AU641085B2 - Detection of bacteria using a nucleic acid amplification - Google Patents

Description

64 10 8

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Boehrlnger Mannhelm GmbH ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

S INVENTION TITLE: Detection of bacteria using a nucleic acid amplification The following statement is a full description of this invention, including the best method of performing it known to me/us'- S S *5e 1A The present patent application concerns a method for the specific detection of bacteria using a nucleic acid amplification.

The detection of bacteria in clinical diagnostics by means of nucleic acids and in molecular biology has recently become more and more important compared to the :o classical immunological tests. This is related to the :fact that advances have been made in the investigation of the nucleotide sequence of nucleic acids from a variety of sources.

The introduction of amplification methods has led to a considerable increase in the sensitivity of nucleic acid '.tests, for example in testing for sickle-cell anaemia.

Such a method is described for example in EP-A-0 200 362. The amplification effect is based mainly on the fact that with the aid of a primer and mononucleotide trinosphates, an extension product of the primer is formed from a so-called target nucleic acid as a template and this extension product is either detected or can itself again be used as a template nucleic acid. In this process a detectable nucleotide c, ?,lso be incorporated into the extension product. The extension product formed can be detected electrophoretically.

2 However, this method of detection is disadvantageous because of the complicated and time-consuming electrophoresis step.

The non-labelled extension product can also be detected according to EP-A-0 201 184 by hybridization with a detectably labelled nucleic acid probe. However, the separation of the hybrid of extension product and probe from the non-reacted probe using the method described in this application is either inefficient because of nonspecific interactions or involves many washi steps which reduce the sensitivity.

A method is proposed in WO 89/11546 in which amplified nucleic acids bound to a solid phase are reacted with a .primer and labelled mononucleotides whereby the labelled nucleic acids which are formed in this process are detected. A disadvantage of this method is that all relevant reactions proceed on the solid phase which •impairs the reaction rate. Such a method is also proposed in EP-A-0 324 474 in which labelled mononucleotides are incorporated and these labelled "nucleic acids are captured with immobilized nucleic acids which are complementary to the nucleic acid to be detected. Nothing is stated about the method for the denaturation of the amplified nucleic acids or about the hybridization conditions. A further disadvantage of this method is that the production of nucleic acids which are efficiently bound to a solid phase is laborous.

A method is described in EP-A-0 357 011 in which two primers are used in the extension reaction one of which is detectably labelled and the other is suitable for binding to a solid phase. A disadvantage of this method is that it is more laborous to separate detectably 3 labelled oligonucleotides from extension products which include these oligonucleotides. If a separation is not carried out, a reduced sensitivity would be expected because of competing reactions.

A method is described in EP-A-0 297 379 and EP-A-0 348 529 in which an immobilized or immobilizable primer is extended with a detectable mononucleotide using the target nucleic acid as template to form an immobilized nucleic acid which is at the same time detectably labelled. One disadvantage of this method is among others that the specificity of the test is not very high. The method, which is also described in EP-A-0 297 379 in which only one immobilized or immobilizable primer is used whereupon the extension products are reacted with a labelled oligonucleotide, has the drawback described in EP-A-0 357 011 that the oligonucleotide is difficult to separate.

A method is described in WO 89/09281 in which both primers have the same chemical group which is used for immobilization as well as for detection. This adds to the aforementioned disadvantage of poor separability.

A method is also described in Proc. Natl. Acad. Sci.

USA, Vol. 86, pp 6230-6234 in which a detectably labelled primer is extended. The detection is carried out after binding the extension product to a capture probe. In this case it is also not possible to separate the detectably labelled primer withoLt additional steps and this therefore interferes with the detection reaction.

4 A method for detecting bacteria cells is known from EP-A-0 131 052. In this method the bacterial nucleic acids are reacted with a detectably labelled nucleic acid probe which is shorter than the nucleic acid to be detected and subsequently the hybrid which forms is detected. A disadvantage of this method is that it is relatively insensitive and therefore requires an extensive removal of other cell components and the nucleic acids to be detected have to be concentrated.

The object of the present invention was therefore to avoid the disadvantages of the state of the art method and in particular to provide a detection method for bacteria which combines high specificity or selectivity with a low background signal.

e* 9* *o The invention concerns a method for the specific detection of a bacterium in a sample which comprises the following steps: a) lysing the sample to release bacterial nucleic Sacids, b) reacting the lysed sample with one or several labelled mononucleoside triphosphates and one or Sseveral enzymes which catalyze the production of a labelled nucleic acid B which contains this nucleotide, c) reacting the sample with a nucleic ac± probe C which is specific for the bacterium and is sufficiently complementary to the nucleic acid B, 5 d) detecting the nucleic acid hybrid D formed from the labelled nucleic acid B and nucleic acid probe C, e) whereby the nucleic acid probe contains at least one immobilizable group, f) subjecting the reaction mixture to a thermal denaturation directly before step c), g) contacting the nucleic acid hybrid D with a solid phase which can specifically bind the immobilizable nucleic acid probe C, h) separating the liquid from the solid phase and S0* S i) detecting the detectable group bound to the solid phase.

