EP1009862A1 - Schnelle detection ubd identifizierung von mikroorganismen - Google Patents

Schnelle detection ubd identifizierung von mikroorganismen

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Publication number
EP1009862A1
EP1009862A1 EP98940684A EP98940684A EP1009862A1 EP 1009862 A1 EP1009862 A1 EP 1009862A1 EP 98940684 A EP98940684 A EP 98940684A EP 98940684 A EP98940684 A EP 98940684A EP 1009862 A1 EP1009862 A1 EP 1009862A1
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EP
European Patent Office
Prior art keywords
probe
micro
cells
organism
nucleic acid
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EP98940684A
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English (en)
French (fr)
Inventor
Frederik Schut
Paris Som Twan Tan
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Microscreen BV
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Microscreen BV
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Priority to EP98940684A priority Critical patent/EP1009862A1/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the invention relates to the field microbiology, more specifically to the field of detection, identification and quantification or enumeration of micro-organisms.
  • Microorganisms such as viruses, plasmids, bacteria, yeasts, fungi, algae, protozoa, plant or animal cells, and other prokaryotic or eukaryotic cells are in general unicellular organisms with dimensions beneath the limits of vision which thus escape easy detection.
  • Micro-organisms are omni -present , complex (mixed) populations can be present in materials as divers as (sea) water, bodily tissues or fluids, pharmaceutical preparations, foodstuffs, industrial products, waste, soil, plants, animals, other micro-organisms, and often their (specific) presence needs to be detected or monitored.
  • Micro-organisms for example are widely used in fermentation processes, can be involved as pathogen in disease, and may be used in modern (recombinant) biotechnology; here, as elsewhere, it is essential to be able to properly identify and quantify the micro-organism (s) involved.
  • culturing techniques are used to establish the microbial status of a material.
  • Culturing micro-organisms is an established way of isolating, detecting, identifying or quantifying them.
  • a great number of micro-organisms can in general be cultured, propagated and manipulated in the laboratory, there are also micro-organisms for which culturing methods have not been developed or for which laboratory culture may be difficult if not impossible.
  • virus, and other intra-cellular organisms in general, cell-cultures are needed in which said organism grows to detect, identify or quantify the specific organism.
  • Other micro-organisms can be isolated and grown in various culture media under various, often very specific, conditions.
  • Quantifying the number of specific organisms present in starting material via culture techniques is relatively easy when it concerns starting material containing only one specific organism, e.g. in the case of monocultures. Such quantification can then be done using e.g. limited dilution tests.
  • starting material that contains mixed populations of micro-organisms the identity and/or the relative presence or absolute numbers of various organisms need to be known, culturing gets often very cumbersome and tedious, and may even be impractical or impossible for several of the organisms present in the starting material, leading to biased results.
  • Probes such as specific antibodies, labelled directly or indirectly with reporter molecules (chromophores or enzymes and corresponding substrates) and used in e.g. immuno-cytochemistry, generally react with antigens (especially (poly) peptides or (glyco) proteins) that are specific for a certain microorganism.
  • nucleic acid probes or primers reactive with specific nucleic acids find application in in si tu hybridisation techniques, such as FISH (fluorescent in si tu hybridisation) or in in si tu amplification techniques such as in si tu PCR (polymerase chain reaction) .
  • Detection of the labelled antibodies or probes or amplified sequences occurs generally by microscopic observation of the material tested.
  • Cells are first immobilised on slides (fixed and lysed e.g. by drying, ethanol fixation, etc.) and subsequently reacted with antibody or nucleic acid (labelled) probes.
  • cells can be kept in suspension until after the probe reaction, after which the suspension is filtered and the cells remain immobilised on a filter disc for analysis. Subsequent amplification of the probe signal allows detection of the labelled cells by microscope.
  • Semi-automated systems, using digital image analysis and image intensification (image cytometric analysis) have been designed to help study microscopic preparations.
