Improved hybridization assay with stringency buffer dilution

Abstract

The present invention relates to a method for analysis of a sample for the presence of one or more target nucleic acids, the method comprising i) contacting the sample with one or more hybridization agents capable of hybridizing to said one or more target nucleic acids to obtain a mixture of the agent and the sample, ii) diluting the mixture obtained in i with a stringency buffer (such as a high-stringency buffer), in the presence of which specific hybridization between the one or more hybridization agents and their respective target nucleic acids is favored over non-specific hybridization events, iii) detecting, qualitatively or quantitatively, hybridization agent(s) hybridized to target nucleic acids in the mixture, which optionally is further diluted.

Description

[0001] IMPROVED HYBRIDIZATION ASSAY WITH STRINGENCY BUFFER DILUTION

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to the fields of diagnostics and sample testing. More specifically, the invention relates to a method for analysis of a sample for the presence and / or quantity of cellular and / or viral target nucleic acids by means of hybridization between a hybridization agent and the nucleic acid.

[0004] BACKGROUND OF THE INVENTION

[0005] Nucleic acid hybridization is a physiochemical process, aiding the understanding of molecular biology. Probe-based assays use hybridization and hybridization agents for the detection, quantitation and analysis of nucleic acids.

[0006] Nucleic acid-based methods target the RNA or the DNA content of the microorganisms. In case of bacteria these methods could detect the nucleic acids inside the cell or in a free form. Many of the bacteria detection methods target the 16S ribosomal RNA (rRNA) since it is ubiquitously expressed as a structural component of the bacterial ribosome, it has high and constant expression level, it is more stable than messenger RNAs, it has conserved regions which are identical in all bacterial species, but also possesses hypervariable regions which could be used to specifically detect bacteria at the strain level.

[0007] In hybridization techniques the target is detected using a short nucleotide (termed a hybridization agent or probe) which has a nucleotide sequence complementary to the target. Originally the probe was made of RNA or DNA, however it soon turned out that these probes have several limitations (sensitivity to degradation by nucleases, slow hybridization kinetics etc.). These constrains stimulated the development of alternative probes such as locked nucleic acids (LNA) and peptide nucleic acids (PNA).

[0008] Peptide nucleic acid (PNA) is a high affinity artificially synthesized polynucleotide probe which has been used in recent years in molecular biology procedures, diagnostic assays and antisense therapies. Due to the high binding strength of PNA, the oligonucleotide probes typically have a length of down to 10-20 nucleobases, employing common nucleobases (A, C, G, T and U). PNAs can hybridize to complementary nucleic acids with sequence specificity according to the Watson-Crick base pairing rules. PNAs are not easily recognised by nucleases or proteases, making them resistant to degradation by enzymes. PNAs are also stable over a wide pH range. However, in spite of PNA being better suited for penetration into cells that some other oligonucleotides, PNA typically cannot readily cross the cell membrane to enter the cytosol, therefore cell penetrating agents are to be used with PNA to improve cytosolic delivery. PNA's backbone, unlike DNA or RNA is composed of repeating N-(2- aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge (-CH2-) and a carbonyl group (-(C=O)-). PNAs carry a net neutral electrical charge, allowing PNA to form hybrids extremely rapidly and stably with naturally occurring nucleic acids and typically require minimal sample preparation for direct detection of nucleic acids.

[0009] This backbone makes PNA probes superior to DNA probes: a PNA probe is resistant to nucleases and proteases, to a limited extent it is cell-permeable, and most importantly shows fast and salt independent hybridization. PNA probes targeting the hypervariable regions of 16S ribosomal RNA have been successfully applied for the detection of bacteria from blood, environmental samples, and even from milk. Published protocols detect the target inside the cells, work with fixed and permeabilized cells, use long hybridization times, and apply centrifugation-based wash steps. The probes are labelled with fluorophores and detected with fluorescent microscopy or flow cytometry. The usual total assay time without detection is appr. 2.5 hours.

[0010] In the recent years, the use of PNA for the detection of nucleic acids have gained significant interest due to their stability and their ability to rapidly hybridize the naturally occurring nucleic acids.

[0011] Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent labelled probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity.

[0012] Perry-O'Keefe et al. (Journal of Microbiological Methods, 47 (2001) 281-292) reported on methods using FISH and fluorescently labelled PNA probes for identifying microorganisms, such as Escherichia coll, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella as well as probes targeting eubacteria and eucarya. The reported PNA FISH method allowed simultaneous identification of multiple species using the standardized PNA-FISH method.

[0013] Rathnayaka et al. (Int. Journal of Molecular Biology, vol. 3, issue 3 (2018), p. 143-149) reported on the use of FISH in food pathogen detection using PNA or LNA probes. The basic steps of the standardized PNA FISH method are disclosed, i.e. (i) fixation and permeabilization of the specimens, (ii) hybridization of the probe with a target nucleotide, (iii) wash to remove unbound probes and (iv) visualization and enumeration of the result. The authors note that in food microbiology, further steps for the sample preparation, such as homogenization, pre-enrichment procedures or bacterial separation might be necessary.

[0014] Rocha et al. (PLoS ONE 13(5) : e0196522. DOI.org / 10.1371 / journal.pone.0196522) reported on the influence of the fixation / permeabilization step on PNA fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria. The combination of paraformaldehyde and ethanol provided a superior performance for the fixation and permeabilization step of the standardized PNA-FISH method.

[0015] Mach et al (Analyst. 2019 February 25; 144(5) : 1565-1574. DOI: 10.1039 / c8an02194e) reported on the direct detection of Gram-negative bacterial pathogens using bulk fluid assays to provide accurate, timely diagnosis of infectious diseases. For the pathogen detection, the probes were diluted in 2X hybridization buffer to a desired concentration and the probe was mixed with equal volume of media or thawed culture (frozen and stored at -80°C until test) for a final probe concentration. The mixture was heated to 95°C to thermolyze the bacteria followed by incubation at 60°C for 30 mins to allow for probe hybridization using double stranded PNA probes of PNA molecular beacons.

[0016] Young et al. (2020. A technical review and guide to RNA fluorescence in situ hybridization. Peer] 8:e8806 DOI 10.7717 / peerj.8806) reviewed RNA-FISH hybridization techniques and reported on the core processing steps of the FISH methodology: tissue preparation (prehybridization), hybridization and washing (post-hybridization), which the authors reported to be essential to a FISH protocol.

[0017] US 2005 / 0032091 discloses a method for the analysis of a target sequence in a sample, the method comprising contacting the sample with at least one pair of probes (Probe A and B), wherein Probe A and B are PNA probes and wherein Probe A comprises a fluorophore and Probe B comprises a quencher for the detection, identification or quantitation of the hybridization of Probe A to the target sequence. Here, the quenching is performed in an attempt to avoid performing stringent wash. The method for analysis is fluorescence in situ hybridization using the standardised PNA-FISH method.

[0018] Zand et al. (Foods 2021, 10, 3112. D01.org / 10.3390 / foodsl0123112) reported on the flow cytometric approaches and FISH for rapid microbial detection and characterization in the food industry. It is noted by the authors that prior to observation of the hybridized cells, a washing step is necessary to remove unbound and excessive probes to minimise falsepositive detection and background "noise" in natural samples is generally considered a challenge. Nevertheless, Zand et al. shows relevant examples of microbial detection of L. monocytogenes in ground beef, ground pork, milk, lettuce, and cooked shrimp; S. Enteritidis in egg, milk, mayonnaise; Escherichia coli 0157 in ground beef and unpasteurized milk; Salmonella spp. in powdered infant formulae; Lactobacillus spp. in milk; Dekkera bruxellensis in wine; S. enterica / L. monocytogenes / Escherichia coli biofilm on glass, polypropylene polyethylene, polyvinyl chloride, copper, silicone rubber, NS stainless steel; and H. pylori in drinking water at polyvinyl chloride coupons.

[0019] There is however still a need to improve the standardized hybridization in order to simplify and / or improve the detection of nucleic acids.

[0020] It is therefore an object of the invention to provide an improved method for the detection nucleic acids that reduces the complexity, costs and time associated with the detection while retaining high specificity and rapid detection of nucleic acids.

[0021] SUMMARY

[0022] It has been found by the inventors that it possible to successfully perform the stringency stress of a bound hybridization agent in a single simple automation friendly dilution step of the hybridization components, thus avoiding the multiple protocol steps of a traditional stringent wash. This novel stringency dilution step may be performed at the same temperature as the hybridization step without the need for removal of liquid by e.g. centrifugation or filtration. In simple terms, the invention resides in adding a volume (diluting), preferably with a dedicated buffer, directly to the hybridized cells, whereby the read-out clearly separates bound hybridization agent from unbound hybridization agent.

[0023] It has also surprisingly been found by the present inventor(s) that traditional pre-treatment and hybridization steps may be combined into a single step, thereby reducing the need for the removal of the enzyme and / or hybridization agent prior to the analysis of the sample.

[0024] Finally, the inventors have realized that the simplified stringency buffer application of the invention can be combined with this advantageous combination of the pre-treatment and hybridization steps.

[0025] So, in a first aspect the present invention relates to a method for analysis of a sample for the presence of one or more target nucleic acids, the method comprising i) contacting the sample with one or more hybridization agents, such as an agent comprising or consisting of a polynucleotide or analogue thereof, capable of hybridizing to said one or more target nucleic acids to obtain a mixture of the agent and the sample; ii) diluting the mixture obtained in step i) with a diluent, which may be in the form of a stringency buffer, in the presence of which specific hybridization between the one or more hybridization agents and their respective target nucleic acids is favored over non-specific hybridization events, to obtain a diluted mixture; and iii) qualitatively or quantitatively detecting in the diluted mixture, hybridization agents hybridized to target nucleic acids.

[0026] It is noted that when referring to a hybridization agent or a probe in the singular tense herein, this does not exclude the presence of several hybridization agents or probes that have different target sequences. It will be understood that the technology taught herein is applicable for both single-probe / hybridization agent assays as well as assays utilizing multiple probes / hybridization agents.

[0027] LEGEND TO THE FIGURES

[0028] Fig. 1 : Graphs showing positive events in flow cytometric counting of bacteria using specific (left-hand panel) and non-specific (right-hand panel) PNA probes.

[0029] A: Staphylococcus aureus.

[0030] B : Pseudomonas aeruginosa.

[0031] Fig. 2: Graphs showing positive events in flow cytometric counting of bacteria.

[0032] A: Detection of Staphylococcus aureus.

[0033] B: Detection of Pseudomonas aeruginosa with and without enzyme.

[0034] Fig. 3: Dot plots derived from flow-cytometry of PNA-stained bacteria.

[0035] A: Time-chase of hybridization events. Left column shows results for Escherichia coil detection (EC PNA), middle column shows results for Staphylococcus aureus detection (SA2 PNA), and right column shows results for Pseudomonas aeruginosa detection (PA PNA). Rows 1-6: results after 5, 10, 15, 20, 25, and 30 minutes, respectively.

[0036] B: Gating selection.

[0037] Fig. 4: Graphs showing use of different solvents and solvent concentrations for stringency dilution.

[0038] Results in different solvent concentrations are shown in rows 1-5 with 5%, 10%, 20%, 30%, and 40% organic solvent, respectively.

[0039] Fig. 5: Graphs showing positive events in flow cytometric counting of bacteria in different matrices at varying concentrations using probes specific for Escherichia coli, Staphylococcus aureus, and a negative control (ECPNA, SA2PNA, and NOPNA, respectively); in Fig. 5C, "SAPNA" denotes SA2PNA.

[0040] A: Detection of Escherichia coil sample in PBS using lOx stringency dilution.

[0041] B: Detection of Staphylococcus aureus sample in PBS using lOx stringency dilution.

[0042] C: Detection of Staphylococcus aureus sample in raw milk using 40x stringency dilution.

[0043] Fig. 6: Graphs showing family specific detection of Gram-negative Enterobacteriaceae family bacteria.

[0044] Fig. 7: Genus specific detection of Gram-positive Lactic acid bacteria.

[0045] Fig. 8: Graphs showing detection of bacteria using probes specific for Staphylococcus aureus Escherichia coli and a negative control (SA2PNA, ECPNA, and NOPNA, respectively).

[0046] A: Staphylococcus aureus (SA) at about 104bacteria / ml and 20x Stringency dilution.