The invention is not intended to encompass methods for the detection of bacteria according to DE-A-39 29 030.

Thus methods are excluded in which the bacterial nucleic acids are reacted with at least two adaptors per nucleic acid strand, at least one of which contains a nucleotide sequence which is specific for a replication system, to form a nucleic acid which is essentially complementary to the nucleic acid to be detected which in addition contains at least one adaptor. Detection methods are preferably excluded which are based on a protein-primed replication system.

The method according to the present invention is a special way of carrying out the so-called hybridization 6 tests, the main features of which are known to one skilled in the area of nucleic acid diagnostics. As far as experimental details are concerned which are not set forth in the following, reference is made in its entirety to "Nucleic Acid Hybridization", edited by B.D.

Hames and S.J. Higgins, IRL Press, 1986, in chapters 1 (Hybridisation Strategy), 3 (Quantitative Analysis of Solution Hybridisation) and 4 (Quantitative Filter Hybridisation), Current Protocols in Molecular Biology, Edt. F.M. Ausubel et al., J. Wiley and Son, 1987, 2.9.1.

2.9.10 and Molecular Cloning, Edt. J. Sambrook et al., CSH, 1989, 9.4.7. 9.5.8. The known methods also include the production of labelled nucleoside triphosphates such as those described in EP-A-0 324 474, the chemical synthesis of modified and unmodified oligonucleotides, the cleavage of nucleic acids by means of restriction enzymes, the choice of hybridization conditions by which means a specificity can be achieved which depends on the extent of homology between the nucleic acids to be hybridized, on their GC content and Stheir length, as well as the formation of nucleic acids from nucleoside triphosphates using polymerases, if desired using so-called primers.

A label within the scope of the present invention is comprised of a directly or indirectly detectable group L. Examples of directly detectable groups are radioactive 32 coloured, or fluorescent groups or metal atoms. Examples of indirectly detectable groups are immunologically or enzymatically active compounds such as antibodies, antigens, haptens or enzymes or enzymatically active parts of enzymes. These are detected in a subsequent reaction or reaction sequence.

Haptens are particularly preferred since nucleoside triphosphates which are labelled with them can generally 7 be used very well as substrates for polymerases and a subsequent reaction with a labelled antibody against the hapten or against the haptenized nucleoside can be easily carried out. Examples of such nucleoside triphosphates are bromonucleoside triphosphates or nucleoside triphosphates coupled to digoxigenin, digoxin or fluorescein. The steroids mentioned in EP-A-O 324 474 and the detection thereof have proven to be particularly suitable. Reference is made in this connection to EP-A-0 324 474 for their incorporation into nucleic acids.

Nucleoside triphosphates (NTP) are ribonucleoside triphosphates (rNTP) or deoxyribonucleoside triphosphates (dNTP).

A target nucleic acid is understood as a bacterial nucleic acid which is the target for the test and the starting material for the method according to the present invention.

A template nucleic acid A is a nucleic acid on which a O" nucleic acid strand is newly formed which is essentially S complementary to it. With reference to the sequence Sinformation, the template nucleic acid serves as a template and contains the sequence information which is transcribed in reaction b) into the nucleic acid B. The nucleic acid A is either the target nucleic acid or a nucleic acid derived therefrom. It can for example be a part of the target nucleic acid or contain a part of the target nucleic acid in addition to other parts, for example highly complex nucleic acids. It can also contain a part of the strand which is complementary to the target nucleic acid.

8 Denaturation means separation of nucleic acid double strands into single strands. A multitude of variants are available to one skilled in the art.

A specific detection is understood as a method by means of which certain bacteria can, if desired, be detected selectively even in the presence of other bacteria.

However, it is also possible to detect a group of bacteria using nucleic acids with partially corresponding or similar nucleotide sequences. Either of thr two complementary strands can be used to detect double-stranded nucleic acids.

A nucleic acid or nucleic acid sequence which is essentially complementary to a nucleic acid is understood as nucleic acids or sequences which can hybridize to the corresponding nucleic acid and have a nucleotide sequence in the hybridizing region which is 0:60% either exactly complementary to the other nucleic acid or differs by a few bases from the exactly complementary nucleic acid. The specificity of this depends on the degree of complementarity as well as on the S: hybridization conditions.

S

Liquid phases are the aqueous phases which are usually used in nucleic acid tests with dissolved organic or inorganic constituents, e.g. hybridization buffer, excess nucleotides, nucleic acids of further bacteria :which are not to be detectei, proteins etc.

