WO2006018311A2 - Determination of nucleic acid analytes by treatment with microwaves - Google Patents

Abstract

The present invention relates to processes and devices for determining a nucleic acid analyte in a biological sample by hybridisation, particularly by fluorescence in situ hybridisation (FISH), wherein the sample is treated with microwaves.

Description

Determination of nucleic acid analytes by treatment with microwaves

Description

The present invention relates to processes and devices for determining a nucleic acid analyte in a biological sample by hybridisation, particularly by fluorescence in situ hybridisation (FISH).

Infectious diseases remain to be one of the major cause of deaths and economic burden in terms of disability adjusted life years (DALYs) (45, 46, 47). Speed and effectiveness of treatment depends on the speed in which the identification of a pathogen can be performed. Without identification the initial treatment must be with expensive broad spectrum antibiotics. Inadequate or unspecific treatment increases the risk of the development of antibiotic resistance. The impact of these facts is well documented (48). Rapid identification of the causative pathogen and evaluation of its susceptibility to antimicrobial agents can clearly reduce such risks.

In contrast to other laboratory disciplines, where only precise requests for the determination of already-specified analytes are submitted, the challenge facing a microbiologist is to identify a pathogen out of some 2000 potentially clinically-relevant organisms with differing characteristics and growth requirements. The current procedure is to culture and isolate organisms on growth media, to identify the organism from its biochemical characteristics and then (in an increasingly important step) to carry out antibiotic susceptibility testing. This procedure can take from two to several days, depending on the organism's growth rate.

Modern DNA-probe or DNA-probe analogue based assays utilizing amplification methods have made little impact on the routine bacteriology and on speeding the procedure. Current chip technology would encompass the binding of >2000 different specific DNA-oligomers to one chip. Because such a device would be controlled by laws governing in-vitro diagnostic devices, this chip requires extensive quality control thus generating extensive production cost. Should the chip be reusable, additional quality control is required for accredited laboratories.

At the beginning of the 1990s, a method of in-situ with fluorescence-labelled oligo-nucleotide probes was developed and has been used successfully with many environmental samples (1). This method, which is known as "FISH" (fluorescence in-situ ) is based on the fact that the ribosomal RNA (rRNA) present in each cell includes both highly conserved sequences, i.e. those with a low specificity, and less conserved sequences, i.e. genus-specific and species-specific sequences. By the middle of the '80s, it had been demonstrated that the sequences of the 16S- and 23S-rRNA can be used for identification of micro-organisms (2). In the case of the FISH method, fluorescence-labelled gene probes whose sequences are complementary to a certain region on the ribosomal target sequences, are introduced into the cells. The probe molecules are usually single-stranded deoxyribonucleic acid fragments or analogues thereof, and are complementary with a target range which is specific for a certain species or genus of bacteria. If the fluorescence-labelled gene probe finds its target sequence in a bacterial cell, it binds to it, and the cells can be detected on the basis of their fluorescence in a fluorescence microscope.

Whole Cell Hybridisation technology was initially applied mainly to environmental studies, where the localisation of physiological clusters (13,14) and the influence of chemicals on a population were of primary interest (27). The technology also had an impact in the detection of spoilants in food hygiene (4). Further, the application of this technology to clinical samples had a significant impact on both diagnostic and therapeutic procedures.

FISH applied to tracheal swabs revealed Haemophilus influenzae (15), Candida species could be detected in artificially infected animals (15,16), pathogenic Yersinia species were detected biopsy specimens, stool, and tracheal swabs (17) as well as Bifidobacteria and other bacteria in stool samples (18, 19). Furthermore, it was possible to demonstrate the advantages of identifying bacterial directly from clinical specimens and blood cultures (21,22,25,26,27,28,29,30,31, 32, 33, 34, 35).

Recent advances have made fluorescent in-situ hybridisation (FISH) a highly sensitive, cost-effective diagnostic procedure that can deliver results in 2-3h from samples such as smears, body fluids, soft tissue aspirates, and formalin-fixed biopsies (36, 37). The application of this technology is of special importance in the support of critically ill patients. It is well shown in the literature that reduction in the time to report bacteriological results has a material impact on survival rates and treatment cost (51 , 52). It is therefore clearly desirable to reduce the time to report from 2-3h to under one hour.

However, the application of the various methods to routine microbiological testing proved to be impractical, as the performance of multiple hybridisations on the same sample is perceived to be cumbersome. The generation of a successful result is often lacking in reproducibility, and sufficient controls had to be built in to achieve a readable result.

While the individual steps of FISH are not complex, the results are often lacking reproducibility, especially when applied to a wide spectrum of samples, particularly clinical samples.

Problems occurring in FISH are reviewed by Amann in (12), for example, and may encompass the lack of target cells in the microscopic field, low signals due to a the number of ribosomes being too low in the target cell (39, 40, 41), inhibition of penetration of target cells by cell wall structures (4, 42), inaccessibility of target sequences (43, 44) and unspecific binding of probes.

Further, the published and available FISH procedures for the identification of bacteria currently carry a turn-around time of 2.5 to 3 hours. While this represents a significant improvement with respect to traditional - A - methodologies, there is a dire need to speed the identification for ethical and financial reasons. Some organisms, especially gram-positive require skill to achieve reproducible results. Attempts to alleviate this problem are also the subject of prior art and patent applications, however without giving entirely satisfactory and reproducible results.

A further problem lies in the fact that in the application of FISH technology requires training as in all molecular biology procedures. Without training and strict adherence to protocols, variable results may be achieved depending on the applicator's skill. This is mainly due to the variables in the fixing of organisms to a slide, and the achievement of optimal hybridisation conditions within a hybridisation chamber. Prior attempts to achieve improved adherence coincided with a significant increase in background noise.

State of the art in-situ hybridisations with DNA-probes or analogues thereof are performed with one or two widely separated fields on a glass slide, covered with a cover-slip. The slides are placed horizontally or vertically into a hybridisation chamber. For the performance of multiple hybridisations on one slide the presence of the cover-slip allows capillary forces to build bridges between the fields, thus enabling unacceptable transfer of probes between fields. For a cost-effective practicable application of the FISH technology, it is therefore necessary to devise hybridisation conditions that will allow multiple hybridisations with DNA-probes or analogues thereof on one slide without a cover-slip.

In order to introduce the FISH technology into routine testing, a standardised and reproducible sample preparation procedure needs to be devised, and the hybridisation procedure needs to be streamlined.