[0047] B: Escherichia coli (EC) at about 104bacteria / ml and 20x Stringency dilution.

[0048] Fig. 9: Graphs showing gating, detection, and identification of a Gram-negative and Grampositive bacteria in a raw milk sample.

[0049] Fig. 10: Graphs showing detection of natural bacteria in raw milk with ethidium bromide and universal bacteria PNA.

[0050] DETAILED DISCLOSURE OF THE INVENTION

[0051] Definitions

[0052] In the context of the present invention, the term 'nucleic acid' refers to biopolymers and / or macromolecules composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. Nucleic acids may in nature define DNA or RNA. The five complementary base pairs are selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U). The nucleic acids may be cellular or viral nucleic acids.

[0053] The terms 'modified nucleic acid' or 'nucleic acid analogue' or xeno nucleic acids (XNAs) refers to nucleic acid-like molecules, which comprise at least one chemical modification compared to RNA and DNA. Typically, such modified nucleic acids exhibit increased binding affinity and / or specificity towards a given RNA or DNA sequence as compared to unmodified RNA or DNA. In brief, the modified nucleic acids fall into at least the following categories: Sugar modified XNAs (such as 2'F RNA, 2'OMe RNA, LNA, FANA, HNA, and 2'MOE), sugar / backbone modified XNAs (such as mirror DNA, ribuloNA, TNA, tPhoNA, and dXNA), backbone modified XNAs (such as PS, boranophosphate, phNA, and PNA), base modified XNAs (such as those comprising C7-modified deaza-adenine, C7-modified deaza-guanosine, C5-modified cytosine, and C5-modified uridine, and XNAs with unnatural base pairing (such as dz-dP, Ds-Px, 5SICSN-aM, dS-dB, Ds-Pa, and TPT3-NaM base pairs). Also, combinations of one of more of these modifications can be included in an XNA.

[0054] The term 'hybridization' refers to nucleic acid hybridization, / .e., to the phenomenon in which single-stranded DNA or RNA molecules anneal to complementary DNA or RNA. The complementary DNA or RNA may also be complementary to other sequences, such as PNA sequences, meaning that the term 'hybridization' also refers to annealing of XNA to DNA, RNA or another XNA.

[0055] The term 'polynucleotide' refers to a biopolymer composed of a plurality of nucleotide monomers covalently bonded in a chain.

[0056] The term 'PNA' denotes peptide nucleic acid, which is an artificially synthesized polymer similar to DNA or RNA. PNA oligomers show greater specificity in binding to complementary DNA or RNA, with a PNA / DNA or PNA / RNA base mismatch being more destabilizing than a similar mismatch in a DNA / DNA or RNA / RNA duplex. PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge (-CH2-) and a carbonyl group (-(C=O)-). PNAs are typically depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the last (right) position. Typically, the backbone of PNA is electroneutral, which in the context of the present invention is useful in certain embodiments due to an ensuing tolerance to changes in pH in the surrounding solvent.

[0057] For detailed reviews relating to the use of PNA in the biosciences, reference is made to Sharma C. and Awashti S K (2016), Chem Biol Drug Res Des: 1-23 (DOI:

[0058] 10. 1111 / cbdd.12833) and to Suparpprom C. and Vilaivan T. (2022), RSC Chem Biol: 3, 648- 697 (DOI: 10.1039 / d2cb00017b).

[0059] The term "sample" is used for any specimen that can contain nucleic acids that can serve as a target for detection in the methods disclosed herein. Thus, the sample will often be a composition (very often complex) obtained from the environment or e.g. from food, feed, or components thereof. The nucleic acid target material in such a sample can be of bacterial origin (meaning that also spores and endospores are relevant targets), fungal (meaning that also fungal spores are targets), viral, or of any other origin (for instance from protozoans, insects, mites, or higher animals such as rodents or humans). In other cases the sample may be non-complex and contain mainly or only the target material.

[0060] The term 'enzymatically degrade the sample' refers to a process wherein one or more enzymes are added to a sample to remove, degrade or digest components of the sample which have properties which are relevant to the analysis performance, e.g. relevant to signal / noise ratio, background, non-specific binding, etc. Also, the enzymatic degradation may contribute to rendering cells, spores, endospores and virus permeable for the reagents disclosed herein. Furthermore the enzymatic degradation through protein digestion reduces autofluorescence generated from intact proteins.

[0061] The term 'hybridization agent' refers to materials which selectively bind to a target cellular and / or viral nucleic acid (e.g. targets such as DNA and RNA).

[0062] The term "permeabilization" or "permeabilize" as used to refer to the process of rendering the cells (cell membranes, cell walls etc.) of a cellular or viral samples permeable to experimental reagents such as nucleic acid probes, antibodies, chemical substrates, etc. See below for a detailed discussion of various permeabilization methods.

[0063] When referring to concentration of substances using percentages (%), this implies weight per volume (g / ml) when the solute is a solid dissolved in a solvent and otherwise volume per volume (v / v) when a liquid is dissolved or in admixture with the solvent.

[0064] Concentrations identified with a times (" x") indication are relative to the standard buffer concentration (l x) for a given buffer type. For instance, a 4x buffer stock solution (which contains 4 times the concentration of the standard buffer concentration) must be diluted four times to arrive at the standard buffer concentration of l x and 8 times to arrive at a 0.5x solution. For each buffer type the standard buffer concentration has a generally accepted composition defining the l x concentration.

[0065] Specific embodiments of the invention

[0066] As set for the above, the present invention relates to a method for analysis of a sample for the presence of one or more target nucleic acids, the method comprising i) contacting the sample with one or more hybridization agents, such as an agent comprising or consisting of a polynucleotide or analogue thereof, capable of hybridizing to said one or more target nucleic acids to obtain a mixture of the agent and the sample, ii) diluting the mixture obtained in step i with a diluent, which may be in the form of a stringency buffer, in the presence of which specific hybridization between the one or more hybridization agents and their respective target nucleic acids is favoured over non-specific hybridization events, and iii) detecting, qualitatively or quantitatively, hybridization agents hybridized to target nucleic acids in the mixture, which is optionally further diluted.

[0067] It will be understood that this procedure of adding a stringency buffer as a diluent provides for a considerable simplification compared to the traditional approach, where the hybridized sample is first concentrated (e.g. by centrifugation), subsequently supplied with stringency buffer in the form of a washing step, followed by yet an addition of liquid before detection takes place.

[0068] The stringency buffer can for instance be obtained from commercial sources. Traditional stringent wash solutions are known in the art. Such solutions may comprise, for example, buffering agents, accelerating agents, chelating agents, salts, detergents, solvents and blocking agents.

[0069] The buffering agents in stringency buffer or hybridization solutions may include SSC, HEPES, SSPE, MABT, PIPES, CHES, MES, TMAC, TRIS, SET, citric acid-disodium citrate, citrate acid- sodium hydrogen phosphate, phosphate carbonate-bicarbonate, a phosphate buffer, such as, e.g., potassium phosphate or sodium pyrrophosphate, etc. The buffering agents may be present at concentrations from 0.01 x to 50x, such as, for example, O.Olx, O. lx, 0.5x, lx, 2x, 5x, lOx, 15x, 20x, 25x, 30x, 35x, 40x, 45x, or 50x. Typically, the buffering agents are present at concentrations from O.lx to lOx. In other words, the concentrations of these buffering agents may be varied considerably relative to the "standard buffer" concentration of lx for a given buffer system.

[0070] The accelerating agents in stringency buffer or hybridization solutions may include polymers such as FICOLL, PVP, heparin, dextran sulfate, sulfonic acid polymer (cf. US 2020 / 0340047), proteins such as BSA, glycols such as ethylene glycol, glycerol, 1,3 propanediol, propylene glycol, or diethylene glycol, combinations thereof such as Denhardt's solution and BLOTTO, and organic solvents and / or chaotrophic agents such as formamide, dimethylformamide, DMSO, alcohols, urea, dimethyl sulfoxide, guanidinium thiocyanate, alkyl diester such as dimethyl succinate, polar aprotic solvents such as e.g. ethylene carbonate, 2-pyrrolidinone, y-butyrolactone, sulfolane, propylene carbonate, N,N-dimethyl-acetamide and isobutyramide etc. The accelerating agent may be present at concentrations from 1% to 80% or 0.1 x to lOx, such as, for example, 0.1 % (or O. lx), 0.2% (or 0.2x), 0.5% (or 0.5x), 1% (or 1 x), 2% (or 2x), 5% (or 5x), 10% (or lOx), 15% (or 15x), 20% (or 20x), 25% (or 25x), 30% (or 30x), 40% (or 40x), 50% (or 50x), 60% (or 60x), 70% (or 70x), or 80% (or 80x). Typically, formamide is present at concentrations from 10% to 75%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 75%, while DMSO, dextran sulfate, sulfonic acid polymer and glycol are present at concentrations from 1% to 20%, such as 5%, 6%, 7%, 8%, 9%, or 10%.

[0071] The chelating agents in stringency buffer or hybridization solutions may include EDTA, EGTA, etc. The chelating agents may be present at concentrations from 0.1 mM to 10 mM, such as 0.1 mM, 0.2 mM, 0.5 mM, 1 maM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. Typically, the chelating agents are present at concentrations from 0.5 mM to 5 mM, such as 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM.

[0072] The salts in stringency buffer or hybridization solutions may include sodium chloride, sodium phosphate, magnesium phosphate, etc. The salts may be present at concentrations from 1 mM to 750 mM, such as ImM, 5mM, lOmM, 20mM, 30mM, 40mM, 50mM, lOOmM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, or 750mM. Typically, the salts are present at concentrations from 10 mM to 500 mM, such as lOmM, 20mM, 30mM, 40mM, 50mM, lOOmM, 200mM, 300mM, 400mM, 500mM or 600 mM.

[0073] The detergents in stringency buffer or hybridization solutions may include Tween detergents (e.g. Tween 20 or Tween 80), SDS, Triton detergents (for instance Triton X-100, Triton X- 114, Nonidet P-40 (NP-40), and Igepal® CA-630), CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate), deoxycholic acid, etc. The detergent may be present at concentrations from 0.001% to 10% (w / v), such as, for example, 0.0001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Typically, the detergents are present at concentrations from 0.01 % to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

[0074] A great variation exists in the literature regarding traditional hybridization wash solutions. For instance, an example of a typical stringent wash is IX SSC (20X SSC=3 M NaCI, 0.3 M trisodium citrate, pH 7.0) at 65°C for 10 minutes. Another example of a typical stringent wash is: first a high stringent wash with 0.4X (or IX) SSC, 0.3% NP-40, pH 7.0 at 73°C for 2 min., followed by a medium stringent wash with 2X SSC, 0.1% NP-40, pH 7.0 at room temperature for 1-10 min. For example, a typical stringent wash for nucleic acids which have more than 100 complementary residues is a 0.1X to 0.2X SSC at 60 to 65°C for 15 minutes. An example a typical medium stringent wash for nucleic acids which have more than 100 complementary residues is 0.5X to IX SSC at 45 to 55°C for 15 minutes. An example a typical low stringent wash for nucleic acids which have more than 100 complementary residues is 2X to 5X SSC at 40 to 50°C for 15 minutes. For shorter probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more typically about 0.01 to 1.0 M, at pH 7.0 to 8.3, and the temperature is typically at least about 30°C, cf. above concerning temperature choices.

[0075] Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For example, for post hybridization washes of single locus FISH with large DNA probes based on, e.g., BAC and Cosmid clones hybridized at 37° C: stringent washes may use 2xSSC and 50% formamide for 3x 5min from 42 to 45° C. In addition, stringent conditions with formamide may be optimized for different types and lengths of probes (e.g. DNA oligos; LNA; PNA) and different targets (e.g. mRNA, virus, gDNA, rRNA).

[0076] The compositions of the disclosure may comprise a stringency buffer solution comprising any of the components of traditional stringent wash components recited above in combination with at least one polar aprotic solvent (including mixes of solvents). The traditional components may be present at the same concentrations as used in traditional wash solutions, or may be present at higher or lower concentrations, or may be omitted completely.