The method according to the present invention for the detection of bacteria is based on the specific detection of a nucleic acid which is specific for the ba,.terium to be detected. These nucleic acids are preferably present 9 in solution. The reaction sequence is usually initiat d by making the bacterial nucleic acid accessible using appropriate reagents, the so-called lysis. For this, physical methods, such as the use of shearing forces hydrodynamic shearing forces, realized by a French press, homogenizer), ultrasound (formation of cavities), osmosis (hydrostatic pressure which is directed against the cell membrane), heat, repetition of extreme changes in temperature (thawing/freezing) and ionizing radiation as well as chemical methods such as the inhibition of cell wall synthesis by antibiotics such as penicillin), enzymatic degradation of the cell wall (by enzymes which specifically attack the cell wall structure, such as lysozyme, lysostatin, proteases) and bacteriophage lysis can be used. Such methods are described for example in Methods in Microbiology, Academic Press, Inc. Vol. 5 B, 1971 and Manual of Methods for General Bacteriology, American Society for Microbiology, chapter 5, 1981.

Since the method according to the present invention is very sensitive and selective it is possible to even detect small amounts of nucleic acids in the presence of other materials such as proteins, cells, cell fragments, as well as nucleic acids which are not to be detected. A purification of samples can therefore be omitted if it can be assured that the nucleic acids to be detected are sufficiently accessible to the reagints used.

When examining foods it is preferable to carry out the release in a multi-step process. Firstly, the sample to be examined is disintegrated in order to release the bacteria to be detected. If the bacteria occur in very small numbers then these are propagated in a culture in a subsequent step. This is not necessary for other 10 bacteria. The bacterial nucleic acids are released by lysis from tne bacteria obtained in this way in a known manner as described above. The nucleic acids can be pretreated. The known pretreatments include for example cDNA synthesis from RNA. In order that the advantages of the method according to the present invention fully come to bear it has proven to be expedient that the nucleic acid has a size of at least 40 bp.

In addition the nucleic acids can be the product of a previous specific or unspecific nucleic acid amplification. Such nucleic acid amplification methods are disclos for example in EP-A-0 201 184, EP-A-0 272 098, DE-A-37 26 934, EP-A-0 237 362, WO 88/10315, WO 90/01069, WO 87/06270, EP-A-0 300 796, EP-A-0 310 229, WO 89/09835, EP-A-0 370 694, EP-A-0 356 021, EP-A-0 373 960, EP-A-0 379 369, WO 89/12696 or EP-A-0 361 983.

The (target) nucleic acids to be detected can be used directly as template nucleic acids A in reaction b) if they fulfil the required conditions for the selected enzyme system. For some enzymes this requires that the nucleic acids are single-stranded, for other enzymes it is necessary that they include recognition sites or promoters for the enzyme system.

If this is not the case then the target nucleic acids must be converted into such template nucleic acids A ir a step prior to reaction b).

A can be a ribonucleic acid or a deoxyribonucleic ac 4 d.

Deoxyribonucleic acids are particularly preferred as nucleic acid A.

11 Within the scope of the invention the enzyme E is an enzyme or enzyme system which catalyzes the templatedependent synthesis of nucleic acids from mononucleoside triphosphates. Preferred enzymes are polymerases and transcriptases, which act to link mononucleotides. Such enzymes are known to one skilled in the art. It is preferable to exclude protein-primed replicases.

In step b) a labelled nucleic acid B is produced from the template nucleic acid A. In principle, this can be carried out in any way or manner provided that the specific information of the nucleotide sequence of nucleic acid A or a part thereof is essentially preserved.

Since the bacterial detection according to the present invention preferably uses ribonucleic acids as target nucleic acids, it is generally not necessary to make them single-stranded befo-e or during reaction b) If a strand separation is desired then this can be carried out by treatment with alKali or thermally, enzymatically or by means of chaotropic salts.

The use of reactions b) which are dependent on the presence of a specific initiator is particularly preferred because of the increased specificity. Such specific initiators have the effect that the enzyme only acts on the nucleic acid which has bound this initiator.

SThe initiator is preferably a so-called specific primer P1 or a promoter.

Specific primers P1 are specially modified or unmodified oligonucleotides which have a nucleotide sequence S which is specially complementary to the nucleic acid A.

12 Modified oligonucleotides can for example contain groups which do not substantially impair the hybridization of the primer with the nucleic acid. Such oligonucleotides can be prepared by chemical or enzymatic means. The target nucleic acid can be used directly as the template nucleic acid A.

The use of such primers P1 therefore requires that at least a part of the sequence of the nucleic acid is known. Moreover the sequence of the primer is selected so that one of its ends, preferably the 3' end, is shorter than the nucleic acid. This therefore has a single-stranded part which extends beyond the 3' end of the primer.

000 Thus it is possible to select primer sequences S on the basis of the published nucleotide sequences of individual bacteria (Salmonella: Inf. Immun. 58: 2651 (1990); Res. Microbiol. 140: 455 (1989); J. Bacteriol.

173: 86 (1991); Listeria monocytogenes: Mol. Microbiol.

