WO1995030025A1

WO1995030025A1 - Detection or assay of target nucleic acids

Info

Publication number: WO1995030025A1
Application number: PCT/GB1995/000971
Authority: WO
Inventors: Ørjan OLSVIK; Jack Andreassen
Original assignee: Dynal As; Dzieglewska, Hanna, Eva
Priority date: 1994-04-29
Filing date: 1995-04-28
Publication date: 1995-11-09
Also published as: GB9408607D0; AU2315595A

Abstract

The present invention provides a method of detection or assay of a target nucleic acid in a sample wherein the sample is introduced into a medium containing one or more amplification enzymes and at least two amplification primers and subjected to at least one cycle of an amplification procedure such that any target nucleic acid present is amplified, characterised in that at least one of said primers is immobilised or provided with means for immobilisation and another is labelled or provided with means for labelling whereby said primers are incorporated into amplified target nucleic acid which is thereby immobilised, if necessary by reaction with a solid phase reactive with said means for immobilisation, and labelled, if necessary by addition of a label reactive with said means for labelling, and the immobilised labelled primers are thereby removed from solution in said medium, followed by examination of the medium to detect the presence or quantity of any label left in solution and hence determine the presence, absence or quantity of target nucleic acid in the sample. A further aspect of the invention provides a sealed vessel containing the components for performance of said assay or detection method.

Description

Detection or Assay of Targe Nucleic Acids

This invention relates to the detection or assay of target nucleic acids, for example to assist identification of nucleic acid variations of medical or forensic interest.

In recent years, nucleic acid analysis has provided a valuable diagnostic or forensic tool by permitting identification of mutant or variant nucleic acids, eg. in diagnosis of disease related mutations or in tissue typing. It is possible to identify extremely small quantities of target nucleic acids, sometimes a single molecule, by initial selective amplification followed by appropriate labelling to permit detection or quantification of the amplified material. The polymerase chain reaction (PCR) has been used extensively in this way; other amplification systems include the Self-Sustained Sequence Replication (3SR) process, the Ligase Chain Reaction (LCR) , and the Nucleic Acid Sequence-based Amplification (NASBA) process.

However, the very great sensitivity of these methods of amplification has provided problems in that false positive reactions are sometimes produced by contamination during the amplification procedure. There is thus a need for methods of amplification and detection which reduce or avoid completely such contamination.

The present invention is based on the concept of carrying out the amplification by introducing the sample into a medium contained in a sealed container comprising all the components necessary for the procedure, including means for immobilising the amplified nucleic acid together with a labelled ligand so that withdrawal of the labelled ligand from solution by immobilisation provides an indication of the presence and/or quantity of target nucleic acid in the sample.

Thus, according to one feature of the invention we provide a method of detection or assay of a target nucleic acid in a sample wherein the sample is introduced into a medium containing one or more amplification enzymes and at least two amplification primers and subjected to at least one cycle of an amplification procedure such that any target nucleic, acid present is amplified, characterised in that at least one of said primers is immobilised or provided with means for immobilisation and another is labelled or provided with means for labelling whereby said primers are incorporated into amplified target nucleic acid which is thereby immobilised, if necessary by reaction with a solid phase reactive with said means for immobilisation, and labelled, if necessary by addition of a label reactive with said means for labelling, and the immobilised labelled primers are thereby removed from solution in said medium, followed by examination of the medium to detect the presence or quantity of any label left in solution and hence determine the presence, absence or quantity of target nucleic acid in the sample.

The term 'nucleic acid' as used herein includes RNA, DNA and analogues thereof such as PNA (Peptide Nucleic Acid) .

Although a single cycle of amplification may be sufficient if the concentration of target nucleic acid is high, in general several cycles will be required, for example 5-20 eg. about 10, cycles.

Whilst any in vitro amplification procedure may be used, eg. PCR or NASBA, the method of the invention may be illustrated in its simplest form in relation to the Ligase Chain Reaction (LCR) . In this method, a point mutation in a target nucleic acid is detected by use of two primers which hybridise to one strand of the nucleic acid over consecutive sequences of DNA such that the point mutation lies at the terminus of one of the primers. Thus, one primer has a terminal base overlying the point mutation and if that terminal base is complementary to the underlying base of the point mutation, it will be possible to ligate the two primers using a suitable ligase. If, however, the terminal base is not complementary to the point mutation, ligation will not take place. If the two primers hybridise with a short gap between the termini, the ligation reaction may be carried out in the presence of one or more nucleotides to fill the gap. For amplification, two further primers are also provided for the complementary strand and, again, will permit ligation if the base of the point mutation is appropriate and there is no mismatch.