  • image cytometry has the disadvantage that, in practical terms, at most some 500 to 1000 individual cells per microscopic preparation can be analysed.
  • a major drawback of above microscopic techniques is the limited sample throughput. The long analysis time required limits the maximum throughput to 20 to 50 samples per day.
  • fixation and lysis of the cells in general suffices to immobilise antigens and nucleic acid to the cytoskeleton or the various organelles present in the cell, the cell in itself disintegrates and gets fragmented due to the fixation and lysis.
  • those antigens and nucleic acids that are for example present (in solution) in the cytosol will disappear during fixation and lysis and cannot be detected.
  • no insight can be gained by above microscopic techniques about (current) enzymatic and/or mRNA activity of the micro-organisms studied.
  • this basic problem in in si tu staining using signal amplification systems relates to the diffusion of large-molecular weight molecules, such as enzymes, antibodies or (strept) avidin, into whole fixed cells.
  • the permeabilization gets more difficult for larger molecules since -the margin between the community of target molecules and the loss of target molecules or complete cell lysis becomes very narrow (Amman et al . , Microbiol . Rev. 59:143-169, 1995; Schonhuber et al , Appl . and Env. Microbiol. 63:3268-3273, 1997).
  • Permeabilization of cells walls by pretreatment with lysozyme (EDTA) in general allow a restricted set of bacteria to be investigated, whereby it must be noted that lysozyme degradation often only permeabilises a minor fraction of the tested cells (causing heterogenous signals) and is in general only applicable with cells fixed on microscopic slides but not with cells in suspension.
  • lysozyme degradation often only permeabilises a minor fraction of the tested cells (causing heterogenous signals) and is in general only applicable with cells fixed on microscopic slides but not with cells in suspension.
  • Rapid techniques to monitor the development of individual species or strains, and/or their (current) enzymatic activity, involved in fermentation processes comprising complex mixtures of species would relay important information related to the management and control of such processes .
  • Rapid flow cytometric techniques such as FACSCAN
  • FACSCAN Fluorescence Activated Cell Sorting
  • PNA peptide nucleic acid
  • Nucleic acid probes used in the method of the invention are provided as well.
  • most nucleic acid molecules in the cell are relatively little or not denatured, thereby greatly hampering hybridisation of most probes that otherwise would hybridise easily with stretches of denatured nucleic acid molecules .
  • An example of such probes provided by the invention is given in Table 3.
  • a further embodiment of the invention entails a method wherein said probe is labelled with a reporter molecule, for example a reporter molecule selected from any of the group of fluorochromes and enzymes .
  • a preferred embodiment of the invention is a method wherein the reporter molecule is horseradish-peroxidase, preferably linked with a sulfo-SMCC linker to said probe.
  • a further embodiment of a method provided by the invention is a method wherein the presence of said (bound) probe is detected by microscopy, image cytometry, fluorometry or flow cytometry, preferably be detecting fluorescence.
  • a preferred embodiment of the invention is a method wherein the fluorescence of tyramine- fluorchromes is detected.
  • a preferred embodiment of the invention is the use of a method of in si tu staining provided by the invention wherein the micro-organism is not immobilized, for example to a slide or filter, but wherein detection of the stained micro-organism occurs in suspension.
  • a composition comprising a cell -wall degrading reagens for use in in si tu staining.
  • a cell wall degrading reagens comprises at least one detergent, such as sodium taurocholate and at least one enzyme, such as a lipase and/or aditionally comprises a carbohydrate degrading enzyme such as lysozyme, finizym, or mutanolysin.
  • said composition comprises at least one os oticum, such as saccharose, sucrose, or lysine betaine, to regulate osmotic pressure and/or at least one cation, such as Ca ++ or Li + (but nor Mg ++ ) .
  • Said composition may be optionally buffered, for example with Tris-HCl.
  • a nucleic acid probe linked with horseradish-peroxidase Said probe and horseradish-peroxidase are preferably linked with a sulfo-SMCC linker.