4: 1091 (1990); Infect. Imran. 55: 3225 (1987).

In reaction step one or several specific primers can be used per nucleic acid single strand to be detected.

In the case of several primers, the regions on the nucleic acid with which the primers can hybridize .00 'preferably do not overlap and it is particularly preferred that there are single-stranded regions of the •nucleic acid A between these regions. In addition to a .i 'part which is essentially complementary to the template nucleic acid A, the primer can also include another nucleotide sequence S1 at the 5' end which cannot hybridize with the nucleic acid in particular not with the region which is adjacent to the complementary region.

13 This sequence S1 can for example be single-stranded or double-stranded and also contain a recognition sequence for an enzyme. This can for example be a restriction cleavage site. With regard to effective priming, the primer can for example also contain a protein in bound form which is recognized by an enzyme E which is preferably a polymerase.

In order that the reaction b) can proceed, the complementary region of nucleic acid A and of primer P1 must firstly be separated from complementary strands which may be present. This separation can be carried out by known methods, for example thermally or nonthermally. The non-thermal denaturation is preferred.

Reaction a) is then started using conditions under which "A and P1 can hybridize with one another.

If a primer is used as the initiator, an enzyme E is also added to the sample that can synthesize a nucleic acid complementary to A in a primer-dependent reaction.

These include in particular polymerases. Their substrate *specificity depends on the nucleic acid A. If A is RNA, then RINA-ependent DNA polymerases such as reverse transcriptase from AMV or M-MuLV) come into special consideration. If A is DNA, then DNA-dependent DNA polymerases, such as Klenow enzyme or Taq DNA polymerase are preferred.

Deoxyribonucleoside triphosphates (dNTP) are also added to the sample, at least one of which is labelled.

The product of the polymerase reaction is a labelled deoxyribonucleic acid B in which the primer P1 as well as the labelled deoxyribonucleoside triphosphate are 14 incorporated. This nucleic acid B is of the same size as or smaller than the template, but has a region which is at least in part essentially complementary to the template.

This nucleic acid B can now be used directly in step c).

However, it is preferable to subject it again as a template nucleic acid to a reaction analogous to step For this, a primer P2 is added to the sample which is essentially complementary to a part of nucleic acid B, preferably to a part of the new region formed from dNTPs. The primer pair P1 and P2 preferably fulfil the conditions described in EP-A-0 201 184. It is particularly preferred that their 3' ends to be extended are 100 complementary to A or B. Pi and P2 are preferably added simultaneously to the template A.

oeooo In order to achieve an even higher sensitivity, it is eoeoe possible to carry out reaction b) several more times, preferably 1-60, particularly preferably 20-60 times whereby each time the products of the reaction are used again in reaction This results theoretically in an almost exponential increase in the number of labelled nucleic acids. In this process both nucleic acid strands ***are formed whereby the length is determined by the nonextendable ends of the primers P1 and P2. It is possible in analogy to EP-A-0 201 184 to also use so-called nested primers in the later cycles of reaction b) whose sequence is chosen so that the nucleic acids which form are smaller than those produced first. Before each cycle of reaction b) it is expedient to separate the double strands formed from A and B in reaction This can be carried out thermally or non-thermally. The thermal separation is preferred. n each case primers P1 and P2 must again be present.

15 The initiator can also be a promoter. A promoter is a nucleotide sequence which is recognized by a RNA polymerase and causes this to synthesize a complementary nucleic acid strand B on the nucleotide sequence which follows the promoter. In this process the labelled NTP is incorporated in addition to non-labelled NTPs into the nucleic acid strand which forms.

Suitable promoters are known, for example RNA-polymerase binding sites from bacterial phages such as T3, T7 or SP6 (Melton et al.: NAR 12, 1984, 7035-56; Pfeiffer Gilbert: Protein Sequences and DNA Analysis I, 1988, 269-280; Uhlenbeck et al.: Nature 328, 1987, 596-600).

In a preferred embodiment of the test procedure, the promoter is part of a specific primer for the production of the template nucleic acid A from the target nucleic acid. This primer has a nucleotide sequence S which is essentially complementary to a part of the target nucleic acid and a nucleotide sequence 1S which is recognized by a RNA polymerase and includes at least one promoter sequence. In a first reaction, a nucleic acid strand Al which is at least partially complementary to the target nucleic acid and which includes the primer with the promoter is formed using the primer and a DNA polymerase which is dependent on the type of target nucleic acid and dNTPs. If it is not already joined to the primer, the newly formed piece of nucleic acid is linked covalently to the primer by addition of a further enzyme, preferably a ligase e.g. E. coli DNA ligase. The ligase can also be thermally stable. Al is preferably shorter than the target nucleic acid. In this transcription reaction a second primer P3 complementary to the target nucleic acid strand is preferably used and the gap between both primers is closed, preferably by a 16 gap filling reaction. The use of labelled dNTPs is possible but not necessary in this case since this measure only results in a slight amplification of the measurement signal in the method according to the present invention.

