionas aer ginosa , GenEMBL Accession No X06684; Pseudomonas testo ⁇ teroni, GenEMBL Accession No M11224) .
  • a colon is used to indicate sequence conservation between the 16S rRNA sequence of these species and E . coli .
  • the last two sequences are from two species of the same genus, and illustrate that variable regions remain variable even between two closely related species.
  • Examples of the sequences of oligonucleotides used as primers for the polymerase chain reaction (PCR) are indicated by overlying arrows, and are termed Primer A and Primer B.
  • Examples of the sequences of oligonucleotides used as probes in the hybridisation are underlined as Probe 1 (GACCCTCG, S . coel . ) and Probe 2 (CTGAGACAC, present in all species shown) .
  • Oligonucleotides were synthesised on an Applied Biosystems 380A machine and labelled with [ ⁇ - 32 P]ATP (3000 Ci/mmol "1 ; Amersha International) and T4 polynucleotide kinase.
  • the sequence of probes p3 and p4 were 5 '-CCACGTTG-3 ' and 5 '- TGCATGGC-3 ' , respectively.
  • Hybridisation of probes to filters Filters were prehybridised for 30 minutes at room temperature in hybridisation solution (4X SSC, 7% sarkosyl) , then hybridised with probe at a concentration of 3nM for 3 hours at 80°C in a volume of 250 ⁇ l per 28 cm 2 filter. Filters were washed for one hour at 12°C in two changes of 100 ml of hybridisation solution, then exposed to either film or storage phosphor screens.
  • Figure 2 is a schematic illustration of the hybridisation information obtained using four oligonucleotide probes (two as indicated in Figure 1) against a nylon filter membrane onto which the PCR products were spotted.
  • Two separate regions of the 16S rRNA gene were amplified independently using primer pair A and B (as indicated in Figure 1) , or primer pair C and D.
  • Probes pi and p2 were designed to be used as probes for nucleic acid samples from the region amplified using primer pair A and B; similarly, probes p3 and p4 for DNA from primer pair C and D.
  • Hybridisations were done in .duplicate, and averages were used.
  • Hybridisation signals were quantified by storage phosphor screen autoradiography, and signals from variable region probes (pi and p3) normalised against signals from conserved region probes (p2 and p4) .
  • the four organisms classified by these means are shown to have been assigned four different hybridisation signatures.
  • Figure 3 is a diagrammatic representation of the concept of assigning hybridisation signatures based on the accumulation of positive or negative assignments to each of six probes assayed against an array of nucleic acid samples.
  • the number of different hybridisation signatures possible in any one set of experiments is, for experiments where only presence or absence is measured, 2 N where N is the number of probes used.
  • the use of 20 probes in such an experiment yields 2 20 , ie over one million different (in this case, binary) hybridisation signatures.
  • Each type of nucleic acid sample will reproducibly be assigned one of these signatures.
  • the assay detects either 0, 1, or 2 copies of the probe in the nucleic acid of the organisms tested.
  • Figure 4 shows nucleotide sequence alignments of portions of the genomes of several different human papillomaviruses, including two associated with a high risk of developing cervical cancer (Pphl6 and Pphl8) .
  • a colon indicates nucleotide conservation between the sequences shown.
  • Sequences for Pphll, Pphl6, Pphl ⁇ , and Pph33cg are from GenEMBL (Accession Numbers M14119, K02718, X05015, and M12732, respectively) .
  • a pair of potential PCR primers are marked as primers E and F, and probes for both a non- conserved (probe 5) and a conserved (probe 6) region are indicated. The probes are prepared and used as in Example 1 above.
  • Figure 5 shows nucleotide sequence alignments of portions of four human rhinovirus genomes as they might appear after reverse transcription. Sequence from human rhinoviruses types 14, 89, IB, and 2 are shown (from GenEMBL sequences, Accession Numbers X01087, M16248, D00239, and X02316, respectively) . As before, potential primers are indicated (Primers G and H) and an example is shown of both a probe from a variable region (probe 7 and a conserved region (probe 8) . The probes are prepared and used as in Example 1 above.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On crible l'acide nucléique provenant d'organismes (amplifié généralement par une réaction de polymérase en chaîne -PCR) au moyen d'une banque de sondes courtes, afin de déterminer si chaque sonde s'hybride avec l'acide nucléique et, dans ce cas, combien de fois. On crée, de ce fait, une "signature d'hybridation" comportant un point (0, 1, 2, etc) pour chaque sonde. On peut utiliser le procédé pour effectuer un criblage initial de bactéries recueillies au hasard pour éliminer celles de type indésirable et celles étudiées précédemment, ainsi qu'également pour déterminer des organismes présentant un intérêt médical ou industriel.
EP91916841A 1990-09-21 1991-09-20 Identification d'organismes Withdrawn EP0550531A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB909020621A GB9020621D0 (en) 1990-09-21 1990-09-21 Identification of organisms
GB9020621 1990-09-21