[0077] The compositions of the disclosure comprise salts such as NaCI and / or phosphate buffer, the salts may be present at concentrations of 0-1200 mM NaCI and / or 0-200 mM phosphate buffer. In some embodiments, the concentrations of salts may be, for example, 0 mM, 15 mM, 30 mM, 45 mM, 60 mM, 75 mM, 90 mM, 105 mM, 120 mM, 135 mM, 150 mM, 165 mM, 180 mM, 195 mM, 210 mM, 225 mM, 240 mM, 255 mM, 270 mM, 285 mM, or 300 mM NaCI and 5 mM phosphate buffer, or 600 mM NaCI and 10 mM phosphate buffer. In other embodiments, the concentrations of salts may be, for example, the concentrations present in 0.1X, 0.2X, 0.3X, 0.4X, 0.5X, 0.6X, 0.7X, 0.8X, 0.9X, IX, 2X, 3X, 4X, 5X, 6X, 7X, or 8X SSC.

[0078] If the compositions of the disclosure comprise accelerating agents such as dextran sulfate, glycol, or DMSO, the dextran sulfate may be present at concentrations of from 5% to 40%, the glycol may be present at concentrations of from 0.1 % to 10%, and the DMSO may be from 0.1% to 10%. In some embodiments, the concentration of dextran sulfate may be 10% or 20% and the concentration of ethylene glycol, 1,3 propanediol, or glycerol may be 1% to 10%. In some embodiments, the concentration of DMSO may be 1%. In some embodiments, the aqueous composition does not comprise DMSO as an accelerating agent. In some embodiments, the aqueous composition does not comprise dextran sulfate as an accelerating agent. In some embodiments, the aqueous composition does not comprise formamide as an accelerating agent, or comprises formamide with the proviso that the composition contains less than 25%, or less than 10%, or less than 5%, or less than 2%, or less than 1%, or less than 0.5%, or as little as or less than 0.1%. If the compositions of the disclosure comprise citric acid, the concentrations may range from 0.2 mM to 200 mM and the pH may range from 3.0 to 9.0. In some embodiments the concentration of citric acid may be 10 mM and the pH may be 6.2.

[0079] If the compositions of the disclosure comprise Tris (tris(hydroxymethyl)aminomethane), the concentrations may range from 0.2 mM to 200 mM and the pH may range from 6.5 to 10.5. In some embodiments the concentration of Tris may be 10 mM and the pH may be 9.

[0080] The compositions of the disclosure may comprise agents that reduce non-specific binding to, for example, the cell membrane, such as salmon sperm or small amounts of total human DNA or, for example, they may comprise blocking agents to block binding of, e.g., repeat sequences to the target such as larger amounts of total human DNA or repeat enriched DNA or specific blocking agents such as PNA or LNA fragments and sequences. These agents may be present at concentrations of from 0.01-100 pg / pL or 0.01-100 pM. For example, in some embodiments, these agents will be 0.1 pg / pL total human DNA, or 0.1 pg / pL non-human DNA, such as herring sperm, salmon sperm, or calf thymus DNA, or 5 pM blocking PNA. The repetitive elements may be deleted from the used probes by silico design, e.g. SureFISH probes, available from Agilent Technologies, and thereby minimizing repetitive sequence binding. Fast (F)ISH hybridization buffers, such as IQFISH, may furthermore be used without the need of blocking repetitive sequences, cf. WO 2009 / 144581.

[0081] One aspect of the disclosure is a composition or solution for use in a stringency dilution step in a hybridization application. Compositions for use in the disclosure include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature non-complementary double-stranded nucleotide sequences. One way to test for whether the amount of polar aprotic solvent is effective to denature non-complementary sequences in a hybridization product is to determine whether the polar aprotic solvent, when used in the methods and compositions described herein, yields a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay.

[0082] Non-limiting examples of effective amounts of polar aprotic solvents include, e.g., about 1% to about 95% (v / v). In some embodiments, the concentration of polar aprotic solvent is 5% to 60% (v / v). In other embodiments, the concentration of polar aprotic solvent is 10% to 60% (v / v). In still other embodiments, the concentration of polar aprotic solvent is 30% to 50% (v / v). Concentrations of 1% to 5%, 5% to 10%, 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% (v / v) are also suitable. In some embodiments, the polar aprotic solvent will be present at a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5% (v / v). In other embodiments, the polar aprotic solvent will be present at a concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v / v).

[0083] In one embodiment, a composition of the disclosure comprises a mixture of 20% polar aprotic solvent (v / v) (e.g., ethylene carbonate, "EC") and 2X SSC at pH 7.0. Another exemplary composition of the present disclosure comprises a mixture of 50% EC and 2X SSC at pH 7.0.

[0084] Different polar aprotic solvents may impart different properties on the compositions of the disclosure. For example, the choice of polar aprotic solvent may contribute to the stability of the composition, since certain polar aprotic solvents may degrade over time. For example, the polar aprotic solvent ethylene carbonate breaks down into ethylene glycol, which is a relatively stable molecule, and carbon dioxide, which can interact with water to form carbonic acid, altering the acidity of the compositions of the disclosure. Without being bound by theory, it is believed that the change in pH upon breakdown of ethylene carbonate and DNA damage from long storage makes the compositions of the disclosure less effective. However, stability can be improved by reducing the pH of the composition, by adding citric acid as a buffer at pH 6.2 instead of the traditional phosphate buffer, which is typically used at about pH 7.4, and / or by adding ethylene glycol at concentrations, e.g., between 0.1% to 10%, or between 0.5% to 5%, such as, for example, 1%, 2%, 3%, etc. For example, with 10 mM citrate buffer, the compositions of the disclosure are stable at 2-8°C for approximately 8 months. Stability can also be improved if the compositions are stored at low temperatures (e.g., -20°C).

[0085] In addition, certain polar aprotic solvents may cause the compositions of the disclosure to separate into multi-phase systems under certain conditions. The conditions under which multi-phase systems are obtained may be different for different polar aprotic solvents. Generally, however, as the concentration of polar aprotic solvent increases, the number of phases increases. For example, compositions comprising low concentrations ethylene carbonate (i.e., less than 20%) may exist as one phase, while compositions comprising higher concentrations of ethylene carbonate may separate into two, or even three phases. For instance, compositions comprising 15% ethylene carbonate exist as a single phase at room temperature, while compositions comprising 40% ethylene carbonate consist of a viscous lower phase (approximately 25% of the total volume) and a less viscous upper phase (approximately 75% of the total volume) at room temperature. On the other hand, compositions comprising greater than 20% EC, for example 40% or 50% EC, and 2X SSC may be present as a single phase at room temperature. The purpose of post-hybridization washes is to separate nonspecific hybrids and remove unbound probe molecules from the sample to minimize background signal. Samples are typically subjected to increasingly stringent washes in SSC buffer containing formamide (or no solvent at higher stringent wash incubation temperatures) and a detergent. Increased stringency can be achieved through sequential washes with incrementally reduced salt concentrations while the wash temperature is matched to the hybridization temperature. At the end of washing, the goal is to allow only the specific and stable hybrids to remain. A wash progression that finishes with a higher concentration of salt (or at a lower temperature, that is, lower stringency) will be less likely to denature and remove nonspecific hybrids, but also may preserve greater intensity of specific labelling.

[0086] Generally, the stringency buffer is applied to dilute by a factor of 2-100 times. Hence, the factor can be about or exactly 2, about or exactly 3, about or exactly 4, about or exactly 5, about or exactly 6, about or exactly 7, about or exactly 8, about or exactly 9, about or exactly 10, about or exactly 11 , about or exactly 12 :, about or exactly 13, about or exactly 14, about or exactly 15, about or exactly 16, about or exactly 17, about or exactly 18, about or exactly 19, about or exactly 20, about or exactly 21, about or exactly 22, about or exactly 23, about or exactly 24, about or exactly 25, about or exactly 26, about or exactly 27, about or exactly 28, about or exactly 29, about or exactly 30, about or exactly 31, about or exactly 32, about or exactly 33, about or exactly 34, about or exactly 35, about or exactly 36, about or exactly 37, about or exactly 38, about or exactly 39, about or exactly 40, about or exactly 41, about or exactly 42, about or exactly 43, about or exactly 44, about or exactly 45, about or exactly 46, about or exactly 47, about or exactly 48, about or exactly 49, about or exactly 50, about or exactly 51, about or exactly 52, about or exactly 53, about or exactly 54, about or exactly 55, about or exactly 56, about or exactly 57, about or exactly 58, about or exactly 59, about or exactly 60, about or exactly 61, about or exactly 62, about or exactly 63, about or exactly 64, about or exactly 65, about or exactly 66, about or exactly 67, about or exactly 68, about or exactly 69, about or exactly 70, about or exactly 71, about or exactly 72, about or exactly 73, about or exactly 74, about or exactly 75, about or exactly 76, about or exactly 77, about or exactly 78, about or exactly 79, about or exactly 80, about or exactly 81, about or exactly 82, about or exactly 83, about or exactly 84, about or exactly 85, about or exactly 86, about or exactly 87, about or exactly 88, about or exactly 89, about or exactly 90, about or exactly 91, about or exactly 92, about or exactly 93, about or exactly 94, about or exactly 95, about or exactly 96, about or exactly 97, about or exactly 98, about or exactly 99, or about or exactly 100 times.

[0087] The one or more hybridization agents are typically nucleic acids or nucleic acid analogues; i.e. often, the one or more hybridization agents is or comprises a polynucleotide being modified to exhibit higher binding affinity and binding specificity for natural nucleic acids than does both (unmodified) DNA and RNA. It is to be understood though that DNA and RNA are not excluded from being the hybridization agent used in the invention, even though modified nucleic acids are preferred, cf. below.

[0088] In many embodiments, the one or more hybridization agents hybridize(s) specifically to a nucleic acid not naturally associated with cells present in the sample, meaning that they are designed to target material that is the result of contamination or infestation or infection in the biologic source from where the sample is derived. Hence, in some embodiments, when the sample is from a multicellular organism, the hybridization agent does not hybridize to normal cells of said multicellular organism, meaning that the detection using the hybridization agent targets the invading organism such as a pathogen or from mutations in cells related to a pathology, such as mutations in cancer cells.

[0089] One example of hybridization agents that are useful in the practice of the present invention are hybridization agents that are specific to rRNA, such as bacterial rRNA or fungal rRNA. Such probes will hence be useful when assaying biological samples for the presence of bacterial or fungal material, typically in cases where such presence is undesired.

[0090] In important embodiments, the one or more hybridization agents has / have an uncharged backbone. This entails the advantage that the hybridization agent can be applied in settings of salt concentrations. In this connection, a preferred type of hybridization agent is or comprises peptide nucleic acid(s) (PNA).

[0091] The one or more hybridization agents further typically comprise(s) a detectable moiety. Such a detectable moiety is typically selected from the group consisting of luminescent, fluorescent, chromogenic, radioactive, and ligand or receptor moieties.

[0092] In an embodiment according to the invention, the hybridization agent comprises a selfquenching portion, which provides an unquenched signal if attached to the target nucleic acid while being quenched while not bound, or vice versa. Thus, this approach covers the use of 'beacons' (stem-loop-forming probes containing a fluorescent moiety and a quencher) or double complimentary probes, where one probe contains the fluorescent moiety and the other contains the quencher.

[0093] In some embodiments, two or more hybridization agents are present in the agent, which differ from each other to simultaneously detect two or more different nucleic acids, and optionally two or more different cells, spores, endospores, or virus, present in the sample. In an embodiment according to the invention, the sample is selected from the group consisting of mammalian body fluids, such as blood, saliva, semen, vaginal fluids, mucus, urine, raw milk or other types of milk, such as milk for human consumption, condensed milk, powdered milk, soya milk, almond milk, other plant-based milk products, and dairy products (e.g. cream, butter, yoghurt, kefir, ice-cream, cheese); an animal tissue sample; and a plant-derived sample. Alternatively, the sample can be from a consumer product or other product, where it is of relevance to detect cells (which can be both undesired and desired as constituents in the product). Examples of such sources for samples can e.g. be beverages, alcoholic or non-alcoholic.

[0094] In an embodiment according to the invention, the sample is milk, preferably cow's milk or other milk for human consumption (e.g., buffalo milk, goat, sheep, camel, llamas, alpacas, yaks, water buffalo, horses, reindeer).

[0095] In the case of milk and milk-derived samples, the degradation by enzymes of the milk sample matrix is particularly useful, since it markedly improves the signal to noise ratio by removing, degrading, and / or digesting the particles that otherwise have properties that resemble the properties of the microorganisms relevant for the analysis.