The present invention is the result of systematic examinations which uses microwaves for the processing of biological samples in FISH analysis. From the experimental data, a protocol has been prepared which enables the use of conventional microwave devices for FISH analysis.

A first aspect of the present invention relates to the use of microwave technology in whole cell in situ hybridisation techniques, particularly to a process for determining a nucleic acid analyte in a biological sample by hybridisation with a labelled probe, comprising treating the sample with microwaves.

In the state of the art, microwaves have been used to denature double stranded chromosomes in different biological samples in order to generate single stranded DNAs which are subsequently used for hybridisation with labelled probes to detect specific sequences in a chromosome or for primers to initiate synthesis of specific DNA fragments, e.g. by polymerase chain reaction (PCR). In another application microwaves have been used to dissociate purified double stranded DNA in order to achieve binding of the single stranded products to a solid matrix, e.g. a nylon membrane, which is subsequently subjected to hybridisation reaction.

No instructions were found to use microwaves for the FISH analysis of ribosomal RNAs (rRNA) in whole cell preparations. The analysis of rRNAs by FISH demands the integrity of both the target cell and the individual ribosomes or polysomes inside the target cell. If the target cells or the intra¬ cellular ribosomes become destroyed, the ribosomes or rRNAs are depleted from the cell in the various washing steps of the FISH protocol which finally results in false negative results. In addition, the shape of the cell may be used as an additional criterion for the characterisation of a biological sample.

The first critical step in whole cell FISH analysis comprises the binding of the biological sample to a solid matrix, the carrier. The binding of the biological sample to the carrier has to resist the various manipulations which are needed to achieve specific binding of the hybridisation probe to the nucleic acid analyte inside the target cells and to detect the binding of the labelled hybridisation probe. In conventional FISH analysis, the biological samples are bound to solid matrices, e.g. glass slides, by exposing the biological sample to elevated temperatures of about 40 - 5O⁰C in the air of an incubator.

A further aspect of the present invention therefore relates to a process for determining a nucleic acid analyte in a sample comprising covalently attaching components of the sample suspected of containing the nucleic acid analyte, e.g. whole cells, to a carrier without previous purification and determining the nucleic acid analyte by in situ hybridisation with a labelled probe.

The attachment of components of the sample to the carrier may comprise microwave treatment for about 2 to 7 min, preferably for about 3 to 6 min, at about 130 to 300 W, preferable at about 200 to 250 W.

The suitability of microwave treatment in whole cell in situ hybridisation procedures is indeed surprising, in particular in fixing cells to slides. Since the typical cell volume of an average bacterium is in the order of about of 1 picolitre, a skilled person would have expected that the supply of microwave energy to a bacterial cell would be detrimental to the cellular morphology resulting in exploded cells as seen when overfixing a bacterial cell by treatment with a burner flame. Surprisingly, however, the application of high amounts of microwave energy does not destroy cells but causes them to adhere to the carrier surface.

It was also found that the fixing of bacteria to carriers with microwave energy significantly reduces background- and autofluorescence. Further, when blood cells were used as samples, the microwave energy destroys the autofluorescence of haemoglobin in such a way that it is possible to detect specific fluorescence, even under a solid layer of erythrocytes. Furthermore, it was possible to detect not only bacteria, but other intracellular pathogens, such as viruses, e.g. hepatitis B viruses in hepatic cells prepared from needle aspirates. Furthermore, it was found that microwave treatment significantly increased the reproducibility of Staphylococcus determination which is known to be very problematic. It was even more surprising that the species specific hybridisation of DNA probes or analogues thereof to bacteria in the presence of microwaves were completed within minutes without loss of the sensitivity and specificity required to detect point mutations with oligo-nucleotides. This allows a reduction of the overall assay time from 2.5 hours to 1 hour and less, without the modification of regions and probes.

In the present invention the biological probe may not be used directly after isolation from a patient but from a transport media where the biological probe has been deposited in a fixed or unfixed mode. Most of the transport media contain agar agar which is auto-fluorescent in conventional FISH protocol. In the present invention, the auto-fluorescence of agar agar disappears after microwave treatment, thereby reducing background fluorescence when detecting a hybridisation probe which may be fluorescently labelled.

The process of the present invention preferably comprises an in situ hybridisation, wherein a nucleic acid analyte is determined in a cell. The hybridisation probe may be a nucleic acid molecule, particularly a DNA oligonucleotide or a nucleic acid analogue such as a locked nucleic acid (LNA), a peptide nucleic acid (PNA) or another nucleic acid analogue capable of forming hybrids with a DNA or RNA molecule within a cell. The probe molecule preferably has a length of 12 to 30, particularly 15 to 25, more particularly 17 nucleotides or nucleotide analogue building blocks and is usually in a single stranded form. The probe carries at least one detectable labelling group, preferably a fluorescent group. The probe may contain one or several labelling groups as known in the art.

The biological sample in which a nucleic acid analyte is determined may be a clinical sample derived from a patient, e.g. a body fluid such as blood, serum, plasma, urine, saliva, etc, a sample from body tissues, a stool sample, etc obtained from a human or a non-human animal. Further, the biological sample may be an environmental, e.g. agricultural sample from plants, soil, sediments or a fresh or salt water sample. Furthermore, the sample may be derived from foodstuffs or drinkable liquids, from cell cultures, medical devices, etc.

By the process of the present invention, the nucleic acid analyte is preferably determined in a microbial cell, such as bacteria and yeast. More preferably, the microbial cell is a bacterial cell. Even more preferably, the microbial cell is a Gram negative bacterial cell, preferably selected from the phylogenetic orders Bacteroides, Burkholderiales, Neisseriales, Campylobacterales, Enterobacteriales, Pasteurellales, Pseudomonadales, Legionellales, Spirochaetales, more preferably selected from the genera Bacteroides, Prevotella, Burkholderia, Neisseria, Helicobacter, Escherichia, Shigella, Klebsiella, Yersinia. In an alternative even more preferred embodiment, the microbial cell is a Gram positive bacterial cell, preferably selected from the order Actinomycetales, Bacillales, Lactobaci Hales from which the genera Mycobacterium, Propionibacterium, Listeria, Streptococcus, Staphylococcus, Clostridium, Enterococcus and Peptostreptococcus are more preferred. In a further even more preferred embodiment, the microbial cell is selected from microbial cells not sensitive for Gram, staining, in particular from Chlamydiales and Mycoplasmataceae.