  • a diagnostic test kit for use with in si tu staining.
  • Such a kit provided by the invention at least comprises a cell wall -degrading composition and/or nucleic acid probe provided by the invention, and can additionally at least comprise fixative, substrate, or buffer provided by the invention.
  • the HRP-nucleic acid probe provided by the invention is also provided for use in other tests systems than in si tu assays alone.
  • Such a probe can for example be used in enzym-linked assays , such as a dip-stick but other formats are also possible, for the rapid detection of genes or gene-products in for example lysed bacterial samples.
  • the deposited reporter molecules can additionally serve as hapten for, e.g., anti-fluorescein-AP, antibo ' dies via which an extra amplification step and the detection of very low numbers of target molecules is possible.
  • image cytometric analysis is in general terms an appropriate tool for the extraction of morphological features or localisation parameters (e.g., as in genome-mapping of eukaryotic cells) , signal amplification is ultimately desirable when natural mixed populations of bacteria (such as those occurring in seawater) are to be investigated.
  • Image cytometry has the disadvantage that in practical terms, at the most, some 500 to 1,000 individual cells per microscopic preparation can be analysed. Furthermore, the long analysis time required limits the maximum throughput to about 50 samples per day.
  • the relatively small number of samples that can be tested render the automation of the fluorescence in si tu hybridization technique practically and economically unattainable.
  • Flow cytometric analysis of samples would enable large scale analysis, if technically feasible.
  • the present invention enables rapid flow cytometric acquisition of quantitative data from probe-labelled cells and the possibility to automate methods for in si tu identification based on nucleic acid probes through generation of unequivocally high signals from such labels.
  • the level of cellular (enzymatic) activity of cells is of considerable interest.
  • the possibility to attach to individual cells not only a name but also an activity in a routine procedure cq. a practical protocol, will greatly advance microbiology.
  • the present invention allows for controlled degradation of for example bacterial cell walls in combination with an amplification technique for the measurement of rRNA in micro- organisms thus allowing in si tu staining and detection, for example by flow cytometric detection.
  • the present invention provides a signal amplification of at least 30-50 fold, and even 8O fold, as compared with conventional fluorescent probes.
  • the invention also provides a method to determine the amount of messenger RNA in micro-organisms and mammalian cells.
  • the commercial impact of the any molecular analysis method stands or falls with possibilities to enlarge the scale and reduce the cost. For large scale analysis, the ability to automate methodologies is crucial. In the ideal case, a sample is taken, directly inserted into an instrument and the data are printed.
  • ISH si tu hybridisation
  • FISH Fluorescence in situ hybridisation
  • RNA's the multi-copy transcripts of the DNA, are naturally amplified within each living cell, as such, they represent ideal targets for genetic labelling techniques.
  • each bacterial cell contains some 20.000 identical ribosomal RNA (rRNA) molecules and some 200 identical messenger RNA (mRNA) molecules. If each of these molecules was tagged by one fluorochrome, signals would not be sufficient to automate the analysis.
  • mRNA molecules represent the "coding" intermediates between DNA and proteins for which the genetic code is contained in the genome.
  • mRNA-ISH messenger RNA in si tu hybridization
  • the invention provides specific protocols, means and methods that are needed to permeabilize the (bacterial) cell wall. Furthermore, the invention provides a method to couple the HRP molecule directly to the nucleic acid probe via a C 6 -thiol linker. Such a nucleic acid probe is derived from a thiolated probe linked to horseradish-peroxidase with a sulfo-SMCC linker, or a linker equivalent thereto.
  • the invention herewith provides a method for linking a nucleic acid probe to horseradish peroxidase comprising mixing a thiol oligonucleotide with a SMCC-HRP complex, incubating said mixture and purifying it by ultrafiltration.
  • a labelled probe provided by the invention, the invention provides a probe molecule which is small enough to penetrate a cell wall when this barrier is properly and controllably degraded.