If the target nucleic acid is RNA, then this is preferably selectively degraded in a subsequent step.

Known methods are suitable for this, for example treatment with alkali or RNAases. Subsequently, a nucleic acid strand A2 is formed which is complementary to Al. For this a primer P2 is preferably added to the reaction mixture that is complementary to a part, preferably to a newly formed part, of the strand Al. The S"primer P2 preferably also contains the promoter sequence S2. This is extended as described above for Al to form A2. Such procedural steps are described for example in •EP-A-0 329 822, DE-A-37 26 934, WO 88/10315, oWO 87/06270, EP-A-0 310 229 and EP-A-0 373 960 which is why reference is made to these disclosures in their entirety.

Reference is made to these disclosures in particular with regard to details which are useful and necessary for the reverse transcription of RNA or the transcription of DNA.

.i In this embodiment it is particularly preferred that the sample additionally contains the strand complementary to the target nucleic acid whereby P1 and P2 are extended simultaneously.

In the said embodiment, a DNA-dependent RNA polymerase under promoter-specific control is subsequently added 17 according to the present invention to the sample pretreated in this way. Such polymerases are e.g. T3, T7 or SP6 RNA polymerase. Using them, labelled nucleic acids B are formed from NTPs and the labelled NTP. In this case the double-stranded nucleic acid A1/A2 serves as the template nucleic acid, preferably several times.

Preferred NTPs are ribonucleotide triphosphates. Since in this variant of reaction b) single-stranded nucleic acids B are formed, a denaturation is not absolutely necessary to separate the strands but it is possible.

A preferred manner of carrying out the invention is the procedure according to DE-A-4010465, however; using S* detectably labelled ribonucleotide triphosphates.

After the last cycle of step the reaction mixture is subjected to a thermal denaturation (reaction f) in the method according to the present invention. Even if step b) is carried out several times, a thermal denaturation is carried out directly before step In this process, especially nucleic acid double strands are separated from one another. Thermal denaturation means in particular denaturation in a temperature range of ca.

50-95 0 C, preferably 85-950C. The denaturation is preferably carried out for 1-15 min.

An advantage of the thermal denaturation is that no additional reagents, such as sodium hydroxide solution have to be used. Thus pipetting steps can be omitted, the method is simplified and the reproducibility of the test is increased.

18 In the subsequent reaction step nucleic acid B is reacted with the probe C in such a way that they together form a nucleic acid hybrid D.

Oligonucleotides or polynucleotides having a length of 6 to 5000, preferably 15 to 2000, come into particular consideration as nucleic acid probe C. The probe C can be a plasmid, a nucleic acid fragment or an oligonucleotide. It can be RNA or DNA. Nucleic acid probe C is added in excess of the expected amount of nucleic acid B to the reaction mixture which is preferably in the form of an aqueous solution. The probe C has a nucleotide sequence which is essentially complementary to B and which is specific for B, and it does not hybridize with r: only to a very slight '"extent with nucleic acids present in the sample or which are newly formed that are not intended to be detected.

The combination of the use of specific primers and hybridization with a specific probe makes the method according to the present invention particularly selective.

o C can be a double-stranded nucleic acid, one strand Cl of which is complementary to a part of B. The other strand C2 of the nuclic acid probe is preferably complementary to other nucleic acids B, in particular to those which are formed when carrying out reaction b) with B as the template nucleic acid. In this case the denaturation of C can be carried out separately from B.

It is, however, preferred that C be denatured together with B.

It is preferred that C is a single-stranded nucleic acid probe C1 and that the strand C2 complementary to Cl is not added. It is then also possible to add C1 before 19 denaturing B. The solution containing the singlestranded nucleic acid Cl also preferably contains reagents which aid in the hybridization such as e.g.

SSC, formamidc or blocking reagents for nucleic acids that are not to be detected. Additional pipetting steps for the addition of the hybridization solution can be omitted by this means.

Single-stranded nucleic acid probes C1 crn for example be produced by chemical nucleic acid synthesis according to DE-A-39 16 871 or also according to EP-B-0 184 056.

Probe C contains one or several (immobilizable) groups I capable of immobilization per nucleic acid strand.

*0 4 The groups I-capable of immobilization are for example chemical groups which can be bound covalently to a solid phase for example by means of a chemical or photoreaction, or groups or parts of molecules which can be bound or recognized by another molecule or part of a molecule via group-specific interactions. Such groups are therefore e.g. hapt.ens, antigens and antibodies, nucleotide sequences, receptors, regulation sequences, S. glycoproteins such as lectins, or even the binding partners of binding proteins such as biotin or iminobiotin. Vitamins and haptens are preferred, biotin, fluorescein or steroids such as digoxigenin or digoxin 4 S* are particularly preferred. It is important for the invention that in each hybrid D the immobilizable group of the prr Iffers from the detectable group of the nucleic acd The mixture that contains the nucleic acid hybrid B when the nucleic acid to be detected was present in the 20 sample is subsequently contacted with a solid phase which can specifically bind the hybrid D via the immobilizable groups of the nucleic acid probe C.