In the process of the present invention the biological sample or an aliquot thereof, e.g. an amount of 5 to 100 μl, preferably 5 to 20 μl containing a sufficient number of cells to be analysed is fixed on a carrier, e.g. a chip or a slide. Preferably the carrier contains a plurality of sample application fields in order to allow parallel testing of different samples and/or parallel testing of a sample with different hybridisation probes. It is further preferred that individual sample application fields are separated from each other by inert materials. The carrier surface may be a glass, metal or metal oxide surface. The carrier may be a planar carrier or a microfluidic device containing channels or any other suitable type of carrier.

After applying the sample to the carrier, e.g. to a predetermined surface area of 10 to 10 000 μm² of the carrier, the sample is dried, e.g. by drying at elevated temperatures of about 40 to 50⁰C in moderately heated air. Subsequently, the cells contained in the sample are fixed on the carrier, e.g. by conventional methods or, preferably, by microwave treatment, e.g. treatment for 2 to 7 minutes at 130 to 300 Watts.

A preferred device for carrying out the process of the invention is shown in Figures 1 to 3. Figure 2 shows the fixing of the cells to the carrier in a microwave oven. The slide, e.g. an 8-fιeld slide as shown in Figure 1, is placed in the chamber of the microwave oven, preferably without a cover and subjected to microwave radiation sufficient to achieve the fixing of the cells on the carrier. After fixing the hybridisation procedure is carried out as explained in detail below. Figure 3 shows that the hybridisation is preferably carried out in a closed hybridisation chamber within the microwave oven. The chamber may be a water tight container, e.g. a plastic container. The hybridisation chamber preferably also contains a fluid, preferably an aqueous medium. The amount of the aqueous medium in the hybridisation chamber is preferably at least 50 ml, more preferably at least 100 ml, e.g. about 250 ml, to obtain an improved temperature control.

In a further aspect of the invention the fixing of the cells on the carrier takes place without previous purification of the sample. In this embodiment, the carrier is preferably pre-coated with a cationic polymer such as poly-lysine. In this embodiment, the cells are preferably fixed via a heterobifunctional linker.

Several heterobifunctional linkers are conceivable and available from commercial suppliers (50) and can also couple micro-organisms to a chip.

A list of preferred heterobifunctional cross linkers is given below:

An especially preferred linker is 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimide-ester (ATFB-SE).

After fixing, the cells on the carrier are dehydrated, e.g. by placing in an organic solvent such as methanol or ethanol for preferably 5 to 30 minutes.

If a determination of analytes in a gram-positive prokaryotic cell is desired, an enzymatic digestions of cell walls is preferably carried out.

Gram negative organisms can be hybridised in-situ without further manipulations after dehydration. However, it is frequently required to identify organisms in their natural environment and methods must allow the simultaneous hybridisation of both gram-negative and gram-positive organisms. This can be achieved by partial digestion with enzymes lysing the cell walls, e.g. Lysozyme (4,5) or a mixture of Lysozyme and Lysostaphin (11 ); or a two step procedure with a.) detergence (Saponin; Triton X) b) a complex mixture of Lysozyme, Lysostaphin and acetylmuramidase-SG or acromopeptidase in PBS-buffer with Saponin (10).

A list of other cell wall digesting enzymes which may achieve comparable results is given below.

Surprisingly, a further increase in hybridisation signal may be achieved by the addition of individual or combinations of cationic antimicrobial peptides such as cathelicidins, defensins, or bacteriocins as a stabilising reagent. These peptides are key components of the innate immune system. It is widely believed that the killing mechanism of these peptides on bacteria involves an interaction with the cytoplasmic membrane (54). The minimal inhibitory concentrations (MIC) for organisms such as Staphylococci are known to be between 1 and 1000 μg/ml at a pH<6 (53). The concentrations of the peptides in the present invention are preferably above the highest MIC value of the relevant microorganisms, e.g. at about 20-40 μg/ml or higher. The MIC values of the exemplary antimicrobial peptides nisin A, nisin Z and pediocin produced by lactic acid bacteria for some gram-positive bacteria are shown below.

a Reported values are means of five replicate trials; nd, not determined; R, resistant; AU, arbitrary units determined using the micro-assay method with indicator strains P. acidilactici UL5 for nisins A and Z, and L. ivanovii HW 28 for pediocin.

In a preferred embodiment a nisin is applied. Nisin is a 34-amino-acid polypeptide bacteriocin (lantibiotic) produced by Lactococcus lactis subsp. lactis. Since its discovery in 1920s, nisin has proven to be an effective inhibitor against a broad spectrum of gram-positive bacteria. Nisin forms pores in the cell membrane of gram-positive bacteria, destroying the EMF and letting small cytoplasmic molecules leak. Size of pores are 0,1 -1nm (Ribosome = >10nm) The application of nisin and other antimicrobial peptides leads to an enhanced hybridisation performance preferably in the presence of Lysozyme/Lysostaphin and a buffer, e.g. a Tris/HCI buffer with a pH >8.

In a further step, the hybridisation probe or a mixture of hybridisation probes is applied to the fixed sample. Hybridisation probes are chosen for having a sufficient degree of complementarity to the nucleic acid analytes in order to allow a specific hybridisation thereto. Preferably, the hybridisation probes are capable of the determination of microorganisms, including bacteria, parasites and viruses. The hybridisation probes may be selected from species-specific or strain-specific hybridisation probes, i.e. probes capable of distinguishing a microorganism species or strain from another species or strain, genus-specific hybridisation probes, i.e. hybridisation probes capable of identifying a genus of a microorganism but not capable of distinguishing between different strains or species within the genus, non-specific probes, e.g. probes capable of identifying the presence of eubacteria in general or hybridisation probes capable of identifying antibiotic resistant microorganisms, particularly microorganisms resistant against macrocyclic antibiotics. Preferred examples of suitable hybridisation probes are disclosed (36) and (37).

The nucleic acid analyte is complementary to the hybridisation probe. Preferably, a nucleic acid analyte is chosen which is present in multiple copies within a cell. More preferably, the nucleic acid analyte is a ribosomal RNA molecule, e.g. a 23S or 16S bacterial ribosomal RNA molecule which is present in several 1000 copies per microbial cell. It should be noted, however, that the nucleic acid analyte may also be a eukaryotic, e.g. protozoal ribosomal RNA molecule or a viral nucleic acid (DNA or RNA). When several different hybridisation probes are used in a single analysis, they usually contain different labelling groups which can be detected in the presence of each other, e.g. different fluorescence labelling groups.