  • various cell wall degrading reagents and compositions are provided by the invention that allow the penetration of a HRP enzyme linked to an oligonucleotide by a C6-thiol linker into bacterial cells.
  • tyramide-fluorochro es are constructed that act as substrate for the HRP.
  • the resulting tyramide-fluorochrome radicals attach randomly in the cell whereby fluorescence is attained for very long periods (up to months) .
  • the methods and means provided by the invention allow for example the direct detection of specific bacterial species (through rRNA-probing) in environmental, clinical or industrial samples via microscopic observation, automated image analysis or flow cytometric methods. Also, potential enzymatic activity determined by the quantification of mRNA species can be measured in si tu in whole cells. Also, the method and means provided by the invention allow the detection of single genes within individual bacterial cells by using microscopic (image) analysis or flow cytometry.
  • the invention allows for the use of any fluorochrome (fluorescein, rhodamine, coumarine, etc.) that can be amino- esterified. This enables the use of multiple-colors, and thus the detection of various parameters simultaneously.
  • measuring mRNA in whole bacterial cells by in si tu hybridisation to detect expression of certain genes is cumbersome, when not using the methods and means provided by the invention.
  • the reported method uses large multiple- labelled transcript probes (some 200 nucleotides long) instead of the small synthetic oligonucleotide probes and is not of great commercial interest since the production of the large transcript-probe used is very tedious and cannot be performed on a routine basis.
  • ELFTM Molecular Probes, Eugene, USA
  • a fluorescent precipitate is formed that will not diffuse away.
  • this methodology cannot be used for bacteria since the necessary enzyme (alkaline phosphatase) is more than twice as large as peroxidase and cannot enter the bacterial cell wall without significant damage to cellular integrity and morphology.
  • the method linking HRP to nucleic acid probes as is provided by the invention can be used to label for example peptide nucleic acid (PNA) -probes as well as oligonucleotide-probes .
  • PNA peptide nucleic acid
  • the methods and means provided by the invention can be used to screen large amounts of samples for the presence of cells that harbour bacteriophage or virus .
  • the HRP-nucleic acid probe provided by the invention can also be used in other tests systems than in si tu assays alone.
  • Such a probe can for example be used in enzym-linked assays , such as a dip-stick but other formats are also possible, for the rapid detection of genes or gene-products in lysed bacterial samples.
  • the deposited reporter molecules can additionally serve as hapten for, e.g., anti- fluorescein-AP, antibodies via which an extra amplification step and the detection of very low numbers of target molecules is possible.
  • the methods and means provided by the invention find application for example as diagnostic kit in the broad field of microbiology.
  • a (non-extensive) list may include several possibilities, such as: To study the effect of novel foods (prebiotics) , probiotics, and functional foods on the intestinal microflora of both man and animal . To monitor the development of mixed bacterial populations that are essential for industrial fermentation processes in classical (cheese and other dairy fermentation, beer, wine, etc.) as well as in modern biotechnology (heterologous (over) expression of proteins, etc.). To rapidly detect microbial contamination in product or production tools from raw material to end product (hygiene control) . To identify and quantify the presence and activity of micro-organisms in wastewater treatment systems.
  • the permeabilization procedure allows for the first time the performance of in si tu hybridization experiments with all of the bacteria which contain rigid polysaccharide capsules, extensive peptidoglycan layers, extraordinary cross-linking moieties in their peptidoglycan layer and/or that can prevail in the form of impermeable spores. Cells need not be brought to the vegetative state prior to in si tu hybridization.
  • the invention allows for the first time the direct detection of non-growing cells or cells obtained directly from the natural environment by in si tu hybridization.
  • the present method ensures sufficient signal amplification as to yield fluorescence signals strong enough to allow the in si tu detection by fluorescence in situ hybridization of low copy numbers of rRNA molecules.
  • the present invention provides a methodology to detect, e.g., Mycobacterium tuberculosis directly in sputum samples or bacteria obtained from soil or bacteria obtained from watersamples by in situ hybridization.