[0096] In an embodiment according to the invention, the milk is raw milk. Further, in an embodiment of the invention, the milk is heat treated (such as by pasteurization, UHT, ESL, or treated by other means of thermal processing).

[0097] In an embodiment of the invention, the liquid processing involves non-thermal processing treatment such as UV light treated milk by e.g. raslysation™.

[0098] In important embodiment according to the invention, the cells to be detected are prokaryotic cells, such as bacteria and archaea.

[0099] In other embodiments of the invention, the cells are eukaryotic cells - these may be nonmammalian (e.g. protozoan, fungal), or mammalian, such as those somatic cells found in milk samples or other samples from a mammal. As detailed herein, such samples include tissue samples.

[0100] In an embodiment according to the invention, the nucleic acids are present in eukaryotes (including vertebrates and mammals), microorganisms, spores, endospores or virus.

[0101] In an embodiment according to the invention, the microorganisms are selected from pathogenic bacteria; pathogenic fungi; and bacteria or fungi, which cause products to attain undesirable characteristics, such as unpleasant odor, taste, texture, color, or feel, or reduced commercial life-span. When referring to pathogenic bacteria or fungi, this entails human pathogens but also pathogens in animals.

[0102] Pathogenic bacteria may be present in humans and animals (including, wild and domesticated animals) or in both humans and animals.

[0103] In an embodiment according to the invention, the bacterium is selected from the group comprising Staphylococcus aureus (SA), Escherichia coli (EC) and Pseudomonas aeruginosa (PA). Other bacteria may be detected by the method of the invention, such as non-limiting examples of bacteria belonging to any of the following genus and species, which are of particular interest due to their relevance as pathogens or due to their relevance in food spoilage:

[0104] Acetobacter aurantius, Acinetobacter baumannii, Actinobacillus actinomycetemcomitans, Actinomyces Israelii, Aeromonas spp, Agrobacterium radiobacter, Agrobacterium tumefaciens, Azorhizobium caulinodans, Azotobacter vinelandii, Anaplasma, Anaplasma phagocytophilum, Anaplasma marginale, Bacillus spp, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, , Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Bacillus wiedmannii, Bacteroides, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus (now known as Prevotella melaninogenica), Bartonella, Bartonella henselae, Bartonella quintana, Bordetella, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brevibacillus agri, Brochothrix spp, Brucella spp, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia, Chlamydia trachomatis, Chlamydophila, Chlamydophila pneumoniae (previously called Chlamydia pneumoniae), Chlamydophila psittaci (previously called Bacillus pumilus, Bacillus sonorensis, Bacillus sporothermodurans, Chlamydia psittaci), Clostridium spp, Clostridium botulinum, Clostridium butyricum, Clostridium difficile, Clostridium perfringens (previously called Clostridium welchii), Clostridium sporogenes, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus, Enterococcus avium, Enterococcus dura ns, Enterococcus faecal is, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus, Lactobacillus acidophilus, Lactobacillus easel, Lactococcus lactis, Lederbergia galactosidilytica, Legionella pneumophila, Leuconostoc spp, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium spp, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracell ulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Paenibacillus lactis, Pasteurella, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Plesiomonas shigelloides, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella spp., Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella spp, Shigella dysenteriae, Staphylococcus, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus spp., Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema, Treponema pallidum, Treponema denticola, Vibrio spp, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Weissella spp, Wolbachia, Yersinia, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis, and various lactic acid bacteria.

[0105] A number of the bacteria above are, apart from being potential pathogens, responsible for off-odors and off-flavors, discolorations, gas production, slime production, and decreases in pH in food and feed.

[0106] Likewise, a number of fungi are in the same manner known as food spoilants: The most common food spoilage causing molds are Mucor, Aspergillus spp, Rhizopus spp, Penicillium spp, Alternaria spp, Botrytis, Byssochlamys, and Fusarium spp. These molds cause off- flavors, mycotoxin contamination, discoloration, rotting, and are externally visible in the food.

[0107] Most commonly food spoilage causing yeasts are Zygosaccharomyces spp, Saccharomyces spp., Candida spp, Dekkera spp. These yeasts cause a change in color, a change in texture, an unpleasant odor, or an undesirable taste in food. Yeasts and molds can enter e.g. a dairy plant from the air, water, packaging and personnel and are often difficult to remove. They thrive in the moist environments and living on improperly cleaned and sanitized surfaces. The main target of molds is cheese, especially pre-prepared grated cheese, and fermented dairy products. The chief genera involved in cheese spoilage are Penicillium, Cladosporium, and Phoma, which mainly attack the product during ripening. Other genera involved are Aspergillus, Cephalosporium, Cladosporium, Geotrichum, Mucor, Scopulariopsis, and Syncephalastrum. Yeast can spoil and grow undetected in e.g. yoghurt. Among the genera growing in yoghurt are Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces , and Sporobolomyces. There is a need to be able to rapidly detect the wide variety of yeasts and molds that trouble the dairy and in other food industries, both for plant hygiene and product analysis.

[0108] The most common foodborne parasites (all pathogenic) are Giardia lamblia, Entamoeba histolytica, Cyclospora cayetanensis, Toxoplasma gondii, and Trichinella spiralis.

[0109] The algae that cause poisoning are e.g. Gonyaulax catenella, Gonyaulax tamarensis, Gambierdiscus toxicus, Ptychodiscus brevis, Microcystis aeruginosa, and Blue-green Algae.

[0110] Some of the foodborne viruses are Norovirus, Hepatitis A virus (HAV), Hepatitis E virus (HEV), Astrovirus (AstV), Rotavirus (RV), Coronavirus, Sapovirus (SaV).

[0111] Hence, detection of any of the above microorganisms or virus is desirable for one or more reasons in medicine, in the food industry and elsewhere. Generally, microbial food spoilage can be determined physically by the following changes: Change in appearance: It is characterized by the cloudiness and liquid formation in the food; Change in texture occurs due to an accumulation of microbial cells and tissue degradation resulting in slime formation and rotting; Color change: It mainly occurs due to the chlorophyll breakdown and mycelial growth; Change in taste and odor: The taste and odor of the food changes due to the oxidation of nitrogenous compounds, sulfides, organic acids etc. by the microbial enzymes.

[0112] Additionally, there is a need for fast detection of needed or desirable microorganism such as e.g. in raw milk for raw milk natural produced cheese; in yoghurt, cheese, ham, pickles, sauerkraut, Kambucha, Kimchi, Miso or in wine, juice and beer and in other food and beverage production. There is a broad need for microorganism detection in e.g. food, drug or biofuel fermentation processes. Furthermore, there is a need for determining numbers of probiotic microorganism such as of e.g. Lactobacilli, Bifidobacterium, Bacillus species and Saccharomyces cerevisiae in food, drugs and dietary supplements production. In the practice of the invention, steps i and ii in combination typically takes less than 360 minutes, such as less than 150 mins, such as less than 140 mins, such as 130 mins, such as 120 mins, such as 110 mins, such as 100 mins, such as 90 mins, such as 80 mins, such as 70 mins, such as 60 mins, such as 50 mins, such as 40 mins, such as less than 20 mins, such as less than 10 mins. Also, steps i and ii in combination typically takes at least 10 seconds, such as at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 60 seconds, such as at least 1.5 minutes, such as 2 minutes, such as 2.5 minutes, such as 3 minutes, such as 3.5 minutes, such as 4 minutes, such as 4.5 minutes, such as 5 minutes, such as at least 10 minutes, such as 20 minutes, such as 30 minutes, such as 40 minutes, such as 50 minutes, such as 60 minutes, such as 70 minutes, such as up to 80 minutes.

[0113] In some embodiments, the stringency buffer comprises one or more detergent(s) and / or step ii entails addition of one or more detergent(s). The detergents can for instance be ionic, nonionic or zwitterionic detergents, cf. details below.

[0114] In important embodiments, the temperature in at least step ii is controlled to optimize that specific hybridization by the one or more hybridization agents is favoured over non-specific hybridization.

[0115] In an embodiment according to the invention, the detection is a multiplex flow cytometrybased detection, but any other useful method may be used, such as manual or automated counting using microscopy and potentially using filters or spectral analysis.

[0116] The stringency buffer can comprise an agent selected from one of more of a pH buffer; a chelating agent; a detergent or a mixture of detergents; a salt or salts; a solvent or solvents; and a DNA staining component.

[0117] In addition to the inventive dilution approach, the detection method can be further improved if it - prior to step i) - comprises providing the sample by obtaining an original sample suspected of containing cells or virus and subsequently a) rendering cells or virus in the original sample permeable to the one or more hybridization agents and 1) adding one or more enzymes to enzymatically degrade the sample and / or to contribute to rendering the cells or virus permeable to the one or more hybridization agents, b)carrying out step i-ii, whereby the mixture in step i comprises the sample, the added one or more enzymes and the added one or more hybridization agents, and c) carrying out step ill.

[0118] By providing an enzyme that can enzymatically degrade the sample, the signal-to-noise ratio of the detection method can be significantly improved, thereby reducing the possibility for false-positive detection of cellular and / or viral nucleic acids. Such components to be degraded may be e.g. relevant biomolecules.

[0119] In an important embodiment according to the invention at least one of the one or more enzymes is one or more proteases, such as one or more proteases has / have broad substrate specificity. This latter feature reflects that the intention behind the use of the enzymes is to effect a general degradation of material, meaning that enzymes having a very narrow substrate specificity (such as some of the proteases being part of the coagulation cascade) would be of very limited usefulness, if any at all.

[0120] In an embodiment according to the invention, the one or more proteases is selected from the group consisting of serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases.

[0121] In an embodiment according to the invention, the one or more proteases is / are selected from the group consisting of Trypsin (3.4.21.4), Chymotrypsin (3.4.21.1), Endoproteinase Asp-N (3.4.24.33), Endoproteinase Arg-C (cp), Endoproteinase Glu-C (V8 protease) (3.4.21.19), Endoproteinase Lys-C (3.4.21.50), Pepsin (3.4.23.1), Thermolysin (3.4.24.27), Elastase (3.4.21.36), Papain (3.4.22.2), Proteinase K (3.4.21.64), Subtilisin (3.4.21.62), Clostripain (endoproteinase-Arg-C) (3.4.22.8), Carboxypeptidase A (3.4.17.1), Carboxypeptidase B (3.4.17.2), Carboxypeptidase P (cp), Carboxypeptidase Y (3.4.16.5), Cathepsin C (3.4.14.1), Acylamino-acid-releasing enzyme (3.4.19.1), and Pyroglutamate aminopeptidase (3.4.19.3), as well any proteolytically active fragments or mutants thereof.

[0122] The one or more enzymes, such as a protease, may be a mix of different enzymes specifically chosen or as commercially provided (e.g., Pronase, cat. No. 10 165 921 001 - a non-specific protease by Roche, distributed by Sigma-Aldrich).

[0123] In an embodiment according to the invention, the at least one of the one or more enzymes is a lipase or an esterase. Esterases catalyze for instance the hydrolysis of short-chain fatty acids while the lipases catalyze the hydrolysis of long chain fatty acids and lipids. Esterases mainly acts on water-soluble substrates while the lipases mainly act on water-insoluble substrates. Esterases are typically more water soluble than lipases. Suitable examples are e.g., Esterase (EC 3. 1.1.1) and Lipase (EC 3.1.1.3).

[0124] Esterases, hence typically function in water, while lipases require to act in a hydrophobic- hydrophilic interphase such as oil-water. In an embodiment according to the invention, the at least one of the one of more enzymes is a carbohydrate degrading enzyme.

[0125] In an embodiment according to the invention, the carbohydrate degrading enzyme is selected from one or more of a cellulase, hemicellulase, a pectinase, and an amylase.

[0126] The enzymatic techniques on plant material in a sample depend primarily on the ability of enzymes to hydrolyze components of the cell wall like cellulose, pectins, and hemicellulose. Degradation of these polymers disrupts the structural complexity of the cell wall and improves the mass transfer, particle size reduction, increased contact surface and enhanced solvent / reagent distribution. Pectinases break down pectin, a polysaccharide in the plant cell wall and is routinely used for clarification in e.g., fruit juices and the wine industry.

[0127] Other suitable enzymes of may also be used, e.g. glycoside hydrolases (glycosidases), e.g., Lysozyme (N-acetylmuramide glycanhydrolase), amylases, cellulases, Chitinase. Lysozyme shows good efficiency towards Gram-positive bacterial cell wall by hydrolyzing the glycosidic bonds of the peptidoglycan residues and thereby making the cell wall permeable.