The hybridisation probe may be suitable to detect point mutations in a cell, i.e. the sequence of the hybridisation probe is complementary to a sequence differing by a point mutation from a wild type sequence. The process of the present invention allows for specific hybridisation of the hybridisation probe with the point mutated sequence, but the probe does not hybridise with the wild type sequence.

A point mutation may be an alteration of a single nucleotide with reference to a wild type nucleic acid, e.g. by insertion or deletion of a nucleotide or by replacement of one nucleotide by another nucleotide.

It is particularly preferred that the hybridisation probe suitable for detecting point mutations has a length as discussed above.

After applying the hybridisation probe, the sample is subjected to conditions which allow a hybridisation of the probe to the nucleic acid analyte in the sample. According to the present invention, these conditions are established by microwave treatment. The hybridisation temperature is preferably in the range of 42-50⁰C, depending on the respective hybridisation probes. A hybridisation temperature of about 46⁰C is particularly preferred..

The hybridisation may comprise microwave treatment for about 0.1 to 10 min, preferably for about 0.5 to 5 min, at about 40 to 800 W, preferably about 60 to 500 W, more preferably at about 80 to 250 W. Most preferred is a microwave treatment for about 1, about 2, about 3, about 4 min at about 240 W (continuous microwave heating) or at about 80 W (pulsed microwave heating, see below).

The hybridisation may comprise microwave treatment in the presence of hybridisation buffer or/and in the presence of wash buffer. Heating, in particular by microwave energy, may be applied continuously or in a pulsed protocol. A pulsed protocol of the present invention comprises successive periods of heating and non-heating (or heating with reduced power). Heating during the pulses or/and reduced heating during the intercepts can be preformed by any method suitable for regulating or/and adjusting the temperature of the sample. Suitable methods are known by those skilled in the art. A pulsed protocol of the present invention allows for precisely maintaining the temperature in a small temperature range with predetermined upper and lower limit. The difference between the upper limit and the lower limit may be smaller than 5 K, preferably smaller than 2 K, more preferably smaller than 1 K.

A pulsed protocol according to the present invention comprises at least two heating pulses and an intercept between the pulses in which heating is reduced or switched off.

The duration of a single pulse may be about 0.05 to 5 min, preferably about 0.25 to 3 min. In a single step of the in situ hybridisation protocol of the present invention (e.g. fixation, hybridisation or washing), the sum of the durations of the pulses may be about 0.1 to 10 min, preferably about 0.5 to 6 min. Most preferred is a duration of about 1 , about 2, about 3, about 4 min.

The length of a single intercept may be about 0.05 to 10 min, preferably about 0.1 to 5 min. In a single step of the in situ hybridisation protocol of the present invention, the sum of the durations of the intercepts may be about 0.1 to 20 min, preferably about 0.2 to 10 min. If the heating is not switched off during the intercepts, reduction of heating refers to a reduction to a value of preferable at the maximum 20%, more preferably at the maximum 50%, even more preferably at the maximum 80% of the pulse power.

It is preferred that heating during the pulses or/and reduced heating during the intercepts is performed by microwave treatment. Any method for microwave treatment of the sample as described herein may be employed. Alternatively, it is preferred that heating during the pulses or/and reduced heating during the intercepts is performed by a cooling or/and heating block thermostat, e.g. a block thermostat comprising Peltier elements. In such a method, the biological sample on the carrier, e.g. glass slide or microfluidic system, is deposited in the block thermostat. Appropriate cooling/heating block thermostats are described elsewhere, e.g. HLC GmbH, Germany. Especially preferred is microwave treatment.

It has been demonstrated that specificity and reproducibility of PCR was improved by touchdown annealing, as published by Don et al (58). Touchdown annealing in the context of PCR refers to incremental decrease of the annealing temperature from cycle to cycle, allowing to give an advantage to the correct annealing product compared with incorrect products. This process is employed when the precise melting temperature is not known and improved specificity and yield (59) is required.

One might expect that due to the fundamental differences in the reaction kinetics of PCR and FISH, touchdown annealing was generally not found suitable for FISH analysis. In a conventional PCR reaction the original nucleic acid analyte is continuously amplified whereas in FISH the nucleic acid analyte remains constant. Hence in the present invention, it was surprising that a pulsed heating protocol reduces hybridisation time by an order of magnitude without loosing specificity. Thus, the repeated touchdown hybridisation cycles gave unprecedented specificity in the short time allowing the detection of point mutations.

A further aspect of the present invention is therefore a process for determining a nucleic acid analyte in a biological sample by hybridisation with a labelled probe, wherein hybridisation between the nucleic acid analyte and the labelled probe comprises decrease of the temperature from a predetermined starting temperature to a predetermined end temperature. The temperature decrease may be performed continuously or stepwise. Temperature control may be performed by any method suitable for regulating or/and adjusting the temperature of the sample. Suitable methods are known by those skilled in the art. The starting temperature may be a temperature above the calculated melting temperature of the probe/analyte hybrid, preferable 15 ⁰C at the most, more preferable 10 ⁰C at the most, most preferable 5°C at the most above the calculated melting temperature of the probe/analyte hybrid. The end temperature may be a temperature below or equal to the calculated melting temperature of the probe/analyte hybrid, preferably 5 ⁰C at the most, more preferably 2 ⁰C at the most, even more preferably 1⁰C at the most below the calculated melting temperature of the probe/analyte hybrid, and most preferably about the calculated melting temperature.

Calculation of the melting temperature can be performed by methods known to a person skilled in the art. The melting temperature of e.g. a DNA-RNA hybrid may be calculated according to Yilmaz and Noquera (2004). L. S. Yilmaz and D. R. Noquera (2004) Mechanistic approach to the problem of hybridization efficiency in Fluorescence In situ Hybridization. Applied and Environmental Microbiology 70 (12):7126-7139.

Stepwise decrease is preferably performed in steps ranging from about 0,2 to 5 K₁ more preferably in steps ranging from 0,5 to 3 K and most preferably in steps ranging from about 1 to 2 K.