  • Such cells normally contain only 50-100 ribosomes and can not be detected by conventional fluorescence in si tu hybridization.
  • the invention allows for the first time the direct detection of messenger RNA's by in si tu hybridization.
  • the present method ensures sufficient signal amplification as to yield fluorescence signals strong enough to allow the in si tu detection by fluorescence in situ hybridization of the naturally (very) low copy numbers of mRNA. Therefore, the present invention provides a methodology to detect, e.g., protease gene expression in Lactococcus lactis cells by in si tu hybridization.
  • Such cells normally contain only 50-100 mRNA molecules that can not be detected by conventional fluorescence in si tu hybridization.
  • the invention allows for the first time the direct detection of streptococci by in si tu hybridization.
  • the present method provides for a permeabilization formula which can permeabilize streptococcal cell-walls without the need of prior formaldehyde fixation.
  • the present invention thereby provides a methodology to detect, e.g., streptococci by in situ hybridization. Such cells can not be detected by conventional fluorescence in si tu hybridization techniques.
  • the invention allows for the first time the quantitative measurement of cells stained by in si tu hybridization on a flow cytometer.
  • the present method provides for a methodology with which cell lysis is prevented. Due to such a prevention, post-hybridization washing of cells is now effective (no background signal) and cells can be measured quantitatively by using methods such as flow cytometry.
  • the invention provides for the first time an optimized methodology of in si tu hybridization staining by which cells from natural samples, such as occur in feacal flora, can be measured on a flow cytometer.
  • the present method provides for a methodology in which the pH of the buffer is adjusted such that FISH-derived fluorescence is optimal . Due to such an optimization, post-hybridization fluorescence is high enough to ensure that cells can be measured quantitatively by using methods such as flow cytometry.
  • the invention describes a washing procedure that allows an effective post -hybridization washing of hybridized cells while maintaining sufficient cell density for flow cytometric measurement .
  • the present invention provides for a permeabilization formula that allows for the first time the performance of in si tu hybridization experiments with all of the bacteria which either contain rigid polysaccharide capsules, extensive peptidoglycan layers, extraordinary cross-linking moieties in their peptidoglycan layer and/or that can prevail in the form of impermeable spores and that breaks down all structural cell wall components in a controlled fashion.
  • One of the embodiments of the present invention is a permeabilization formula
  • another embodiment of the present invention is a signal amplification methodology which, when used in combination ensure i) the formation of pores or gaps in the cell wall of sufficient size as to facilitates the entering of bulky molecules such as horseradish peroxidase enzymes conjugated to oligonucleotides and ii) that facilitates the formation through such molecules of sequence-specific fluorescent precipitates thus allowing the microscopic or flow cytometric detection of cells stained by fluorescence in situ hybridization.
  • Example 1 Cell wall -degrading composition
  • Solution A 20 ml 48% (w.v) sucrose solution (9.6 g sucrose in 20 ml of Milli-Q water) ; sterilize by autoclaving;
  • Solution B 1 ml 1% (w/v) sodium taurocholate solution
  • Tris (hydroxymethyl) -aminomethane in 90 ml of Milli-Q water add cone. HCl to pH 7.5, add
  • Solution E 1 ml 20% (w/v) lysozym solution (200 mg lysozym in 1 ml of sterilized Milli-Q water) ;
  • Solution F 1 ml 1% (w/v) lipase (10 mg of lipase in 1 ml of sterilized Milli-Q water) ;
  • Solution G finizym, no preparations needed; purchase commercially from Novo Nordisk France;
  • Solution H 1 ml 10.000 U/ml mutanolysin (10.000 U mutanolysin in 1 ml of sterilized Milli-Q water) ;
  • LD-Bos cheese starter culture by mixing 1 ml of culture with 30 ml of 0.2% EDTA. Incubate for 30 min on a roller-bench. Pellet the cells by centrifugation for 10 min at 10.000 rpm. Wash the cell pellet in 15 ml of PBS (1000 ml of Milli-Q water, 8 g of NaCl , 0.2 g of KC1, 1.81 g Na 2 HP0 4 *2H 2 0 and 0.24 g of KH 2 P0 4 , pH 7.4) . Pellet the cells by centrifugation for 10 min at 10.000 rpm.