The type of solid phase depends on the groups I capable of immobilization. It preferably has an immobilizing group R which can enter into a binding interaction with I. If the immobilizable group is for example a hapten, then a solid phase can be used which has antibodies against this hapten on its surface. If the immobilizable group is a vitamin, such as e.g. biotin, then the solid phase can contain binding proteins such as avidin or streptavidin in an immobilized form. Particularly preferred groups I and R are biotin and streptavidin.

Immobilization via a group on the modified nucleic acid is particularly advantageous since this can be carried Sout under milder conditions than for example hybridization reactions.

0 For the immobilization of the nucleic acids which are formed, the reaction mixture is preferably dispensed into a vessel after formation of the nucleic acid hybrids D, the surface of this vessel being able to react with the immobilizable group. The hybridization reaction with the probe preferably takes place at the same time as the immobilization. The vessel can for example be a cuvette, a tube or a microti.tre plate. It is, however, also possible to use a solid phase in the form of a porous material 'such as a membrane, a tissue or a pad on which the reaction mixture is applied. It is also possible to use so-called beads or latex particles.

The solid phase should have at least as many binding sites for the immobilizable group of the probe as nucleic acid hybrids D and thus nucleic acids B present.

21 The production of a preferred solid phase is described in EP-A-0 344 578 which is referred to in its entirety.

After an incubation period during which the immobilization reaction takes place, the liquid phase is removed from the vessel, the porous material or the pelleted beads. The solid phase can subsequently be washed with a suitable buffer since the binding of the hybrids D to the solid phase is very efficient. In this connecti-- the method according to the present invention allows the use of particularly few washing steps since, in contrast to the detectable probes used in the state of the art, the probes which are difficult to separate do not necessarily have to be completely removed, or leads to comparatively low background signals.

4 The amount of modified nucleic acids bound to the solid Sphase can in principle be determined in a known manner, whereby the steps which have to be carried out depend on the type of the detectable group. In the case of directly detectable groups, for example fluorescent labels, the amount of label is determined fluorometrically. If the detectable group is a hapten, then the modified nucleic acid is preferably reacted with a labelled antibody against the hapten as described analogously in EP-A-0 324 474. The label can also be an enzyme label such as -galactosidase, alkaline phosphatase or peroxidase. In the case of an enzyme label, the amount of nucleic acid is measured by means of the usually photometric, chemiluminometric or fluorometric monitoring of a reaction of the enzyme with a chromogenic, chemoluminogenic or fluorogenic substrate. It is, however, also possible to monitor the reaction electrochemically if a redox enzyme is used as the label or to monitor a change in pH by mean- of a pH 22 electrode. The measurement signal is a measure of the amount of target nucleic acid which was originally present and thus of bacteria to be detected. In initial experiments it was found that even 1-5 genome equivalents/reaction can be detected.

The detection of the nucleic acid can be carried out qualitatively as well as quantitatively. In the case of a quantitative analysis, it has proven to be expedient to carry out a comparitive experiment with a sample of known nucleic acid content. It is possible to establish a calibration curve and this is recommended.

In an embodiment of the method using PCR, oligonucleotides are added to the sample as primers P1 and P2. In this case P1 is complementary to a part of *o.

the nucleic acid single strand A which represents both the target and the template nucleic acid. P2 is homologous to a part of A which is at a distance from this. The mixture is now treated as described in EP-A-0 201 184, whereby however, for example a digoxigenin-labelled or fluorescein-labelled deoxymononucleotide triphosphate is also used in addition to the unmodified deoxymononucleotide triphosphates. 20-30 amplification cycles are preferably carried out. Afterwards single-stranded biotin-labelled probe C is added. The mixture is incubated at a temperature between 50 and 95°C, preferably between and 950C, particularly preferably between 85 and 95 0

C

for ca. 1 to 15 min. An advantage of the thermal denaturation at this stage, if desired a'ter several amplification steps, is that it is desirable to add as few reagents as possible. In the case of a chemical denaturation it may in extreme cases be necessary to add very high concentrations of such reagents, for example

I

23 in order to avoid buffering effects also caused by the reagents necessary for the lysis and their neutralization. This can also be an advantage in the subsequent wall binding of the hybrids. The mixture is transferred to a streptavidin-coated vessel, preferably after cooling the reaction mixture to ca. 37 0 C, and incubated again. The solution is removed and the vessel is washed. A conjugate of antibodies against digoxigenin and an enzyme is added and it is incubated again. After removing the solution and washing the vessel, it is reacted with a chromogenic substrate for the enzyme and the formation of colour is observed. Previously detectably labelled probes have always been used in prior art methods. A complete separation of the nonhybridized probes was necessary for the accuracy of the measured result but was relatively laborous.