During the temperature decrease, the hybridisation between the nucleic acid analyte and the labelled probe may be monitored by a means for determining hybridisation between the nucleic acid analyte and the labelled probe, e.g. by fluorescence measurement. Suitable means and methods for determination of hybridisation are known to a skilled person. If a hybridisation signal is detected, temperature decrease may be stopped.

In a preferred embodiment, temperature control during temperature decrease is performed by treatment of the sample with microwaves. Any method of microwave treatment of the sample as described herein may be employed. In an alternative preferred embodiment, temperature control during temperature decrease is performed by a cooling or/and heating block thermostat, e.g. a block thermostat comprising Peltier elements. In such a method, the biological sample on the carrier, e.g. glass slide or microfluidic system, is deposited in the block thermostat. Appropriate cooling/heating block thermostats are described elsewhere, e.g. HLC GmbH, Germany. Especially preferred is microwave treatment.

The pulsed protocol of the present invention may comprise temperature decrease as described above, in particular during the intercepts.

The hybridisation further comprises contacting the sample with a washing solution/buffer in order to remove unhybridised probe molecules. The stringency of the washing conditions may be chosen according to the desired hybridisation specificity. Preferably, the washing step is also carried out by applying microwave treatment. Appropriate conditions for microwave treatment during hybridisation and/or washing may be determined by the skilled person, e.g. according to the charts in Tables 1 and 2.

The invention also relates to a device for determining a nucleic acid analyte in a biological sample, comprising

(a) a hybridisation chamber comprising a carrier on which at least one sample can be attached,

(b) means for applying microwave energy to the hybridisation chamber,

(c) optionally, means, e.g. channels for applying the sample, probes and/or reagents to the carrier, and

(d) optionally, means for determining hybridisation between the nucleic acid analyte and a labelled probe.

The hybridisation chamber (a) is preferably a closed chamber which contains fluid, e.g. an aqueous medium at least during the hybridisation step. Thus, the sample and and the fluid (b) may be uniformly heated by microwave energy in order to avoid the occurrence of undesired energy bursts within the sample chamber and to obtain an improved temperature control. The hybridisation chamber is preferably a water-tight sealable plastic container, e.g. a Tupperware® container.

The carrier in the hybridisation chamber preferably contains multiple sample application sites in order to allow testing of a plurality of samples in parallel. Furthermore, the carrier in the hybridisation chamber preferably does not contain a cover slip.

Further, the invention relates to a device for determining a nucleic acid analyte in a sample comprising

(a) a hybridisation chamber comprising a carrier suitable for covalent attachment of components of the sample,

(b) optionally, means, e.g. channels for applying the sample, probes and/or reagents to the carrier, and

(c) optionally, means for determining hybridisation between the nucleic acid analyte and a labelled probe.

It should be noted that the features of both aspects of the invention (microwaves and covalent coupling) can be combined in order to obtain particularly good results.

The invention is explained in more detail by reference to a specifically preferred embodiment as described below, without being limited thereto.

In this embodiment an aliquot of a sample is taken and placed onto an optically inert carrier, preferably a glass slide, and air dried preferably at 46⁰C to remove extracellular water. The preferred embodiment is to take a 10 μl aliquot and place it onto a rectangular field of a multi-field slide. The slide and field dimensions may be constructed to hold multiples of a defined volume, and in particular to prevent carry-over of probe solution or/and samples from one field to the other. The preferred slide does not contain a cover slip and holds a plurality, e.g. eight rectangular fields with 9 x 7 x 0.16 mm, giving a volume of 10 μl each. Slides holding more and smaller fields, with surface areas down to the size of one microscopic field are feasible.

Preferably the fields are separated from one another with a hydrophobic material, e.g. Teflon^®, to avoid carry-over of sample and probes from one field to the other. Due to the hydrophobic character of the separation material, even a two-fold overfill will not cause cross-talk of probes from one field to the other. With two or more differentially labelled DNA-probes or analogues thereof per field it is thus possible to run multiple hybridisations in parallel, without a carry-over from field to field, on the same slide. In addition, several slides may be processed in parallel. The ability to enable multiple and simultaneous processing of DNA-probes or analogues thereof and sample, without carry-over of probe or sample from field to field, is important for the adaptation of ISH technology to routine diagnostic testing.

The slide must remain horizontal until the completion of the step. In contrast to state of the art ISH procedures, the fields need not be covered with a cover-slide. This invention provides guidance as to how the hybridisation with DNA-probes or analogues thereof is achieved while omitting the cover- slip. The invention allows the simultaneous hybridisation of multiple samples with the same probe, or the same sample with multiple probes and permutations thereof. State of the art ISH literature describes hybridisation steps requiring from 60 minutes to over night procedures for individual probe hybridisations(12).

Further a stable liquid/vapour phase transition suitable for stable hybridisation conditions may be generated.

In the context of the present invention, the microwave power or energy refer to commercial standard microwave ovens used e. g. in household.

Preferably, the cells are fixed by energising sample and carrier with microwaves, i.e. electromagnetic waves of 10⁶ to 10¹⁴ Hertz. In a preferable embodiment microwaves with a frequency of 2450 MHz (± 50 Hz) as provided by commercial microwave ovens are utilised (Figure 1 ). Other wavelengths, especially in the infra-red spectrum, may also be applicable to this technology. The amount of energy applied is a function of time and power at levels ranging from above 10 KJ to below 140 KJ according to Table 1. In the preferred embodiment a slide is exposed to 230 Watts for five minutes, equivalent to 69 000 watt seconds or 69 kJ.

To dehydrate, the slides are placed in organic solvent such as ethanol or methanol for 5 to 30 minutes. The preferred embodiment is to place the slides in pure methanol for ten minutes. For the hybridisation of probes towards gram-positive organisms the dehydration of the sample is stopped after seven minutes, dried and digested with a suitable reagent, e.g. Lysozyme/Lysostaphin optionally together with a lantibiotics for 5 to 10 min in a humidified chamber at 46⁰C. Then, the digestion is stopped, e.g. by placing the slide back into methanol for further three minutes.

Subsequently, a hybridisation mix containing at least one hybridisation probe is applied to each respective field. The slides are then placed into a hybridisation chamber containing 2 ml hybridisation buffer at ambient temperature, and the chamber is sealed watertight. The slides remain horizontal and no cover slip is added. The hybridisation chamber in turn is placed in a sealable plastic container containing wash buffer at ambient temperature, and exposed to irradiation in the micro-wave oven (Figure 2), optionally following an irradiation protocol setup in a dedicated procedure of the present invention. In the preferred embodiment a 1000 ml plastic container is filled with 250 ml wash buffer together with a hybridisation chamber a volume of 40 to 50 ml. Depending on the ambient temperature, the design of hybridisation chamber and plastic container the container is exposed to microwaves according to Table 2.