  • PBS 1000 ml of Milli-Q water, 8 g of NaCl , 0.2 g of KC1, 1.81 g Na 2 HP0 4 *2H 2 0 and 0.24 g of KH 2 P0 4 , pH 7.4
  • the thus treated cells of the LD-Bos cheese starter culture can be used for in situ hybridization with rRNA, mRNA or DNA-targeted deoxyribonucleic acid (DNA) oligonucleotide probes, ribonucleic acid (RNA) oligonucleotide probes, peptide nucleic acid (PNA) probes that are for their subsequent detection labelled with reporter molecules such as:
  • fluorescent labels e.g., fluorescein, rhodamine, methylcoumarine , tertramethylrhodamine ,
  • enzymatic labels e.g., oxidases, peroxidases, phosphatases, luciferases, dehydrogenases, hexokinases or papain
  • - proteinaceous labels e.g., streptavidin, antibodies, Fab fragments or aequorin
  • chemiluminescent labels e.g., acridinium esters, isoluminol, luminol or ruthenium tris [bipyridyl] )
  • hapten e.g., biotin, digoxigenin, ethidium, glucosyl, sulfone, either
  • the thus treated cells are permeable to large biomolecules and the procedure can be used to introduce large (bio) molecules of any kind in the bacterial cell.
  • the procedure can be shortened by combining separate successive steps, e.g., (para) formaldehyde fixation can be combined with ethanol dehydration in one step.
  • the (bio) chemicals in the protocol can be substituted for any (bio) chemical with comparable activity, e.g., ethanol can be replaced by methanol or propanol and the enzymes can be changed for enzymes with comparable biological catalytic activity provided that the environment of the reaction is modified according to routine skills to suit the new (bio) chemistry.
  • any (bio) chemical with comparable activity e.g., ethanol can be replaced by methanol or propanol
  • the enzymes can be changed for enzymes with comparable biological catalytic activity provided that the environment of the reaction is modified according to routine skills to suit the new (bio) chemistry.
  • Step 1 De-protection of commercially derived 5 ' - thiol labelled oligonucleotides :
  • Deoxyoligonucleotides (10-30 mers) with a protected thiol (SH) group attached to the 5-prime end via a C-6 spacer arm were commercially derived (Eurogentec, Seraing, Belgium) .
  • the thiol -groups were de-protected by incubating the oligonucleotide (2 mg/ml) in DP buffer (50 M (Na) phosphate, pH 7.4; 5 mM EDTA; 100 M NaCl; 50 mM hydroxylamine-HCl) for 30 min at 30°C.
  • Step 2 Linking of the horseradish peroxidase enzyme to a sulfo-SMCC linker:
  • Solution 1 50 mg/ml of sulfo-SMCC (sulfosuccinimidyl 4- [N-maleimidomethyl ] -cyclohexane-1- carboxylate; Pierce, Rockford, IL, USA) in DMSO (dimethylsulfoxide) .
  • Solution 2 10 mg/ml of enzyme (HRP; Sigma Chemical
  • PEN buffer pH 8.0 (50 mM (Na) phosphate, pH 8.0; 5 mM EDTA; 100 mM NaCl) .
  • a volume of 10 ⁇ l of freshly prepared solution 1 was mixed with 200 ⁇ l of solution 2 and the mixture was incubated at 30°C for 1 h.
  • 790 ⁇ l of PEN buffer, pH 6.7 (50 mM (Na) phosphate, pH 6.7; 5 mM EDTA; 100 mM NaCl) was added and the SMCC-HRP complex was purified via ultrafiltration using Centricon 30 (Amicon, Beverly, MA, USA) .