Publications (1)

Publication Number Publication Date
EP0550531A1 true EP0550531A1 (fr) 1993-07-14

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP91916841A Withdrawn EP0550531A1 (fr) 1990-09-21 1991-09-20 Identification d'organismes

Country Status (4)

Country Link
EP (1) EP0550531A1 (fr)
JP (1) JPH06501385A (fr)
GB (1) GB9020621D0 (fr)
WO (1) WO1992005280A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9210916D0 (en) * 1992-05-21 1992-07-08 Isis Innovation Nucleic acid quantification
ES2158854T3 (es) * 1992-10-13 2001-09-16 Hoffmann La Roche Oligonucleotidos derivados de la familia de genes sod.
CA2150986C (fr) * 1994-06-17 1999-11-02 Mary Kathryn Meyer Amorces et sondes d'oligonucleotides pour la detection de bacteries
US5693467A (en) * 1995-05-19 1997-12-02 The American Type Culture Collection Mycoplasma polymerase chain reaction testing system using a set of mixed and single sequence primers
DE19732086C2 (de) * 1997-07-25 2002-11-21 Univ Leipzig Verfahren zur quantitativen Bestimmung von Eubakterien
GB9719044D0 (en) * 1997-09-08 1997-11-12 Inst Of Ophthalmology Assay
US6821770B1 (en) 1999-05-03 2004-11-23 Gen-Probe Incorporated Polynucleotide matrix-based method of identifying microorganisms
EP2365097A1 (fr) * 1999-05-14 2011-09-14 Enterprise Ireland (trading as BioResearch Ireland) Analyses à base de sondes d'acide nucléique pour organismes procaryotes et eucaryotes
CN103451313B (zh) * 2013-09-27 2016-03-09 中国科学院上海微系统与信息技术研究所 一种基因芯片的金沉积检测方法

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FI71768C (fi) * 1984-02-17 1987-02-09 Orion Yhtymae Oy Foerbaettrade nykleinsyrareagenser och foerfarande foer deras framstaellning.
AU5698286A (en) * 1985-01-08 1987-04-24 Biotechnica Limited Determination of identity between two organisms
US4900659A (en) * 1986-01-30 1990-02-13 Enzo Biochem, Inc. Nucleotide sequence composition and method for detection of Neisseria gonorrhoeae and method for screening for a nucleotide sequence that is specific for a genetically distinct group
US5310893A (en) * 1986-03-31 1994-05-10 Hoffmann-La Roche Inc. Method for HLA DP typing
WO1989005359A1 (fr) * 1987-12-01 1989-06-15 Integrated Genetics, Inc. Detection de salmonelle
WO1989006704A1 (fr) * 1988-01-11 1989-07-27 Microprobe Corporation Sondes d'oligonucleotides servant a la detection de pathogenes du periodonte
GB8810400D0 (en) * 1988-05-03 1988-06-08 Southern E Analysing polynucleotide sequences
JPH02203800A (ja) * 1988-04-15 1990-08-13 Innogenetics Sa:Nv ナイセリア菌株を検出するための交雑プローブ
FR2636075B1 (fr) * 1988-09-07 1991-11-15 Biotechnologie Ste Europ Procede de detection des bacteries, levures, parasites et autres eucaryotes, notamment dans les produits alimentaires
IE81145B1 (en) * 1989-04-20 2000-05-03 Thomas Gerard Barry Generation of specific probes for target nucleotide sequences

Non-Patent Citations (1)

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Title
See references of WO9205280A1 *

Also Published As

Publication number Publication date
JPH06501385A (ja) 1994-02-17
GB9020621D0 (en) 1990-10-31
WO1992005280A1 (fr) 1992-04-02

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