In the preferred embodiment the temperature is then maintained by reducing the energy to a sufficient level, e.g. to about 20-30KJ, preferably to about 27KJ ( "keep warm or warm hold cycle") for a period of about 1 to 10 min, preferably about 5 min. After a further about 1 to 10, preferably about 5 minutes with no energy input, the slide is removed from the hybridisation chamber and completely immersed in the wash buffer. Again, sufficient energy for maintaining the temperature, e.g. about 27KJ is supplied ("keep warm" level for five minutes). After a further about 1 to 10 minutes, preferably about five minutes, the slides are removed, briefly dipped into deionised water and air-dried. Mounting medium is added, and the slide is read out under an epifluorescence microscope.

In a preferred embodiment, a predetermined temperature during sample attachment, hybridisation or/and washing is adjusted by duration or/and power of microwave treatment. Hybridisation may be performed in a chamber placed in a predetermined volume of fluid serving as an "energy buffer" for preventing undesired temperature change, which fluid may for instance be a wash buffer.

The duration or/and the power of microwave treatment can be determined by a calibration protocol which determines the irradiation time that is required at a predetermined level of power (or vice versa) to elevate a defined fluid volume (e.g. an aqueous solution) from ambient temperature to a predetermined temperature, e.g. several degrees above the melting temperature. In a preferred embodiment the temperature is raised to 48⁰C using 240Watts. By taking stepwise measurements the time required to hit the annealing temperature is determined in a second calibration. A third and fourth calibration the time required to elevate the temperature to several degrees above the annealing temperature is determined with and without opening the plastic container.

In a preferred embodiment the following procedure is performed: The aqueous solution is 250ml wash buffer and the hybridisation chamber is floated in the wash buffer within the plastic container for hybridisation. After the hybridisation cycle the slide is removed from the hybridisation chamber and immediately placed into the now preheated wash buffer. The predetermined wash cycle is then started. After the wash period the slide is briefly dipped into deionised water and air dried. Mounting medium is added, and the slide is read under an epifluorescence microscope.

In general, the calibration of the microwave irradiation power or/and duration of the present invention comprises

(a) determining the microwave irradiation time required at a predetermined level of power or determining the microwave irradiation power required at a predetermined irradiation time to elevate a predetermined volume of a fluid from a first predetermined temperature to a second predetermined temperature, or/and

(b) determining the time required without microwave irradiation or at a predetermined microwave power which does not increase or maintain the temperature, or determining the microwave power which does not increase or maintain the temperature required in a predetermined time to decrease the temperature of a predetermined volume of a fluid from a third predetermined temperature to a fourth predetermined temperature.

The fluid may be any fluid which can be heated by microwave irradiation, in particular an aqueous medium.

In steps (a) and (b), calibration curves may be obtained which allow for determination of the irradiation power and duration to reach temperatures in a range from the first predetermined temperature to the second predetermined temperature or/and the third predetermined temperature to the fourth predetermined temperature.

The calibration curves may be almost linear relationships between the temperature depending upon the duration or/and the temperature depending upon the microwave power. The calibration curves may be fitted by a straight line.

In steps (a) and (b) additional heating or/and cooling may be introduced to speed up heating or/and cooling.

In step (a), the first and second temperature may be selected from temperatures comprised by the range of 1O⁰C to 80⁰C, wherein the second temperature is larger than the first temperature. The first temperature may be the ambient temperature. The larger the ambient temperature, the smaller the irradiation time or the irradiation power to reach a particular second temperature (see e. g. Table 2).

In step (b), the third and fourth temperature may be selected from a temperatures comprised by the range of 1O⁰C to 80⁰C, wherein the third temperature is larger than the fourth temperature. The fourth temperature may be the ambient temperature.

The ambient temperature is the most variable parameter which may vary from laboratory to laboratory. This temperature may be kept constant and may be checked before every run of an analysis in order to guarantee reproducible results. Alternatively, several calibration curves with different "ambient temperatures" may be prepared. The succeeding temperatures are determined empirically. The increase of the ambient temperature depends on the microwave power used for irradiation and the irradiation time (and the volume of the wash buffer).

Steps (a) or/and (b) may be controlled by a computer. Therefore, subject of the present invention is a computer program product comprising code suitable for executing steps (a) or/and (b), when loaded into the memory of a suitable computer connected to a microwave oven. The computer program product may produce the calibration curves as described above. Further, the computer program product of the present invention may comprise code for calculating microwave irradiation times or/and powers for a given fixation, hybridisation or/and washing protocol, e. g. a fixation, hybridisation and washing protocol as described in the examples.

In order to allow the automation of the FISH procedure on a robotic pipetting instrument, several micro-fluidic devices may be considered. Thus, in a further embodiment samples are applied to a commercially available plastic chip, available e.g. from (49). As the channels in such a chip cannot be dried, and reagents cannot be exchanged without removing the micro¬ organisms from the visual field, conditions had to be found under which micro-organisms could be attached to the channel walls. In this invention the chip was coated with a polymer carrying a strong positive charge. In the preferred embodiment the chip is coated with poly-D/L-Lysine under conditions that will maintain the positive charge of the Θ-amino group of lysine, while enabling the coupling a heterobifunctional reagent as indicated above to the cationic polymer.

The coating of the carrier and the linker coupling reaction is carried out in a buffer in which the positive charge is maintained. Preferably, the buffer maintains a pH of ≥ 8. The coupling of cells in the sample to the cationic polymer, e.g. poly-D/L-Lysine, requires two effectively antagonistic considerations:

(1) to attach one side of the hetero-bi-functional linker (e.g. a succinimide ester) to a de-protonated amino group, e.g. the Θ- amino group of lysine;

(2) to attract micro-organisms carrying negatively charged cell walls, via the positive charges.

Further, the occurrence of an additional problem would have been expected which arises from the sterical conformation of poly-lysine. Below pH 8 poly- L-lysine shows a random conformation suitable for the attraction of micro¬ organism. An increase in the pH, however, will cause an increased α-helix formation, sterically hindering the coupling of the linker. Furthermore, it would have been expected that the coupling of the linker will remove the positive charge and inhibit the attraction of micro-organisms.