[0128] Enzymes and hybridization agent are typically added simultaneously, in a single step process or the enzymes and hybridization agent are added in sufficiently rapid succession to each other to operate simultaneously.

[0129] The removing / destroying / degrading / changing / permeabilizing components of the sample matrix with enzymes, that otherwise may be disturbing or inhibiting the assay performance, while simultaneously hybridizing with specific markers to their specific, the specificity and / or sensitivity of the assay may be increased while decreasing the detection time.

[0130] In an embodiment according to the invention, no substantial removal of the one or more enzyme and / or the hybridization agent occurs prior to detecting the hybridized nucleic acids.

[0131] An advantage of not removing the one or more enzyme and / or the hybridization agent prior to detecting the hybridized nucleic acids is that the detection (and if needed in advance, staining) can be performed directly with the sample, without any further enrichment of the targeted cells, spores and / or viruses. The targeted cells, spores and / or viruses however may of course be enriched from the sample by conventional methods known in the art, such as by filtration, centrifugation, flow sorting, dielectrophoresis, metal hydroxides, magnetic beads or particles, by utilizing different forms and ways of affinity catching etc. Enrichment may also be performed additionally or in combination with growth enrichment of the sample e.g., by using enrichment culturing. Enrichment may in embodiments provide a higher sensitivity of an assay by increasing the analyte or microorganism or virus concentration and by removing components that may give rise to noise.

[0132] In important embodiments of embodiments where enzymes are added in step a discussed above, the hybridization agent is capable of specific hybridization at salt concentration(s) where the at least one enzyme is enzymatically active. In these embodiments, it becomes of high relevance to utilise hybridization agents having a non-charged backbone as is the case for PNA-based hybridization probes.

[0133] In an embodiment according to the invention, a)-b) are carried out at the essentially same temperature, or wherein one or more temperature changes are included. It may e.g. be a two-step or multi-step temperature process, if e.g. using a sample matrix, where the enzyme employed exhibits an optimum activity at temperature lower than the hybridization temperature. The enzyme treatment may then start at a lower temperature (such as 40°C), whereafter the temperature is increased to the optimum for hybridization still without changing liquid or perform washes. An increase in temperature may as well be used for stopping the enzyme activity, potentially, if required, followed by temperature decrease for obtaining an optimum hybridization temperature. Changes in temperature may be employed to facilitate the action of different enzymes e.g. the activity of a first enzyme may be stopped by increasing the temperature while digestion is continued by a second enzyme not sensitive to the temperature increase. Lower and / or higher initial temperatures than the hybridization temperature employed generally allow for different variations of exposure of enzymatic digestion of the sample while e.g. keeping the hybridization temperature and incubation time constant. Hence, different variations of temperature, e.g. without removing components are possible, providing advantages that can be utilized by the skilled person; a possibility is inclusion / addition of new or further components after e.g. a first temperature incubation step.

[0134] In an embodiment according to the invention, the method further comprising incubating the sample after b).

[0135] In an embodiment according, between about 10° and about 109microorganisms / ml are detectable.

[0136] In important embodiments of the present invention, b) is carried out on (the optionally diluted) mixture from a), which comprises the sample, the added one or more enzymes, and the hybridization agent. This is to say, the facilitation of the hybridization reaction is achieved on the material obtained from a), without substantial removal of any components of the mixture. In this case, at the end of b), the sample is in preferred embodiments diluted with a stringency buffer, preferably a high-stringency buffer, the effect of which may be compensated or enhanced by modulating temperature. Thereafter, the detection is performed on the diluted mixture thus obtained. That is, it is preferred that prior to c) no removal takes place of components of the mixture comprising the sample, the added one or more enzymes, and the hybridization agent.

[0137] In an embodiment according to the invention, the sample is selected from tissue from higher animals. This could be tissue from birds (chickens, geese, ducks etc.), fish and other seafood animals, domestic animals, including pets (dogs, cats, ferrets, hamsters, etc.), livestock (cattle, sheep, pigs, goats, etc.), beasts of burden (horses, camels, donkeys, etc.), wildlife creatrues, and humans.

[0138] In an embodiment according to the invention the sample processing and staining includes the use of classical cytogenetic and histology methods. This includes the use of e.g. cryopreservation preparations, cytological preparations, formalin-fixed and paraffin-embedded (FFPE) tissue sections preparations and other similar preparations techniques. See for instance Muller et al. 2009. Pathology. Education Guide. Immunohistochemical (IHC) Staining Methods, Fifth Edition, Chapter: 11. Preparation of FFPE Tissue Slides for Solid Tumor FISH Analysis. Publisher: Dako North America, Carpinteria, California.

[0139] In the case of e.g. pathology FFPE tissue section samples on slides, a pre-treatment preparation is required for performing ISH (in situ hybridization) and FISH staining. The tissue staining benefits from a digestion step, with for instance proteases such as pepsin or Proteinase K to facilitate the (F)ISH probes' access to their target, for permeabilization, and for reducing autofluorescence background from intact tissue proteins. This step is typically followed by a wash step and often a dehydration step prior to initiating the hybridization step. Such separate pre-treatment steps may according to the present invention be combined partly or fully into a single step together with the hybridization step, thereby simplifying the protocol and thus potentially simplifying an automated solution and / or simplify a protocol employed on an instrumental implementation, such as described in WO 2021 / 239761, WO 2013 / 071358, WO 2009 / 009419 and WO 2004 / 001390. The beneficial effects may appear as a simpler staining method, a simpler apparatus scheduling, a higher sample throughput, reduced fluidic handling, use of less reagent bottles / containers, a reduction in wash buffer needed, a reduction in fluid waste, a lower instrument down time etc. Hence, a large variation of potential advantages provided by the invention will become apparent to persons skilled in the relevant technical fields, such as in the field of diagnostic solutions, from a consideration of the present description. The methods and compositions of the invention may be used fully or partly in all types of hybridization applications in the fields of cytology, histology, or molecular biology. According to one embodiment, the target nucleic acid sequence in the methods of the invention is present in a biological sample. Examples of such samples include, e.g., tissue samples, cell preparations, cell fragment preparations, and isolated or enriched cell component preparations. The sample may originate from various tissues such as, e.g., breast, lung, colorectal, prostate, lung, head & neck, stomach, pancreas, esophagus, liver, and bladder, or other relevant tissues and neoplasia thereof, any cell suspension, blood sample, fine needle aspiration, ascites fluid, sputum, peritoneum wash, lung wash, urine, feces, cell scrape, cell smear, cytospin or cytoprep cells. The methods and compositions of the invention may be used fully or partly for diagnostic purposes that includes virus, human cells, microorganisms such as Human Papillomavirus (HPV) mRNA, Epstein-Barr Virus (EBV) mRNA, and Kappa / Lambda mRNA detection with specific probes in cancer diagnostic on e.g. FFPE tissue sections or by the use of flow cytometry. The methods and compositions may be employed in in situ hybridizations probes that detect gene or chromosome aberrations, and mutations such as deletions, amplifications, translocations, substitutions, and single point mutations. Other examples of useful diagnostic applications of the invention are as a fast and simple complementary microorganisms' detection in e.g., urinary tract infections or sepsis for fast selection of the right use of antibiotics for treatment of infection.

[0140] In an embodiment the enzyme or enzymes used are modified and / or redesigned enzymes, e.g. altered to function under different / harsher conditions than non-modified natural enzymes, and / or enzymes isolated from organisms living under extreme conditions such as e.g. lower / higher temperatures, lower / higher salt concentrations, unusual solvents etc. than what most organisms normally are exposed too. It is hence envisaged to employ enzymes that are more tolerant to environmental factors such as the type of salts present, salt concentrations, pH, temperatures, types of solvent, substrate concentrations, inhibitors of enzyme activity etc.

[0141] PREAMBLE TO THE EXAMPLES

[0142] The Examples below describe in detail specific embodiments of the invention. While the invention will be described in conjunction with these embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims. Materials and methods

[0143] BactoScan™ FC Buffer Powder, BactoScan™ FC Enzyme 150, BactoScan™ FC Staining Medium, BactoScan™ FC Detergent were acquired from FOSS Analytical A / S, Denmark.

[0144] Custom Peptide Nucleic Acid with fluorophores was procured from PANAGENE, South Korea.

[0145] Dimethyl Sulfoxide (DMSO) D8418, Formamide 47671, 2-Pyrrolidinone P74370, Sulfolane T22209, 1- Methyl-2- Pyrrolidinone 443778, Ethylenediaminetetraacetic acid (EDTA) E9884, Ethidium bromide E7637 and Sodium dodecyl sulfate (SDS) L3771 were acquired from Sigma-Aldrich.

[0146] Formaldehyde F1635 was obtained from Merck. Tris-HCI 09360-203 and Tris-Base 09350-101 were obtained fom Molar Chemicals, Hungary.

[0147] NaCI 27810 was obtained from VWR International Ltd.

[0148] PNA hybridization test relied generally on the disclosure in Perry-O'Keefe H, Rigby S, Oliveira K, Sorensen D, Stender H, Coull J, Hyldig-Nielsen JJ. Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Methods. 2001 Dec;47(3): 281-92. DOI: 10.1016 / s0167-7012(01)00303-7.

[0149] PNA was obtained in a lyophilized state from PANAGENE (www.panagene.com / _ENG / html / ) and was dissolved in 20 pl DMSO and Ultrapure Nuclease Free water to get a final concentration of 30 pM. The PNA species employed are set forth in the following table:

[0150] Table 1 - PNA probes utilised UPNA labels Eubacteria universally

[0151] SA1PNA labels Staphylococcus aureus specifically

[0152] SA2PNA labels Staphylococcus aureus specifically

[0153] ECPNA labels Escherichia coll specifically

[0154] PAPNA labelling specifically Pseudomonas aeruginosa

[0155] EB1PNA labels the Enterobacteriaceae family members specifically

[0156] EB2PNA labels the Enterobacteriaceae Family members specifically

[0157] LABPNA labels the Lactobacillus genus members specifically

[0158] Bacterial cultures if not present internally, were procured either from www.atcc.org / or www.dsmz.de / and further maintained in house as per the propagation protocol provided from the source.

[0159] Bacterial growth was in the experiments induced in the respective liquid medium and was then spiked in raw milk at a known concentration (CFU / ml) to test the PNA probes.

[0160] The three most common organisms used in the examples were:

[0161] Staphylococcus aureus (SA): Gram positive, obtained from ATCC cell bank (ATCC 27660); cultured in Brain-Heart Infusion media.

[0162] Escherichia coil or Escherichia coil (EC) : Gram negative, obtained from ATCC cell bank (ATCC 25922); cultured in Tryptic Soybean media.

[0163] Pseudomonas aeruginosa (PA): Gram negative, obtained from ATCC cell bank (ATCC 27853); cultured in Tryptic Soybean media.

[0164] Flow cytometers used were CytoFLEX S V2-B2-Y3-R2 Flow Cytometer (9 Detectors, 4 Lasers) with autosampler (Beckman Coulter) and Attune NxT with 3 lasers: blue-green-red, with autosampler (Invitrogen / Thermo Fisher Scientific). On CytoFLEX, typically the following settings was in general used: ethidium bromide yellow / green laser PE DsRed 585 / 42 BP;

[0165] Alexa 405 violet laser PB450 450 / 45 BP; Alexa 488 blue laser FITC GFP 525 / 40 BP, Alexa 647 red laser APC 660 / 10 BP. For Attune NxT the blue laser was used for excitation of the Alexa 488 and the ethidium bromide and for the emission of Alexa 488 with BL1 525 / 50 nm and ethidium bromide with BL2 574 / 26 nm filters. For Alexa 647 the red laser was used and the RL1 670 / 14 filter.

[0166] Raw milk was tested with and without the preservatives Azidiol and Bronopol, frozen at -18°C and non-frozen (kept refrigerated at 4°C) and the treatment of the milk for obtaining improved storage stability did not show significant impact on performance. In general, raw milk with a low natural bacteria count was used, e.g. 5,000-30,000 IBC / ml. However, raw milk with counts above 106IBC / ml have also been tested and it has been shown that the invention works with high cell counts as well.