After ligation of the two pairs of primers, the double stranded DNA so produced may be subjected to strand separation, for example by heating, and the separate single strands may then serve as templates for further ligation primers. If this procedure is repeated, exponential production of ligated DNA takes place and the relevant region of the target DNA is amplified. On the other hand, if there is a mis-match at the site of the point mutation, no amplification will take place. Standard amplification conditions, well known and described in the literature may be used, and this applies to all the amplification procedures.

When adapted for the method of the present invention, using immobilisation of amplified DNA, the means for immobilisation attached to one primer may conveniently be an affinity binding partner such as biotin or a hapten such as digoxigenin which binds to a corresponding binding partner for example avidin, streptavidin or an anti-hapten on a solid phase. Such a solid phase may be, for example, the walls of the vessel containing the medium or a dipstick or, more preferably particles which can readily be separated from the medium. The solid phase may be provided with a "particulate" or porous surface. Superparamagnetic particles, such as Dynabeads (Dynal AS, Oslo, Norway) are especially suitable since they permit the normal kinetics of the reaction to proceed unimpaired while allowing rapid and simple removal from solution using a magnet .

Labels for use in the method of the invention will, in general, be those giving a direct visual signal such as fluorescent molecules eg. fluorescein isothiocyanate (FTC) or chemiluminescent molecules such as acridinium esters. Such labels are well-known or widely described in the literature. A range of suitable labels are commercially available, including fcr example the fluorescent Chro aTide^® labels, consisting of fluorophore- and hapten-labelled NTPs useful for generating labelled nucleic acids (Molecular Probes, Inc., Eugene, OR, USA) or bisbenzimide fluorochromes available from Calbiochem, La Jolla, CA, USA. Other labels include tris (2, 2 ' -bipyridine) -ruthenium (II) chelate (TBR) and synthetic or naturally occurring dyes and pigments. Particles may be used as labels eg. colloidal gold particles in the size range 3-20 nm. The label molecules may be attached directly to the DNA by covalent bonds, for example via 5'-amino or 5 ' -SH groups. Alternatively, the label molecules may be attached to DNA-binding proteins such as the lad repressor which binds to the lac operon; the DNA sequence of the lac operon may thus be attached as a 'handle' to one of the primers, for example by chemical synthesis of the primer and handle, and the labelled repressor protein added to label the primer or the amplified DNA incorporating the primer.

The sample may be any sample containing the target nucleic acid. It may for example be a laboratory sample or a clinical, biological or environmental sample, eg. serum or other body fluid or blood-derived product, faeces, food or allied products, cell suspension, cell culture, tissue homogenate or cell lysate etc. It may be desirable to dilute the sample or to submit a more crude or complex sample to one or more purification steps, for example to remove potential contaminants or substances which may interfere in the assay, such as haemoglobin.

In general, the components of the reaction mixture will be provided in a suitable vessel adapted to permit visual inspection or photometric assay of any labelled primer dissolved in the medium. To avoid contamination, the vessel is preferably sealed with a membrane which may be punctured by a syringe to introduce the sample. However, with appropriate precautions, a simple lid or cover may serve to avoid contamination.

In general, it is preferable for the components to be in dried form, for example lyophilised form, and for water to be added, for example by injection through the above membrane, to dissolve the components. The use of magnetic particles for immobilisation makes it possible to keep these separate from the other components by magnetic attraction to a locus above the level of any fluid in the reaction vessel. In this way, the primers to be immobilised may carry ligands such as biotin and remain in solution throughout the amplification reaction before being contacted with the magnetic beads (coated, for example, with streptavidin) to effect immobilisation and ultimate withdrawal from the medium. Similarly it is possible to coat (with a specific binding agent such as streptavidin) an area of the vessel wall above the liquid level so as to immobilise the amplified nucleic acid at the end of the reaction simply by inverting the vessel .

It may be preferable for the means for immobilisation to be linked to the primer via a region of nucleic acid which does not hybridise to the target. This may be a standardised oligonucleotide, such as oligodA which may readily be hybridised to a complementary oligonucleotide sequence, such as oligodT or an equivalent PNA sequence, which is immobilised or carries means for immobilisation. It will be appreciated that in such a system, when all the components of the amplification mixture are present in a single solution and conditions are appropriate for hybridisation rather than strand separation, the standard oligonucleotide and complementary oligonucleotide will be hybridised to form a primer which is immobilised or provided with means for immobilisation. On the other hand, during the strand separation phase, such oligonucleotides may separate and only recombine when hybridising conditions are restored or even only when the reaction mixture is cooled to room temperature after amplification. However, if the complementary oligonucleotide carrying means for immobilisation is of such a length as to be contiguous with the adjacent primer, the ligation reaction will serve to ligate not only the two primers but also the additional complementary oligonucleotide. Similarly, a gap between the oligonucleotide and the primer may be filled by effecting ligation in the presence of appropriate nucleotides.