Surprisingly, however, the successful attachment of micro-organisms was achieved by coating the carrier, e.g. a micro-fluidic channel with poly-D/L- lysine in a suitable buffer, e.g. a 5 mM to 150 mM buffer between pH 8 and 9.5 e.g. for 5 min: 0.1 to 10 mg/ml of the heterobifunctional crosslinker was dissolved in an organic solvent such as DMF or DMSO and incubated for 5 min to two hours: the channels were then rinsed with the respective buffer and the chips were stored in the dark.

In the preferred embodiment the following steps were taken for the activation of each channel of a chip:

(1 ) A 0.1% poly-L-lysine ( Sigma #P 8920) solution was diluted 1 :10 in 50 mM NaHCO₃ pH 8.0

(2) 15μl of the poly-L-lysine solution was injected into each channel and incubated at room temperature (RT) for 5 minutes

(3) Unbound poly-L-lysine was washed out with 2x20μl 50 mM NaHCO₃ pH 8.0

(4) 1 mg/ml heterobifunctional linker ATFB-SE was dissolved in DMF

(5) 15μl ATFB-SE solution was injected to each channel and incubated at RT for 1 hour

(6) The chip was washed with 2 x 20μl PBS and stored in the dark at 4-8⁰C.

Sample and reagents were pipetted into each channel using standard 10 μl to 1000 μl pipette tips. 1 to 20μl of sample were then applied to each channel and were then irradiated with UV-light as generated by a standard UV-Lamp (9 Watt at 320nm) for one minute up to one hour. The channels were then flushed with 1 x 15μl to 40 x 15μl alcohol and further dehydrated for one to ten minutes. Channels with gram-positive organisms were flushed with 1 to 10 x 20μl Lysozyme/Lysostaphin and incubated for 5 min at 46⁰C. The enzyme reaction was stopped by flushing with 1 to 10 x 20μl alcohol. To remove the alcohol prior to hybridisation the channel was flushed with 1 to 10 x 20μl hybridisation buffer.

For the hybridisation the probes were mixed with hybridisation buffer and 1 to 20μl were applied to each channel. The chip was sealed with Parafilm and floated on pre-heated wash buffer in a water tight container and are subjected to microwave treatment at 80 to 500 W for 0.1 to 5 min. Then, the container was kept in the microwave with no energy input for further 0.1 to 5 min. Then it was rinsed with 1 x to 20 x 20μl wash buffer and incubated at 80 to 500 W for 0.1 to 5min. Then 20μl mounting medium were added and the fluorescence was read out.

In a preferred embodiment the chip and slide are used for the FISH procedure according to the following procedure:

Summary of step:

Although the amount of sample probe employed both on the chip and on the slide were equivalent, the altered alignment in micro-fluidic chambers surprisingly allowed a significant reduction in incubation time.

The standardisation of the procedure, the reduction of the time required for the hybridisation, and the direct heating of the hybridisation chamber via microwaves represents a significant improvement in the determination of analytes by in-situ hybridisation over prior art methods.

The invention is further illustrated by the following figures, tables and examples. Figure and Table legends

Figure 1 : Carrier component for FISH analysis of eight individual samples and hybridisation chamber.

Figure 2: Binding of biological samples on the carrier using a microwave device.

Figure 3: Equipment for hybridisation.

Table 1 : Microwave energy chart for FISH activation.

Table 2: Microwave exposure needed to increase ambient wash buffer temperature. Microwave irradiation time to reach a temperature of 48 ⁰C depending on the ambient temperature (=buffer temperature) at a microwave power of 160 W or 230 W₁ respectively. The larger the ambient temperature, the smaller the irradiation time

Figures 4 to 7: Calibration curves for a Sharp R-234 microwave oven in the temperature range of 20⁰C to 6O⁰C and a volume of 250 ml water.

Figures 8 to 9: Hybridisation protocol based on the calibration data of Figures 4 to 7.

Example 1

Calibration curves are determined for a Sharp R-234 microwave oven providing a maximal power of 800 W.

a) Calibration of heating step 1 5-10 I water were placed into a holding container and equilibrated to RT (22° C). 250 ml aliquots were drawn off for each test measurements and placed into a sealable plastic container. Measurements of the temperature in the wash buffer were performed after 1 , 2, 3, and 4 min irradiation at 240 W. The container was always cooled to RT with running tap water between measurements. The 1^st reading (RT₁ time = 0 min) was taken immediately before irradiation and the 2^nd after irradiation at the end-point. The calibration curve of Figure 4 is the average of two or three measurements at each irradiation time ("data" in Figure 4). "Target" in Figure 4 refers to the desired melting temperature of 48 ⁰C.

b) Calibration of cooling

Using the time and energy determined in the calibration curve of Figure 4 (2,89 min irradiation at 240 W), 250 ml water of RT (22°C) were heated to 48 ⁰C. Then irradiation was stopped (time = 0 min). Measurement of the temperature is performed at the end of each respective time cycle (1 , 2, 3 or 4 min after stopping irradiation) without opening the container from start at RT to measurement. The calibration curve of Figure 5 is the average of two to five measurements at each time in the wash buffer ("data" in Figure 5). "Target" in Figure 5 refers to the desired annealing temperature of 46 "C.

c) Calibration of heating step 2 (container opened before start)

Using the time and energy determined in the calibration curve of Figure 4 (2,89 min irradiation at 240 W), 250 ml water of RT (22°C) were heated to 48 ⁰C. Then irradiation was stopped, and the water was cooled to 46 ⁰C with open lid (time = 0 min). The container was closed and irradiated at 80 W. Measurements of the temperature in the wash buffer were performed after 1 , 2, 3, and 4 min irradiation (Figure 6, "data"). "Target" in Figure 6 refers to the desired melting temperature of 48 ⁰C.

d) Calibration of heating step 3 (container remained sealed) Using the time and energy determined in the calibration curve of Figure 4 (about 2,89 min irradiation at 240 W)₁ 250 ml water of RT (22⁰C) were heated to 48 ⁰C. Then irradiation was stopped. Then the water was cooled to 46 ⁰C using the calibration curve of Figure 5. This is time = 0 min. Irradiation was performed at 80 W and measurements were taken at the end of the respective cycle time without opening the container from start at RT to measurement. The calibration curve of Figure 7 is the average of two measurements at each time in the wash buffer ("data" in Figure 7). "Target" in Figure 7 refers to the desired melting temperature of 48 ⁰C.