[0167] The used raw milk was tested negative, unless mentioned otherwise, by CFU for SA, PA and EC with MSA (Mannitol salt agar), PIA (Pseudomonas isolation agar) and CCA (coliform Chromo Select agar) media from Sigma-Aldrich (sigmaaldrich.com / deepweb / assets / sigmaaldrich / product / documents / 995 / 504 / 63567dat. pdf, sigmaaldrich.com / deepweb / assets / sigmaaldrich / product / documents / 295 / 152 / 17208dat. pdf, and sigmaaldrich.com / deepweb / assets / sigmaaldrich / product / documents / 392 / 035 / 81938dat. pdf) respectively. For total bacteria count CSMA (Plate count Skimmed milk agar) was used from Sigma-Aldrich (sigmaaldrich.com / deepweb / assets / sigmaaldrich / product / documents / 271 / 815 / tnl321en- mk.pdf).

[0168] COMPARATIVE EXAMPLE

[0169] Pre-treatment performed in a step separate form hybridization step and with the use of a non-diluting stringent wash

[0170] This comparative example illustrates the state-of-art pre-treatment of samples.

[0171] Here is shown an example of the present art, where Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) bacteria are detected by an assay based on the paper by Perry-O'Keefe et al 2001. cf. above.

[0172] One ml of log phase growing bacteria 108cells / ml were pelleted by centrifugation, 4,700 G (7000 rpm), for 5 min, and resuspended in PBS (7 mM a2HPCU, 7 mM Na2HPCU, 130 mM NaCI). Cell suspensions were centrifuged again (4,700 G, 5 min), then resuspended in PBS with 5% v / v formaldehyde and fixed for 1 h. The fixed cells were rinsed in PBS, resuspended in 50% (v / v) ethanol, and incubated at -20° C for at least 30 min prior to use. A 100 pl aliquot of each of the fixed cells, approximately 108cells / ml, was pelleted by centrifugation, 4,700 G for 5 min, the pellet rinsed with PBS, and then resuspended in 100 pl hybridization buffer, 25 mM Tris-HCI, pH 9.0; 100 mM NaCI; 0.5% w / v SDS, Ethidium bromide 25 ng / ml, containing 100 and 150 nM Alexa 488 labelled PAPNA and SA1PNA probe, respectively. Next, the cells were incubated at 55°C for 30 min, centrifuged at 4,700 G for 5 min, then resuspended in 1000 pl of stringent wash solution (10 mM Tris pH 9.0, 1 mM EDTA), incubated at 55°C for 10 min, followed by pelleting by centrifugation at 4,700 G for 5 min. The wash was repeated two more times for a total of three washes. Finally, the cells were resuspended in the stringent wash buffer and analyzed in a flow cytometer (Attune NxT). The ethidium bromide positive events were gated, and these were analyzed for PNA binding.

[0173] Fig. 1A shows SA1PNA specifically binding to Staphylococcus aureus containing samples where the nonspecific PAPNA did not show any binding.

[0174] Similarly, when Pseudomonas aeruginosa was used for the assay as above, the specific PAPNA for Pseudomonas detected the Pseudomonas and the nonspecific SA1PNA did not show any binding, Fig. IB.

[0175] The fixation and alcohol incubation prior to the hybridization is required to secure efficient permeabilization of the cells. The procedure is performed on cells in PBS and not in complex sample matrix such as e.g., raw milk. The procedure involves furthermore numerous wash steps where centrifugation is required as well as other steps. The procedure is a multistep procedure.

[0176] EXAMPLE 1

[0177] Combined treatment and hybridization with non-diluting stringent wash and with "open information" of reagents components used

[0178] This example demonstrates efficacy of the combined treatment and hybridization.

[0179] 62.3 pl raw milk spiked with Staphylococcus aureus in log phase (106cells / ml) was added to a combined treatment and hybridization buffer for a total volume of 250 pl and incubated at 55°C for 30 min. No stirring or mixing was used. Next, the cells were centrifuged at 4,700 g for 5 min, then resuspended in 1000 pl of stringent wash solution (10 mM Tris pH 9.0, 1 mM EDTA), incubated at 55°C for 10 min, followed by pelleting by centrifugation at 4,700 G for 5 min. The wash was repeated two more times for a total of three washes. Finally, the cells were resuspended in stringent wash buffer and analysed in a flow cytometer (Attune NxT). The ethidium bromide positive events were gated, and these were analysed for PNA binding.

[0180] The hybridization buffer contained 25 mM Tris-HCI, pH 9.0; lOOmM NaCI; 0.5% W / V SDS; Ethidium bromide 25 ng / ml, 2.9 v / v % BactoScan™ Enzyme or alternatively the serine endopeptidase Subtilisin A from Sigma-Aldrich, and with 150 nM SA1PNA or PAPNA Alexa 488 labelled probes or no probe. Excitation of Alexa 488 and ethidium bromide was made with blue laser. Emission of Alexa 488 with BL1 525 / 50 nm and ethidium bromide with BL2 574 / 26 nm.

[0181] The mean fluorescence intensity measured was Blank: 160; PAPNA: 241; and SA1PNA: 3097. Fig. 2A thus shows SA1PNA specifically binding to Staphylococcus containing samples where the nonspecific PAPNA only showed minor binding compared to no PNA. The observed minor binding suggests that more stringent wash conditions are required.

[0182] This example employs the hybridization staining of the specific bacteria without any pretreatment of the cells or removal of sample matrix components or any stirring / mixing and thereby simplifying the staining of nucleic acids compared with prior art.

[0183] The same experiment performed as above, but with PBS spiked with the Gram-negative Pseudomonas aeruginosa (PA) and with or without subtilisin is shown in Fig. 2B.

[0184] 62.3 pl spiked PBS with PA in log phase (106cells / ml) was added to a combined treatment and hybridization buffer for a total volume of 250 pl, and incubated at 55°C for 30 min. Next the cells were centrifuged at 4,700 G for 5 min, then resuspended in 1000 pl of stringent wash solution (10 mM Tris pH 9.0, 1 mM EDTA), incubated at 55°C for 10 min, followed by pelleting by centrifugation at 4,700 G for 5 min. The wash was repeated two more times for a total of three washes. Finally, the cells were resuspended in stringent wash buffer and analysed in a flow cytometer (Attune NxT). The ethidium bromide positive events were gated, and these were analysed for PNA binding.

[0185] The used hybridization buffer contained 25 mM Tris-HCI, pH 9.0; lOOmM NaCI; 0.5% W / V SDS; Ethidium bromide 25 ng / ml, with or without 2.9 v / v % BactoScan™ Enzyme or alternatively the serine endopeptidase Subtilisin A from Sigma-Aldrich, and with 150 nM SA1PNA or PAPNA Alexa 488 labeled probes or no probe.

[0186] Fig. 2B demonstrates that the permeability of the Gram-negative bacteria benefitted from a combined treatment and hybridization of the sample compared to the situation when the hybridization occurs without e.g., additional enzyme pre-treatment steps (such as alcohol or paraformaldehyde treatment, drying and / or freezing etc.) or a combined treatment and hybridization step. A traditional multiple step stringent wash was used after the combined treatment and hybridization incubation. EXAMPLE 2

[0187] Application of different incubation times in combined treatment and hybridization step in a triple bacteria detection

[0188] This example shows three different bacteria spiked in raw milk labelled with three specific PNA performed in a single experiment and measurement.

[0189] Escherichia coli (EC), Staphylococcus aureus (SA), and Pseudomonas aeruginosa (PA) were used to test different incubation times of hybridization from 5 min to 30 min, see Fig. 3.

[0190] 62.3 pl spiked non-treated raw milk with SA, EC and PA (about 106cells / ml) was added to 187.6 pl Combined Treatment and Hybridization buffer for a total volume of 250 pl and incubated at 55°C for 5, 10, 15, 20, 25 or 30 min. Then the sample was diluted 40x with a dilution stringency buffer prewarmed to 55°C and measured immediately thereafter on the CytoFLEX flow cytometer. No stirring or mixing besides adding the milk and the stringency buffer were performed.

[0191] The Combined Treatment and Hybridization buffer consisted of 5.5 pl BactoScan™ Enzyme, approximately 2.1 pl PNA, 180.1 pl BactoScan™ Buffer Solution with ethidium bromide and detergents.

[0192] A 100 ml stock of BactoScan™ Buffer Solution consisting of 6 g BactoScan™ FC Buffer Powder, 5 ml BactoScan™ FC Detergent and 2 ml BactoScan™ FC Staining Medium was made. A stock solution of 5 ml BactoScan™ Buffer Solution and BactoScan™ Enzyme was made as well before the combined treatment and hybridization. The PNA's were diluted with the BactoScan™ Buffer Solution before added as part of the Combined Treatment and Hybridization buffer to a concentration of 250 nM. The PNA was added to the Combined Treatment and Hybridization buffer prior to use for practical experimental reasons but it was shown (data not shown) that PNA in BactoScan™ Buffer Solution was stable without effecting their performances at room temperature for at least three weeks. The used dilution stringency buffer contains 10 mM Tris pH 9.0, 1 mM EDTA, 20 v / v% formamide and 5% v / v% BactoScan™ detergent. It had no significant influence on the performance if the sample is measured immediately after the addition of pre-heated stringency buffer or if kept at room temperature till measured until at least an hour after dilution. In this experiment the measurement was done right after the dilution step. The raw milk was tested negative (CFU) for the three bacteria using specific plates for each bacteria. This shows that the two Gram-negative cells (EC and PA) already are stained after 5 min and the Gram-positive cell (SA) require longer incubation time, but picks up from 10 min. This is done without the need of steps or pre-steps using e.g. alcohols, paraformaldehyde, drying and / or freezing for permeabilization. It can furthermore be performed without any washing steps and at constant temperature. The time to answer may be very fast such as a few minutes. Purification or enrichment of the cells from a complex sample matrix might be avoided. It can be done with a minimum of interactions which makes both a manual solution and an instrumentation automated solution much simpler than seen in the present art.

[0193] Fig. 3A, shows the time chase of the PNA staining of Gram-negative and Gram-Positive bacteria using ECPNA Alexa 488, SA2PNA Alexa 647, and PAPNA Alexa 405 to label Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa respectively. The positive events are in the lower right quadrant.

[0194] The left-hand dot plot in Fig. 3B is all events shown in an FSC / SSC plot. The second is an EtBr / SSC plot showing the positive ethidium bromide-stained events gated in the lower right quadrant. The two figure plots are showing how the gating was performed in Fig. 3A by using the incubation time of 30 min as example.

[0195] Table 2 below shows the positive cell counts.

[0196] Table 2

[0197] Positive cell counts

[0198] Time / min EC POS SA POS PA POS TOTAL

[0199] 5 810 98 1049 1957

[0200] 10 810 960 1102 2872

[0201] 15 882 2111 1233 4226

[0202] 20 798 2753 1284 4835

[0203] 25 891 3035 1249 5175

[0204] 30 923 3118 1287 5328

[0205] It will be understood by those skilled in the art that performing changes in the used parameters in the example such as e.g. temperatures, lasers, flow cytometer, used filters, measuring set up and data analysis, concentrations, and incubation time and / or the used reagent components could be beneficial and will further improve the results shown. EXAMPLE 3

[0206] Use of stringency dilution with different denaturation solvents

[0207] In this example, four different solvents Formamide, 2-pyrolididone, 1- Methyl-2- Pyrrolidinone (NMP) and Sulfolane were tested from 5% to 40% v / v in the stringency buffer (10 mM Tris pH 9.0, 1 mM EDTA) to demonstrate that a broad variance of nucleic acid denaturation solvents might be used for stringency dilution.

[0208] 108mid log phase Staphylococcus aureus (SA) was spiked into 1 ml raw milk and then added to the specific PNA hybridization reaction consisting of a combined treatment and hybridization solution.

[0209] The hybridization reaction consisted of a total of 250 pl: 192 pl 1.25x Hybl buffer, 50 pl bacteria spiked raw milk, 5.5 pl BactoScan™ FC Enzyme, Ethidium bromide 25 ng / ml and 2.5 pl PAPNA Alexa 488 or SAI PNA Alexa 488 probes or no PNA for final concentration of 300 nM.

[0210] The incubation was performed for at 55°C for 30 min. After the incubation a 40 X dilution with the specific Stringency dilution buffer was performed, 195 pl stringency dilution buffer at room temperature added 5 pl hybridization reaction in 96 well titer plate and measured immediately on the Attune NxT. No mixing or stirring applied.