The same considerations apply to the labelled primer. In general, the labelled primer will hybridise to the strand of the target nucleic acid complementary to that to which the immobilised primer hybridises; in this way all the amplified nucleic acid in double stranded form will be both immobilised and labelled. Since, however, the objective is merely to immobilise labelled primer in order to indicate the presence of the target nucleic acid, it is also possible for the labelled primer to hybridise to the same strand as the immobilised primer, although two primers for the complementary strand are needed for exponential amplification.

Where the solid phase and/or label is to be attached to the primers throughout the amplification reaction, such attachment is preferably covalent, for example via 5'-SH groups on terminal bases of the primers which may be bound to maleimide groups on the solid phase or 5'-amino groups which may bind to activated carboxyl groups on the solid phase.

The same principle may be applied to PCR amplification. An assay system in which PCR is carried out using primers which are capable of being immobilised or labelled is described in WO 90/11369. It is generally known by the acronym DIANA (Detection of Immobilised Amplified Nucleic Acids) . It has not previously been suggested, however, to carry out the procedure in a sealed medium or to detect or determine the presence of the target nucleic acid by removal of labelled antibodies from solution to permit visual estimation in the same reaction vessel. In PCR, only two primers are used for each cycle of amplification, one for each strand of target DNA and thus, for use in the present system, one of these should be immobilised or possess means for immobilisation while the other should be labelled or carry means for labelling. The polymerase used in PCR is usually heat stable; Taq polymerase is most commonly used.

PCR is commonly effected in two series of cycles using nested primers, that is a first series of cycles may be effected with two primers not adapted for immobilisation or labelling to provide an initial amplification while a second series of cycles is effected using at least one further primer which hybridises to the DNA strands between the first primers, thereby amplifying a shorter sequence of the target DNA but with increased sensitivity due to the use of the further primer(s) which must hybridise accurately to the amplified DNA. In the present system, it is possible to perform a second series of cycles using a second immobilising primer and the same labelled primer; in order to prevent this second primer from being involved in the first series of PCR cycles, the second immobilising primer must be held above the level of the medium and only introduced after the first series of cycles has been completed. This can be achieved by immobilising the second primer on magnetic beads which can be held magnetically in the upper part of the reaction vessel until required or by coating the upper region of the vessel with means reactive with the second primer and inverting the vessel at the end of the reaction.

The 3SR method of amplification has the advantage over PCR that it is isothermal (37-42°C) . Double stranded target DNA is transcribed to RNA copies of the separate strands using RNA polymerase and each RNA copy is caused to hybridise to a DNA primer having a 5 ' - polymerase binding site permitting a DNA strand to be synthesised by the action of reverse transcriptase,- the RNA strand in each duplex is then digested away using RNase to leave two DNA strands (thus avoiding the strand separation step used in PCR which is normally effected by heating) . Each separate DNA strand is then caused to hybridise to a primer which is identical to that used to hybridise with the RNA copy generating the complementary DNA strand and a second pair of DNA strands is synthesised by the action of reverse transcriptase. Thus, using only two primers a single double stranded DNA sequence produces two double stranded sequences which may then be transcribed by reverse transcriptase to pairs of RNA copies to begin the cycle again and thus permit exponential amplification. As with PCR and LCR, the primers are incorporated into the amplified DNA and thus, in accordance with the method of the invention, one primer may be immobilised or possess means for immobilisation and one may be labelled or possess means for labelling.

In order to amplify target RNA, one may simply perform one 'limb' of the 3SR method described above, using the same primers, to convert the single stranded RNA to double stranded DNA which will then be transcribed by RNA polymerase as described above, to a pair of RNA copies to initiate amplification.

As indicated above, in order to avoid contamination with extraneous target DNA, the amplification is preferably effected in a sealed vessel into which only the sample and possibly water, aqueous buffer, or other suitable medium, are introduced. However, contamination is less serious after amplification is completed and components such as labelled ligand reactive with one of the primers incorporated into the amplified nucleic acid may be added at this stage. This enables the amplification procedure to be effected in the absence of the label which may be a relatively large molecule which might exert steric effects on the reaction if attached to a primer throughout.