The above calibration curves demonstrate that the temperature of an aqueous medium can be adjusted fast an reproducibly to the exact temperatures required for a hybridisation protocol.

By the strategy described in a) to d), calibration curves may be obtained for any other commercial microwave oven providing a power range of up to about 250 W or even larger, e.g. up to 500 W or 800 W.

Example 2

Based upon the calibration data of Example 1 , the "pulsed" hybridisation protocol as described in Figure 8 was performed in a Sharp R-234 microwave oven. By "pulsed" application of microwave energy according to the calibration data of Example 1 , the temperature can be precisely maintained in a small temperature range of less than 2°C over about 10 to about 20 minutes (hybridisation: total pulse duration about 6 min, total intercept duration about 16 min, washing: total pulse duration about 5 min, total intercept duration about 8 min). Figure 9 describes the corresponding temperature profile of the box containing 250 ml aqueous medium (wash buffer). Prior to hybridisation, fixation of the sample was performed at 240 W for 3:51 , as demonstrated in Figure 2. References

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Claims

Claims

1. A process for determining a nucleic acid analyte in a biological sample by hybridisation with a labelled probe comprising treating the sample with microwaves.

2. The process of claim 1 , wherein the hybridisation is an in situ hybridisation, wherein a nucleic acid analyte is determined in a cell contained in the biological sample.

3. The process of claims 1 or 2, wherein the probe is labelled.

4. The process of any one of claims 1 to 3, wherein the sample is attached to a carrier under microwave treatment.

5. The process of claim 4 wherein microwave treatment is performed for about 2 to 7 minutes at about 130 to 300 Watts.

6. The process of any one of claims 1 to 5, wherein the sample is pretreated in order to remove extracellular liquid before microwave treatment.

7. The process of any one of claims 1 to 6, wherein treatment with microwaves is carried out in that the morphology of cells contained in the sample is maintained.

8. The process of any one of claims 1 to 7 wherein treatment with microwaves is carried out in that intracellular components in the sample are preserved.

9. The process of any one of claims 1 to 8, wherein a nucleic acid analyte is determined in a microbial cell.

10. The process of claim 9, wherein the microbial cell is a yeast cell.

11. The process of claim 9, wherein the microbial cell is a bacterial cell.

12. The process of claim 11 , wherein the microbial cell is a gram negative bacterial cell or/and a bacterial cell not sensitive for Gram staining.

13. The process of claim 11, wherein the microbial cell is a gram positive bacterial cell.

14. The process of claims 1 to 13, wherein the nucleic acid analyte is a ribosomal RNA.

15. The process of claim 13 comprising an enzymatic digestion/treatment of the sample whenever a probe for the detection of a Gram positive bacterial cell is used.

16. The process of claim 15, wherein the enzymatic digestion of the sample is carried out in the presence of a cationic antimicrobial peptide.

17. The process of any one of claims 1 to 16, wherein the hybridisation between nucleic acid analyte and sample is carried out under microwave treatment.

18. The process of claim 17, wherein the hybridisation comprises microwave treatment for about 0.1 to 10 min at about 40 to 800 W.

19. The process of claim 18, wherein the hybridisation comprises microwave treatment in the presence of hybridisation buffer or/and in the presence of wash buffer.

20. The process of claim 18 or 19, wherein during hybridisation, a predetermined temperature is adjusted by duration or/and power of microwave treatment.

21. The method of claim 20, wherein the duration or/and power of microwave treatment is/are determined by a calibration protocol.

22. The method of any of the preceding claims, wherein the hybridisation is executed in a small temperature range with a predetermined starting temperature and a predetermined end temperature.

23. The method according to claim 22, wherein the small temperature range for hybridisation is alternatively provided by a heating/cooling block thermostat.

24. The process of any of claims 17 to 23, wherein the hybridisation is carried out in a device comprising:

(a) a hybridisation chamber comprising a carrier on which at least one sample has been attached, and an aqueous medium,

(b) means for applying a predetermined temperature range to the hybridisation chamber,

(c) optionally, means for applying the sample, probes and/or reagents to the carrier, and

(d) optionally, means for determining hybridisation between the nucleic acid analyte and a labelled probe.

25. The process of claim 24, wherein the sample and the aqueous medium in the hybridisation chamber (a) are uniformly heated.

26. The process of claims 24 or 25, wherein the carrier in the hybridisation chamber does not contain a cover slip.

27. The process of any one of claims 1 to 26, wherein a plurality of samples is tested in parallel.

28. The process of claim 27, wherein the carrier consists of separate sample application areas/fields which prevent carry-over from one area/field to another.

29. The process of any of the preceding claims, wherein a stable liquid/vapour phase transition suitable for stable hybridisation conditions is generated.

30. The process of any one of claims 1 to 29, wherein at least one sample is contacted with a plurality of hybridisation probes.

31. The process of any of the claims 1 to 30, wherein the hybridisation probe has a length of 12 to 30 nucleotides.

32. The method of any of the preceding claims wherein hybridisation between the nucleic acid analyte and a labelled probe comprises decrease of the temperature from a predetermined starting temperature to a predetermined end temperature.

33. The method of claim 32, wherein the starting temperature is a temperature 15 ⁰C at the most above the calculated melting temperature of the probe/analyte hybrid.

34. The method of claim 32 or 33, wherein the end temperature is 5 ⁰C at the most below the calculated melting temperature of the probe/analyte hybrid.

35. The method of any of the claims 32 to 34, wherein the hybridisation is monitored by a means for determining hybridisation between the nucleic acid analyte and a labelled probe.

36. A device for determining a nucleic acid analyte in a sample comprising (a) a hybridisation chamber comprising a carrier on which at least one sample can be attached;

(b) means for applying a predetermined temperature range to the hybridisation chamber,

(c) optionally, means for applying the sample, probes and/or reagents to the carrier, and

(d) means for determining hybridisation between the nucleic acid analyte and the labelled probe.

37. A device for determining a nucleic acid analyte in a sample comprising

(a) a hybridisation chamber comprising a carrier suitable for covalent attachment of components of the sample and

(b) optionally, means for applying the sample, probes and/or reagents to the carrier, and

(c) optionally, means for determining hybridisation between the nucleic acid analyte and a labelled probe.