0 The method according to the present invention circumvents this disadvantage in that instead of hybridizing labelled probes and determining them, the presence of incorporated labelled mononucleotikes is measured and is used as a measure of the presence or the amount of nucleic acids to be detected. The separation of ;non-incorporated labelled mononucleotides can be S.'i achieved simply and completely with the present method *060 since they are neither bound to the nucleic acids nor to the surface. On the other hand, an excess of immobilizable probe C does not interfere with the determination since the immobilizable groups of probe C are not used as a label and thus do not contribute to the measured result. In particular it is easier to calculate the binding capacity of the solid phase than with incorporation of immobilizable NTPs.

24 The method according to the present invention is therefore very sensitive and selective, in addition it can be carried out in a very short time.

The method according to the present invention is suitable for the detection of bacterial species and also for taxonomic groups of bacteria. The method can be used particularly well for the determination of pathogenic microorganisms, such as e.g. of Salmonella species, Listeria monocytogenes, Campylobacter species, Vibrio parahaemolyticus, Vibrio cholerae, E. coli, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum, Bacillus species, Yersinia enterocolytica. The test is preferably possible in foods such as milk, milk products such as cheese, kefir, yoghourt, meat, fish, seafood, vegetables, lettuce, T**o rice, cereals, eggs, poultry, spices, herbs and dried foods. Ribonucleic acids as well as deoxyribonucleic acids can be detected. Ribonucleic acids are preferred as the target nucleic acid such as rRNA; particularly preferably 16 S or 23 S-rNA.

In the method according to the present invention it is possible to select different specificities for the specific initiator and the nucleic acid probe. As a result it is possible to achieve a double specification.

S

Figure 1 shows a diagram of the course of an embodiment using a primer elongation (use of only one primer PI).

Figure 2 shows a diagram of the course of a preferred embodiment using the PCR principle with two primers whereby the nucleic acids B formed first are again used as template nucleic acid.

25 Figure 3 shows a calibration curve which was obtained according to example 1.

Figure 4 shows the nucleotide sequence of the primers used in example 1 and of the probe (DNA; linear; singlestranded; 2 0, 19 or 2 0 bp).

to 6 to 26 Abbreviations: A template nucleic acid Al opposite strand of A P1 primer complementary to A E r-nzyme/enzyme complex El DNA polymerase or reverse transcriptase L detectable group (label) B product of reaction a) C nucleic acid probe I immobilizable group D nucleic acid hybrid of B and C R immobilizing group F solid phase S1 sequence complementary to A Sl' another sequence complementary to A S2 promoter S2' promoter The invention is elucidated in more detail by the following example:

C

0.0.

.0 27 Example The bacteria are lysed with 1 Triton X-100 using a min incubation at 950C. 10 Ml of the lysis preparation are used in a polymerase chain reaction. The primers used bind to positions 689-708 and 2223-2241 in the 23 S rRNA gene of Listeria monocytogenes (Figure i.e. a sequence of 1552 nucleotides is amplified. 5 fg to 5 ng of chromosomal DNA is used in the dilution series used in this case.

PCR cycles (15 sec 94 0 C; 30 sec 60°C; 90 sec 72 0

C)

are carried out in a volume of 100 il containing 200 mM of each primer; 50 mM KC1; 10 mM Tris-HCl, pH 1.5 mM MgCl 2 100 ug/ml gelatin; 200 AM dATP; 200 gM dGTP; 150 MM dTTP; 50 MM digoxigenin-11-2'-deoxyuridine- •5'-dUTP (Dig-[11]-dTP, Boehringer Mannheim); 2.5 U Thermus aquaticus (Taq) DNA polymerase. 20 Ml of the amplification preparation and 40 ng biotin-labelled sample DNA, Figure 4, which binds to position 1191-1210 in the above-mentioned gene are denatured in a total volume of 40 Al for 10 min at 95 0 C. Subsequently, 160 pl hybridization solution (52.5 mM sodium phosphate, pH 6.8; 6.25 x SSC [1 x SSC 0.15 M NaCl; 0.015 M sodium citrate] and 62.5 formamide) is added and it is pipetted into a streptavidin-coated microtitre plate.

The hybridization/wall binding is carried out for three hours at 37°C while shaking gently. After removing the hybridization solution and washing three times with 0.9 NaC1, 200 mU/ml <digoxigenin>-horseradish peroxidase conjugate is added and incubated for 30 min at 37 0 C in 10 mM Tris-HCl, pH 7.5; 0.9 NaCI; 1 BSA; Pluronic T68. After washing three times (conditions see above) it is incubated with 0.1 2,2- 28 azino-di[3-ethylbenzothiazole]-(ABTS) for 30 min at 370C and the absorbance is measured at 405 nm.