Where the primers are provided with means for subsequent attachment of solid phase and label respectively, these must naturally be different. In general, high affinity binding systems such as biotin and avidin or streptavidin are best for immobilisation. One particularly suitable means of attaching a label is via a DNA binding protein as described above. Where the solid phase and label are attached to the primers, however, they are preferably bound covalently in order to survive the heating stages of the PCR and LCR reactions .

The quantity of labelled reagent introduced into the system should, in general, be calculated to be slightly less than that expected to be removed by the intended number of amplification cycles if target nucleic acid is present. However, if the reaction is effected under standard conditions, it may be possible to quantify the amount of target nucleic acid in the sample by using a slight excess of label and determining the amount remaining after magnetic separation.

As mentioned above, standard amplification conditions and primer and label concentrations etc. may be used. Conveniently, an excess, eg. a ten-fold excess of the non-labelled amplification primer may be used to help drive complete extension of the labelled amplification primer. As exemplary of reaction quantities, for an LCR reaction, for example 10 pmoles of the non-labelled amplification primer and 1 pmole of the labelled primer may be used. As regards the detection primer, for the above-mentioned amplification primer quantities, up to 0.1 pmole of the detection primer would suffice. Conditions and quantities may however be varied according to choice and the methods and systems being used or investigated. The important consideration is that all the labelled primer should hybridise to the target sequence, if it is present.

In all of the methods described above, the components for the reaction may be provided in a vessel permitting light to pass through the medium in a region from which the immobilised amplified DNA has been withdrawn. A cuvette of the type used in photometry or a microtitre well (or array of wells) may be suitable. If the illumination is along one axis of the vessel, the amplified DNA may be immobilised (magnetically or otherwise) to one of the walls parallel to that axis to leave a clear light path.

According to a further feature of the invention we provide a vessel containing one or more amplification enzymes, a primer which is immobilised or carries means for immobilisation, a primer which is labelled or carries means for attachment of a label, any further primers which are required and any nucleotide bases which are required. The vessel will preferably be sealed by a membrane permitting introduction of the sample and any water necessary by a syringe. Such a vessel may, for example, be a tube or a microtitre well and may conveniently be transparent along at least one axis, or at one end. The components will preferably be in dry form, eg. lyophilised form. A supply of high purity water or other suitable medium may be provided for introduction into the reaction vessel conveniently in a solid disposable syringe when the vessel is sealed by a membrane.

Where the immobilisation of primers and amplified DNA is by attachment to superparamagnetic particles, a permanent magnet, eg. a neodymium based magnet, may be provided. If the magnetic particles are to be withheld from the initial phases of the reaction, the magnet may be used to draw the particles to an upper region of the vessel .

The invention is now described by way of illustration only with reference to the drawings in which:

Fig. 1 shows the reaction scheme of the invention using the Ligase Chain Reaction (LCR) ; and

Fig. 2 shows diagrammatically the procedure using LCR.

A transparent vessel is provided containing an aqueous buffer, pH 7.0, a thermostable ligase and oligonucleotide primers P¹, P², P³ and P⁴ as well as oligonucleotides P⁵ attached to Dynabeads (Dynal AS, Oslo) via biotin/streptavidin binding and P⁶, attached to a fluorescent label. P⁵ hybridises to the 'handle' (cross-hatched) region of P¹ while P⁶ hybridises to the 'handle' region of P⁴.

A sample containing target double stranded DNA is introduced and subjected to strand separation by heating to 95°C. On cooling to about 60°C, the oligonucleotides P^x-P⁴ hybridise to the target DNA and P⁵ and P⁶ hybridise to P¹ and P⁴. The thermostable ligase then effects ligation to form double stranded DNA. In a second cycle, the double stranded DNA is subjected to strand separation, whereupon the newly ligated strands and the target DNA are available as templates for a further ligation reaction. Ten further cycles of heating and cooling are effected to amplify the original target DNA whereby all the labelled oligonucleotide P⁶ is incorporated into DNA containing P⁵ attached to magnetic beads. Magnetic separation of all the beads removes all labelled material into a region not interfering with the light path of the photometer. Low or zero absorbance shows the removal of dissolved label and hence the presence of target DNA in the sample.

Fig. 2 shows diagrammatically the use of M28 magnetic beads (Dynal AS) to effect magnetic separation after LCR. In A, the label (chromatophor) and M28 beads are distributed throughout the medium and a photometric reading shows the absorbance produced by the chromatophor. In B, the M28 beads and chromatophor are joined by incorporation into amplified DNA and are removed by application of a magnet so that the photometer gives low or zero absorbance. In C, there has been no amplification due to the absence of target DNA and the M28 beads are not bound to the chromatophor which thus remains distributed throughout the medium and gives a similar photometric reading to A. In practice, a positive control tube would probably be run employing a known amount of target DNA and a negative control would be run using irrelevant DNA.