[0211] The HYB1 buffer consisted of 25 mM Tris-HCI, pH 9.0, lOOmM NaCI, 0.5% w / v SDS and 25ng / ml Ethidium bromide. Stringency dilution buffer 10 mM Tris pH 9.0, 1 mM EDTA with different percent v / v of solvents.

[0212] Fig. 4 shows that no PNA and nonspecific SA1PNA peaks are separated (left peaks) from the specific PNA binding peak (peak on the right in each histogram) even at 5% solvent concentration in spite of the fact that it occurred at room temperature.

[0213] In general, the separation improved with increasing solvent concentration at the used conditions. Other organic solvents or a mix of solvents can also supplement the used organic solvents in this example. In the shown examples of this application 20% formamide are in general used as solvent. However, lower percent or above 40% organic solvent or no solvent may be used. It could be beneficial to use a solvent that are less hazardous than formamide to minimize potential health issues. The Sulfolane at 40% v / v could not be measured in the used flow cytometer as the viscosity at room temperature did not allow steady flowrate. Note, that the performance of the peak separation improves significant if the stringency dilution temperature is raised to a higher temperature than room temperature, e.g. to 35, 45, 55 or 60°C. This exemplifies as well that the temperature of the stringency dilution might variate from the in general used temperature of 55°C in this application. In the shown example a combined treatment and hybridization is used, however the stringency dilution works as well on samples that first have had a separated pre-treatment, then followed by hybridization (Data not shown). In this example the stringency buffer did not contain detergents. The use of e.g. Tween 20 or BactoScan™ detergents improved the stringency dilution performance (Data not shown).

[0214] Non-specific PAPNA and Staphylococcus aureus specific SA1PNA was used for the assay. The light grey peak is showing specific SA1PNA binding compared to no PNA and nonspecific PAPNA binding shown in black and darker peaks, respectively. The histograms are merged histograms.

[0215] Similar results may be obtained with the use of other denaturation solvents or a mix of solvents or without using any solvents by e.g., changing the used stringency buffer temperature and / or adding other components such as e.g., detergents, alcohols, permeabilizing proteins or chemicals or other agents.

[0216] EXAMPLE 4

[0217] Stringency dilution work at different dilution degrees

[0218] In this Example it is shown that the invention works on a buffer containing bacteria, a simple sample matrix, with a stringency dilution of 10 times.

[0219] 50 pl spiked PBS with Escherichia coli or Staphylococcus aureus (SA) (about 107cells / ml) was added to 200 pl Combined Treatment and Hybridization buffer for a total volume of 250 pl and incubated at 55°C for 30 min. Then the sample was diluted lOx, 20 pl to 180 pl dilution stringency buffer, and measured thereafter immediately on the CytoFLEX flow cytometer using the blue laser with a flow rate of 25 pl / min. No stirring or mixing besides adding the PBS with cells and the incubated liquid to the stringency dilution buffer were performed.

[0220] 100 ml of BactoScan™ Buffer Solution contained 6 g BactoScan™ FC Buffer Powder, 5 ml BactoScan™ FC Detergent and 1 ml BactoScan™ FC Staining Medium. The Combined Treatment and Hybridization buffer consisted of 192.5 pl BactoScan™ Buffer Solution and 5,5 pl BactoScan™ Enzyme with approximately 2 pl ECPNA Alexa 488 or SA2PNA Alexa 488 or no PNA. The PNA had been pre-diluted with the BactoScan™ Buffer Solution before added as part of the Combined Treatment and Hybridization buffer. The final PNA concentration was 0 nM or 50 nM or 150 nM for no PNA, ECPMA and SA2PNA respectively. The used dilution stringency buffer containing 10 mM Tris pH 9.0, 1 mM EDTA, 20 v / v% formamide and 5% v / v% BactoScan™ detergent.

[0221] Fig. 5A and the Table 3 shows that an effective separation of the specific EC peak from the non-specific NOPNA and SA2PNA peaks could be obtained with lOx stringency dilution. Fig. 5B and the Table 4 shows that SA peak was separated, but not as optimal as the separation under the used condition as the EC. Table 3 Escherichia coil in PBS with lOx stringency dilution

[0222] Population count

[0223] All EtBr PNA events channel channel

[0224] NOPNA 49382 49314 55

[0225] ECPNA 49704 49652 49124

[0226] SA2PNA 49900 49838 174

[0227] Ethidium bromide (EtBr), No PNA (NOPNA), non-specific SA2PNA, and Escherichia coli specific ECPNA were used for the assay. All events are the FSC / SSC events.

[0228] Table 4 Staphylococcus aureus in PBS with lOx stringency dilution

[0229] Population count

[0230] All EtBr PNA events channel channel

[0231] NOPNA 25750 25574 117

[0232] ECPPNA 26308 26237 156

[0233] SA2PNA 26628 26573 16957

[0234] Ethidium bromide (EtBr), No PNA (NOPNA), non-specific ECPNA, and Staphylococcus aureus specific SA2PNA were used for the assay. All events are the FSC / SSC events.

[0235] In Fig. 5C and Table 5 is shown the same experiment as above but with Staphylococcus aureus (SA) spiked into raw milk instead of PBS, the stringency dilution was performed with a 40x pre-heated stringency buffer to 55°C instead of a 10X and a flow rate of 30 pl / min instead of 25 pl / min. The used raw milk had been tested negative for SA. This illustrates that for the Gram-negative SA, a pre-heated 40x dilution worked better than the lOx dilution even though the lOx dilution did function under the used conditions.

[0236] Table 5 Staphylococcus aureus in raw milk with pre-heated 40x stringency dilution

[0237] Population count

[0238] All events EtBr NOPNA / ECPNA SA2PNA

[0239] Pl P2 P3 P4

[0240] NOPNA 12887 12887 12858 5

[0241] ECPNA 12114 12114 12020 61

[0242] SA2PNA 13172 13172 150 13016

[0243] Ethidium bromide (EtBr), No PNA (NOPNA), non-specific ECPNA, and Staphylococcus aureus specific SA2PNA were used for the assay. P4 shows the positive stained events. The histograms are merged.

[0244] EXAMPLE 5

[0245] Demonstration of importance of both detergent and enzyme

[0246] This example is to illustrate the importance of the detergent and enzyme components on assay performance. The protocol in Example 2 was followed, but with the following changes: The Staphylococcus aureus (SA), Escherichia coli (EC) and Pseudomonas aeruginosa (PA) were spiked in raw milk separately with triplex PNA present for each reaction. The Combined treatment and hybridization incubation was performed with or without detergent, BactoScan™ FC Detergent, and / or enzyme, BactoScan™ Enzyme, for 30 min.

[0247] Table 6 shows that to detect the Gram-positive SA, both the presence of detergents and digestive enzymes are necessary to get sufficient mean fluorescence intensity to detect the bacteria. The two Gram-negative bacteria, EC and PA, could be detected without, but with less efficiency then with both detergents and enzymes present. It can therefore be concluded that the best performing assay conditions have both detergents and enzymes present at the combine treatment and hybridization incubation of the sample.

[0248] Table 6 The effect of detergents and enzymes

[0249] Samples No ENZ No ENZ With ENZ With ENZ

[0250] No DET With DET No DET With DET

[0251] SA - - - + + +

[0252] EC + 1 ++ + + + +

[0253] PA + + / - ++ + + + Mean fluorescence intensity: non weak +; moderate ++; and strong +++.

[0254] EXAMPLE 6

[0255] Test of the method of the invention on Gram-negative bacteria and Gram-positive bacteria

[0256] Detection of family or Genus of bacteria using a single PNA probe is shown in this example with the invention.

[0257] Four different bacteria from the Enterobacteriaceae family with two Enterobacteriaceae family specific PNA probes EB1PNA and EB2PNA labelled with Alexa 488 were used. Staphylococcus aureus and Pseudomonas aeruginosa, which does not belong to Enterobacteriaceae family, were used to test the probe specificity. The Escherichia coli ECPNA Alexa 488 probe was used as a specific PNA positive control.

[0258] 62.3 pl spiked untreated raw milk with the tested mid log phase bacteria (about 106cells / ml) was added to 187.6 pl Combined Treatment and Hybridization buffer for a total volume of 250 pl and incubated at 55°C for 30 min. Then the sample was diluted 40x with a dilution stringency buffer prewarmed to 55°C and measured immediately thereafter on the CytoFLEX flow cytometer. The positive ethidium bromide events were used to gate the cells. No stirring or mixing besides adding the milk and the stringency buffer were performed.

[0259] The Combined Treatment and Hybridization buffer consisted of 5.5 pl BactoScan™ Enzyme, approximately 2.1 pl PNA, 180 pl BactoScan™ Buffer Solution with ethidium bromide and detergents, see Example 3 for further details. The dilution stringency buffer contained 10 mM Tris pH 9.0, 1 mM EDTA, 20 v / v% formamide and 5% v / v% BactoScan™ detergent. A PNA concentration of 250 nM was used.

[0260] Figure 6 and Table 7 show that both the Enterobacteriaceae family specific PNA probes were able to detect all tested bacteria from Enterobacteriaceae family and no detection was observed from non-family bacteria.

[0261] Table 7 Family Enterobacteriaceae specific detection

[0262] Bacteria ECPNA EB1PNA EB2PNA

[0263] The Enterobacter is a non-coliform bacteria, where Escherichia coli, Klebsiella, and Serratia are coliform bacteria. The non-Enterobacteriaceae bacteria SA and PA were not recognized.

[0264] Seven Gram-positive bacteria from Genus Lactobacillus (Latic acid bacteria) were then tested with a specific PNA probe LABPNA labelled with Alexa 488. In-silico analysis of the probe sequence indicated that the probe should not bind to the two Lactococcus, but to the other five Lactobacillus. The Gram-positive Pseudomonas aeruginosa (PA) and Gram-negative Escherichia coli, which does not belong to Lactobacillus, were used to test the LABPNA probe specificity. The Enterobacteriaceae EB1PNA Alexa 488 probe was used as a positive control. A PNA concentration of 250 nM was used. No stirring or mixing besides adding the milk and the stringency buffer were performed. The assay was performed as described above for Enterobacteriaceae.

[0265] Fig. 7 and Table 8 show that the Lactobacillus specific LABPNA was able to detect all the expected bacteria from Lactobacillus (1-5) and no detection was observed from non- Lactobacillus bacteria (8-9). This shows that the invention can detect both Gram-positive as well as Gram-negative bacteria. The positive control, EB1PNA, did recognize Escherichia coli (8), but not the Lactobacillus (1-7) or SA (9) as expected.

[0266] Table 8 Specific detection of Gram-positive Lactic acid bacteria.

[0267] Tested bacteria LABPNA EB1PNA The number in the table corresponds to the respective number of the dot blots in Fig. 7.

[0268] The black arrows in Fig. 7 point on the light gray LABPNA Alexa 488 stained dotted events. The dark gray dots are EB1PNA Alexa 488 stained events. The numbers in the dot plots in Fig. 7 correspond to the number in Table 8. The dot blots are merged blots.

[0269] EXAMPLE 7

[0270] 20x dilution in a non-enriched sample matrix (milk) with detection of 104bacteria per ml

[0271] This example shows that a 20x dilution may be used with the invention.

[0272] The used raw milk had a natural IBC of 20,000 / ml measured on BacSomatic™, FOSS Analytical A / S, Denmark. The milk was tested negative (CFU) for the present of both SA and EC bacteria with specific MSA and CCA plates. Skim milk agar plates (CSMA) showed 24,000 CFU / ml.

[0273] Mid log phase Staphylococcus aureus (SA) or Escherichia coll (EC) was spiked in 1 ml raw milk to approximately 4.5xl04bacteria and then added 50 pL of the spiked sample to 200 pL Combined Treatment and Hybridization buffer (see Example 3) containing no PNA, ECPNA Alexa 488 or SA2PNA Alexa 647 for a final concentration of 0, 50 or 150 nM, respectively. The sample was incubated at 55°C for 30 min. Next the sample was diluted 20x with a dilution stringency buffer prewarmed to 55°C and 800 pl was measured thereafter with a flowrate of 200 pl / ml on the CytoFLEX flow cytometer. No stirring or mixing besides adding the milk and the stringency buffer were performed. Dilution stringency buffer: 10 mM Tris pH 9.0, 1 mM EDTA + 20% formamide.