The absorbances for this reaction as well as for the control reaction~ with biolabelled sample are shown in Figure 3.

With the aid of this curve it is now also possible to determ~ine the nucleic acid concentrations present in samples of unknown bacterial concentration and thus the amount of bacteria..

S6* 66

IN:

Claims (12)

1. Method for the specific detection of a bacterium in a sample which comprises the following steps: a) lysing the sample to release bacterial nucleic acids, b) reacting the lysed sample with one or several labelled mononucleoside triphosphates and one or several enzymes which catalyze the production of a labelled nucleic acid B which contains this nucleotide, a c) reacting the sample with a nucleic acid probe C which is specific for the bacterium and sufficiently complementary to the nucleic acid B, and d) detecting the nucleic acid hybrid D formed from the labelled nucleic acid B and nucleic acid probe C, wherein e) the nucleic acid probe contains at least one immobilizable group, f) the reaction mixture is subjected to a thermal denaturation after step a), 30 g) the nucleic acid hybrid D is contacted with a solid phase which can specifically bind the immobilizable nucleic acid probe C, h) the liquid phase is separated from the solid phase and i) the detectable group bound to the solid phase is detected. whereby methods are excluded in which the bacterial nucleic acid probes are reacted with at least two adaptors per nucleic acid strand, at least one of which contains a nucleotide sequence which is *e specific for a replication system, to form a :nucleic acid which is essentially complementary to the nucleic acid to be detected which in addition contains at least one adaptor.

2. Method as claimed in claim 1, wherein reaction b) S. proceeds in the presence of a specific initiator.

3. Method as claimed in claim 1 or 2, wherein the enzyme or the enzyme complex catalyzes the polymerisation of nucleoside triphosphates to a nucleic acid B which is essentially complementary to nucleic acid A.

4. Method as claimed in claim 3, wherein the nucleoside triphosphates include ribonucleoside triphosphates. 31 Method as claimed in claim 2, wherein in reaction a primer P1 is used as the specific initiator, a part of which is essentially complementary to the nucleic acid A, and is extended by the enzyme with incorporation of the mononucleoside triphosphate to form a nucleic acid B which is essentially complementary to nucleic acid A.

6. Method as claimed in claim 5, wherein in addition a primer P2 is used a part of which is essentially complementary to the nucleic acid B.

7. Method as claimed in one of the claims 3 or wherein the nucleoside triphosphates include deoxyribonucleos .e triphosphates.

8. Method as claimed in one of the claims 1 to 3, wherein the nucleic acid to be detected is single- stranded or is made single-stranded, then at least one primer per single strand to be detected is added to the sample, said primer containing a nucleotide sequence a part of which is essentially complementary to the nucleic acid to be detected and containing a transcription initiation sequence, the primer is extended by a nucleotide sequence which is complementary to the nucleic acid to be detected and the nucleic acid formed in this way is used as template nucleic acid in reaction a).

9. Method as claimed in one of the previous claims, wherein the nucleic acid B formed in step a) is used again in reaction a) as template nucleic acid. 32 Method as claimed in one of the previous claims, wherein reaction b) is carried out several times in succession whereby in each case the products of the reaction are used as the starting material of the renewed reaction.

11. Method as claimed in claim 9 or 10, wherein a nucleic acid hybrid is formed from nucleic acid A and nucleic acid B in reaction b).

12. Method as claimed in one of the previous claims, wherein the denaturation is carried out between and 95 0 C.

13. Method as claimed in claim 12, wherein the nucleic acid probe C is a single-stranded nucleic acid Cl :whose opposite strand is not present in the solution.

14. Method as claimed in claim 6, wherein the nucleic acids formed by extension of the primers P1 and P2 are again reacted with P1 and P2 after separation of the strands whereby each time the new strands formed using the one primer serve as a template for the extension of the other primer. 4 e 33 A method as claimed in claim 1, substantially as hereinbefore described with reference to the drawings and/or Example. disclosed herein or referred to or i.ndica e specification and/or cllai application, individuall c ively, and any and all combinations -Mza said sharls or= r-eaba;G-88. *866 06 6O 6 6S 66 66 6 6 0 *660@6 6 6 666666 6 6 666066 6 DATED this TWENTY FIRST day of FEBRUARY 1992 Boehringer Mannheim GmbH by DAVIES COLLISON CAVE Patent Attorneys for the applicant(s) 6 Abstract Method for the specific detection of bacteria in a sample by reacting the sample with one or several labelled nucleotide triphosphates and one or several enzymes which catalyze the production of a labelled nucleic acid B containing this nucleotide, thermally denaturing, reacting the sample with a nucleic acid probe C which is sufficiently complementary to nucleic acid B and contains at least one immobilizable group, contacting the nucleic acid hybrid D which may have formed with a solid phase which recognizes and binds the immobilizable group, removing the liquid from the solid phase and determining the label on the solid phase as a measure for the presence of the bacterium. g 9 9