EXAMPLE 1

The gene encoding the heat-labile enterotoxin (LTh or LTlb) located on plasmid(s) in enterotoxigenic Escherichia coli strains was used as target DNA. The target sequences were located in position (PI) 46 to 65 (and P3 rev) and (LCR P2) 65 to 84 (and LCR P4 rev) using the gene sequences and numbers published by Leong et al. (Infect Immun 48: 73-77, 1985) . The positive control (LTlb pos) was E. coli strain ATCC 43886, and the negative control was E. coli strain ATCC 43922. The control reference assay was a PCR procedure described by Olsvik and Strokbine (pp 271-276 In Persing et al . , Diagnostic Molecular Microbiology, ISBN 1-55581-056-X, 1993) . Test strains (6 positive and 6 negative enteric E. coli isolates) were taken from the laboratory collection of the Enteric Diseases Laboratory Section, Centers for Diseases Control and Prevention, Atlanta, GA.

Oligonucleotide LCR PI had an 18 oligonucleotide tail with sequences from the E. coli lac operon (Homes et al., J. Clin. Microbiol. 29: 2375-2379, 1991) , and P4 a 20 oligonucleotide tail from the E. coli SLT 1 operon, 215-234 (Olsvik and Strokbine supra, pp 271-276) .

A reverse oligonucleotide to the lac tail (P5 trap) was synthesized with a 5 ' -biotin molecule, and a reverse oligonucleotide to the SLT1 tail (P6 signal) was 5'- labelled with fluorescein.

The test organisms (one small colony of each) were added directly to clear glass tubes and preheated for 2 minutes as described by Fields et al. (J. Clin. Microbiol. 30: 2117-2121, 1992) , to effect lysis and DNA liberation. The LCR reaction was then carried out immediately in situ using the ligase, buffers and cycling conditions described by Cernesky et al. (J. Infect. Dis. 170: 1308-1311, 1994) , except that 100 micro gram Dynabeads, Streptavidin coated, was added prior to LCR amplification. The tube was then placed in a magnetic rack to draw the magnetic beads to the side of the tube and the intensity of the fluorescein in the central volume of the tube was observed. In the case of the positive control and the 6 strains containing the target DNA, the central volume of the tube was clear, showing that all fluorescein label had been incorporated into amplified target DNA. In the case of the 6 strains negative for the target DNA and the negative control, the fluorescein colour could be observed in the central volume of the tube.

There was full correlation between the PCR and the LCR assay for the 12 test strains and the controls.

Claims

Cla ims

1. A method of detection or assay of a target nucleic acid in a sample wherein the sample is introduced into a medium containing one or more amplification enzymes and at least two amplification primers and subjected to at least one cycle of an amplification procedure such that any target nucleic acid present is amplified, characterised in that at least one of said primers is immobilised or provided with means for immobilisation and another is labelled or provided with means for labelling whereby said primers are incorporated into amplified target nucleic acid which is thereby immobilised, if necessary by reaction with a solid phase reactive with said means for immobilisation, and labelled, if necessary by addition of a label reactive with said means for labelling, and the immobilised labelled primers are thereby removed from solution in said medium, followed by examination of the medium to detect the presence or quantity of any label left in solution and hence determine the presence, absence or quantity of target nucleic acid in the sample.

2. A method as claimed in claim 1, wherein the amplification procedure is the Ligase Chain Reaction (LCR) .

3. A method as claimed in claim 1 or claim 2, wherein the target nucleic acid is DNA.

4. A method as claimed in any one of claims 1 to 3 , wherein the solid phase comprises particles .

5. A method as claimed in any one of claims 1 to 4 , wherein the solid phase comprises magnetic particles.

6. A method as claimed in any one of claims 1 to 5, wherein the means for immobilisation is biotin or a hapten.

7. A method as claimed in any one of claims 1 to 6, wherein the label is a fluorescent label.

8. A method as claimed in any one of claims 1 to 7, wherein the amplification is effected within a sealed vessel.

9. A vessel containing one or more amplification enzymes, a primer which is immobilised or carries means for immobilisation, a primer which is labelled or carries means for attachment of a label, any further primers which are required and any nucleotide bases which are required.

10. A vessel as claimed in claim 9, wherein the components are in dry form.