[0274] Fig. 8A and B show that even though that the used settings of the used system are on the limit of its detecting capacity, a difference is detected between the specific PNA and no PNA. In the EtBr channel the total bacterial IBC count in SA and EC spiked samples was 563 and 512 (average of three), which are close to the expected number of 520 total IBCs. Therefore, the total bacterial count was in the expected range. Despite using a high dilution factor a sensitivity of about 104bacteria / ml was obtained. It is clear that performing e.g. an preenrichment of bacteria and / or e.g. using quenching technology such as beacons instead of dilutions stringency or changing assay parameters or reagents would make it possible to improve to a much higher sensitivity if this should be required. Combined factor (250 / 50) x(20x dilution) / (0.8 ml measuring) = 125x

[0275] (20,000 + 45,000) / 125 IBC = 520 expected IBC events

[0276] EXAMPLE 8

[0277] Gating strategy and simultaneous Gram-positive and Gram-negative detection of two specific bacteria

[0278] In this example, mid log phase Staphylococcus aureus (SA) and Escherichia coll (EC) were spiked in a raw milk sample. Specific PNA for Staphylococcus and Escherichia coll, labeled with Alexa 647 and Alexa 488, was used for the assay, respectively.

[0279] Spiked milk samples (about 107cells / ml) were hybridized with two specific PNA, SA2PNA and ECPNA, at 250nM concentration with the Combined Treatment and Hybridization buffer, see Example 3, and incubated at 55°C for 30 minutes. Following hybridization, the sample was diluted 40 times to a total volume of one ml in a single step with 55°C pre-heated stringency dilution buffer (10 mM Tris pH 9.0, 1 mM EDTA + 20 v / v% formamide + 5% v / v% BactoScan™ FC Detergent) and then measured 4000 events in the CytoFLEX flow cytometer using Blue (Alexa 488), Yellow (EtBr) and Red (Alexa-647) laser at a flow rate of 10 pl / min. No stirring or mixing besides adding the milk and the stringency buffer were performed.

[0280] Fig. 9 shows to the left of the dotted line the bacterial population differentiated using Forward scattering (FSC) and Side scattering (SSC). When the same population, to the right of the dotted line, are visualized using an SSC and ethidium bromide scattered plot, a better separation from the general noise is obtained. From this plot the two different bacterial populations were detected specifically using the fluorophore labelled PNA labelling. This does not exclude the use of an FSC / SSC gating but exemplifies that it might not be required for discriminating noise from specific bacteria.

[0281] Experiments using specific probes such as e.g. ECPNA and SAPNA, without the use of e.g. EtBr or UPNA to gate positive events, showed that staining of specific cells may be performed with a single specific stain without using a general staining of the present cells (Data not shown). EXAMPLE 9

[0282] Total counts of natural bacteria present in raw milk sample using specific universal PNA probe

[0283] This example shows bacteria detection of a natural high count raw milk sample of about 3xl06IBC / ml (BacSomatic™). Specific PNA for Eubacteria (prokaryotic Bacteria), UPNA labeled with Alexa 647, was used for the assay.

[0284] Raw milk was treated and hybridized with the Combined Treatment and Hybridization buffer, see Example 3, with the specific UPNA at a 250 nM concentration and incubated at 55°C for 30 minutes. Following hybridization, the sample was diluted 40 times to a total volume of one ml in a single step with 55° C pre-heated stringency dilution buffer (10 mM Tris pH 9.0, 1 mM EDTA + 20 v / v% formamide + 5% v / v% BactoScan™ FC Detergent) and then measured on the CytoFLEX flow cytometer using Yellow (EtBr) and Red (Alexa-647) laser. No stirring or mixing besides adding the milk and the stringency buffer were performed.

[0285] Ethidium bromide binds unspecific double stranded nucleic acid where the universal PNA binds specific to a conserved Eubacteria region on the rRNA. The histogram in Fig. 10 shows that the positive signal peak is better separated from the background peak when the specific UPNA probe is used than the unspecific Ethidium. It furthermore showed corresponding count numbers between the two. The use of UPNA for detection and gating showed strong indications for improving sensitivity when compared to the use of a general unspecific nucleic acid staining reagent. It is shown that natural prokaryotic Bacteria can be detected with the invention.

[0286] The black arrows in Fig. 10 point on the background events (noise) in the dot blots and the corresponding background in the histograms. The light grey histogram is positive events, and the black histograms is noise. The numbers in the dot plots show the positive events in the lower right quadrant. The numbers are the raw data coming out of the measured volume after dilution. They are not in terms of IBC / ml.

Claims

CLAIMS1. A method for analysis of a sample for the presence of one or more target nucleic acids, the method comprising i) contacting the sample with one or more hybridization agents, such as an agent comprising or consisting of a polynucleotide or analogue thereof, capable of hybridizing to said one or more target nucleic acids to obtain a mixture of the agent and the sample; ii) diluting the mixture obtained in step i) with a diluent, which may be in the form of a stringency buffer, in the presence of which specific hybridization between the one or more hybridization agents and their respective target nucleic acids is favored over non-specific hybridization events, to obtain a diluted mixture; and iii) qualitatively or quantitatively detecting in the diluted mixture hybridization agents hybridized to target nucleic acids.

2. The method according to claim 1, wherein the one or more hybridization agents are nucleic acids or nucleic acid analogues.

3. The method according to claim 1 or 2, wherein each of the one or more hybridization agents is or comprises a polynucleotide being modified to exhibit higher binding affinity and binding specificity for natural nucleic acids than does both DNA and RNA.

4. The method according to any one of the preceding claims, wherein the one or more hybridization agents hybridize(s) specifically to a nucleic acid not naturally associated with cells present in the sample.

5. The method according to claim 4, wherein the sample is from a multicellular organism and the hybridization agent does not hybridize to normal cells of said multicellular organism.

6. The method according to any one of the preceding claims, wherein the one or more hybridization agents is / are specific to rRNA, such as bacterial rRNA or fungal rRNA.

7. The method according to any one of the preceding claims, wherein the one or more hybridization agents has / have an uncharged backbone.

8. The method according to any one of the preceding claims, wherein the one or more hybridization agents is / are or comprise(s) peptide nucleic acid(s) (PNA).

9. The method according to any one of the preceding claims, wherein the one or more hybridization agents further comprises a detectable moiety.

10. The method according to claim 9, wherein the detectable moiety is selected from the group luminescent, fluorescent, chromogenic, radioactive, and ligand or receptor moieties.

11. The method according to any one of the preceding claims, wherein the one or more hybridization agents comprise(s) a self-quenching portion, which provides an unquenched signal if attached to the target nucleic acid while providing a quenched signal while not being bound, or vice versa.

12. The method according to any one of the preceding claims, wherein two or more hybridization agents are present in the agent, which differ from each other to simultaneously detect two or more different nucleic acids, and optionally two or more different cells, spores or virus, present in the sample.

13. The method according to any one of the preceding claims, wherein the sample is selected from the group consisting of mammalian body fluids, such as blood, saliva, semen, vaginal fluids, mucus, urine, raw milk or milk, condensed milk, powdered milk, dairy products; an animal tissue sample; and a plant-derived sample.

14. The method according to claims 13, wherein the sample is milk, preferably cow's milk or other milk, including plant-derived milk, for human consumption.

15. The method according to claim 14, wherein the milk is raw milk.

16. The method according to claim 4, wherein the cells are prokaryotic cells.

17. The method according to claim 4, wherein the cells are eukaryotic cells.

18. The method according to any one of the preceding claims, wherein the target nucleic acids are present in eukaryotes, microorganisms, spores, endospores, or virus.

19. The method according to claim 18, wherein the microorganisms are selected from pathogenic bacteria; pathogenic fungi; and bacteria or fungi, which cause humans, animals and / or products thereof to attain undesirable characteristics, such as unpleasant odor, taste, texture, color, or feel, or reduced life-span or commercial lifespan.

20. The method according to any one of the preceding claims, wherein steps i and ii in combination takes less than 360 minutes, such as less than 150 mins, such as less than 140 mins, such as 130 mins, such as 120 mins, such as 110 mins, such as 100 mins, such as 90 mins, such as 80 mins, such as 70 mins, such as 60 mins, such as 50 mins, such as 40 mins, such as less than 20 mins, such as less than 10 mins.

21. The method according to any one of the preceding claims, wherein steps i and II in combination takes at least 10 seconds, such as at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 60 seconds, such as at least 1.5 minutes, such as 2 minutes, such as 2.5 minutes, such as 3 minutes, such as 3.5 minutes, such as 4 minutes, such as 4.5 minutes, such as 5 minutes, such as at least 10 minutes, such as 20 minutes, such as 30 minutes, such as 40 minutes, such as 50 minutes, such as 60 minutes, such as 70 minutes, such as up to 80 minutes.

22. The method according to any of the preceding claims, wherein the stringency buffer comprises one or more detergent(s) and / or wherein step ii entails addition of one or more detergent(s).

23. The method according to any one of the preceding claims, wherein the temperature in at least step ii is controlled to optimize that specific hybridization by the one or more hybridization agents is favored over non-specific hybridization.

24. The method according to any one of the preceding claims, wherein the detection is a multiplex flow cytometry-based detection or a detection via microscopy.

25. The method according to any one of the preceding claims, wherein the stringency buffer is a high-stringency buffer.

26. The method according to any one of the preceding claims, wherein the stringency buffer comprises an agent selected from one of more of- a pH buffer;- a salt or salts;- a solvent or solvents;- a chelating agent;- a detergent or a mixture of detergents; and- a DNA staining component.

27. The method according to any one of the preceding claims, wherein the stringency buffer is applied to dilute by a factor of 3-100 times.

28. The method according to any one of the preceding claims, which prior to step i comprises providing the sample by obtaining an original sample suspected of containing cells or virus and subsequently a) rendering cells or virus in the original sample permeable to the one or more hybridization agents and adding one or more enzymes to enzymatically degrade the sample and / or to contribute to rendering the cells or virus permeable to the one or more hybridization agents, b) carrying out step i-ii, whereby the mixture in step i comprises the sample, the added one or more enzymes and the added one or more hybridization agents c) carrying out step ill.

29. The method according to claim 28, wherein the one or more enzymes is / are selected from one or more of a protease, a lipase, an esterase, and a carbohydrate degrading enzyme.

30. The method according to claim 28 or 29, wherein the enzymes and hybridization agent are added simultaneously, in a single step process or wherein the enzymes and hybridization agent are added in sufficiently rapid succession to each other to operate simultaneously.

31. The method according to any of claims 28-30, wherein no substantial removal of the one or more enzyme and / or the hybridization agent occurs prior to detecting the hybridized nucleic acids.

32. The method according to any one of claims 28-31, wherein at least one of the one or more enzymes is one or more proteases.

33. The method according to claim 32, wherein the one or more proteases has / have broad substrate specificity.

34. The method according to claim 33, wherein the one or more proteases is selected from the group consisting of serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases.

35. The method according to any one of claims 28-34, wherein the at least one of the one or more enzymes is a lipase or an esterase.

36. The method according to any one of claims 28-35, wherein the at least one of the one of more enzymes is a carbohydrate degrading enzyme.

37. The method according to claim 36, wherein the carbohydrate degrading enzyme is selected from one or more of a cellulase, a hemicellulase, a pectinase, and an amylase.

38. The method according to any one of claims 28-37, wherein the hybridization agent is capable of specific hybridization at salt concentration(s) of between 0 to 1200 nM where the at least one enzyme is enzymatically active.

39. The method according any one of claims 28-38, wherein a)-b) are carried out simultaneously in a single step process.

40. The method according to any one of claims 28-38, wherein a)-b) are carried out in rapid succession to each other without any intermediate purification to operate simultaneously.

41. The method according to any one of claims 28-40, wherein a)-b) are carried out at essentially the same temperature, or wherein one or more temperature changes are included.

42. The method according to one of claims 28-41, further comprising incubating the sample after b).

43. The method according to any of claims 28-42, wherein between about 10° and about 109microorganisms / ml are detectable.

44. The method according to any one of claims 28-43, wherein b is carried out on the optionally diluted mixture from (a) comprising the sample, the added one or more enzymes, and the hybridization agent.

45. The method according to claim 44, wherein, prior to (c) no removal takes place of components of the mixture comprising the sample, the added one or more enzymes, and the hybridization agent.

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