  • the complex was washed 3 times with PEN buffer, pH 6.7 in Centricon 30 ultrafilters and the supernatant of the final wash was made up to 200 ⁇ l with PEN buffer, pH 6.7.
  • a volume of 67 ⁇ l of the deprotected thiol oligonucleotide (step 1) was mixed with 200 ⁇ l of the purified SMCC-HRP complex (step 2) and the mixture was incubated at 30°C for 1.5 h.
  • the cross-linked enzyme/oligonucleotide complex was purified via ultrafiltration using Centricon 30 ultrafilters. The complex was washed 3 times with PEN buffer, pH 6.7 in Centricon 30 ultrafilters and the supernatant of the final wash was made up to 67 ⁇ l with PEN buffer, pH 6.7.
  • the enzyme-labelled probes were stored at 4°C. For hybridization, the probe-solution was diluted 10 to 100 times in PEN buffer, pH 6.7.
  • the (bio) chemicals in the protocol can be substituted for any (bio) chemical with comparable activity, e.g., the cross-linker sulfo-SMCC can be replaced by any cross- linker (such as sulfo-GMBS or DTBP) provided that the environment of the reaction is appropriately modified to suit the new chemistry.
  • the cross-linker sulfo-SMCC can be replaced by any cross- linker (such as sulfo-GMBS or DTBP) provided that the environment of the reaction is appropriately modified to suit the new chemistry.
  • linkers are succinimidyl- , salicylamido- or hydrazide- derivatives that contain at least two reactive sites that are homobifunctional or heterobifunctional reactive towards (-NH2) amines [e.g., 1, 5-Difluoro-2,4-dinitrobenzene (DFDNB) , Dimethyl adipimidate 2HC1 (DMA) ; or Succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (SMCC) ] (-SH) sulfhydryls
  • ASBA 4- [p-Azidosalicylamido] butylamine
  • EDC l-Ethyl-3- [3-dimethylaminopropyl] -carbodiimide Hydrochloride
  • reaction product A enzyme-labeled oligonucleotides
  • reaction product B un-reacted enzyme
  • reaction product C enzyme with a SMCC moiety attached to it
  • the present invention therefore represents an important improvement of the art.
  • the proposed method for preparing the HRP-labeled oligonucleotides is therefore one of the embodiments of the present invention.
  • Example 3 HRP-Labelling and use of Fluoresceine-Tyramide in whole cell fluorescence in situ hybridization with Lactococcus lactis cells.
  • Fluoresceine-Tyramide (FT) substrate Tyramine (Sigma) was linked to Fluoresceine-NHS (Boehringer) by mixing 15 mg of fluoresceine-NHS in 200 ⁇ l DMSO with 150 ⁇ l tyramine (20 mg/ml H 2 0) and using a 4 h incubation at
  • Cells of Lactococcus lactis subsp. cremoris were grown in overnight culture. Cells from two ml of culture were washed in PBS and resuspended in cell wall degrading reagens (50 mM Tris-HCl, pH 7.0; 0.03% (w/v) Na-taurocholate ; 5 mM CaCl 2 , 20% (w/v) sucrose; 0.5% (w/v) lysozym; 0.003% (w/v) pancreatic lipase; 1% v/v finizym.
  • cell wall degrading reagens 50 mM Tris-HCl, pH 7.0; 0.03% (w/v) Na-taurocholate ; 5 mM CaCl 2 , 20% (w/v) sucrose; 0.5% (w/v) lysozym; 0.003% (w/v) pancreatic lipase; 1% v/v finizym.
  • Hybri di sat ion Hybridisation was carried out by mixing 10 ⁇ l of the cell suspension in ethanol with 90 ⁇ l of hybridisation solution
  • washing solution 100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% v/v Tween 20
  • cells were either used immediately for flow cytometric analysis or were filtered onto a 0.2 mm pore-size IsoporeTM polycarbonate membrane filter (Millipore Corporation, U.S.A.) for epifluorescence microcopic observation. Filters were mounted on microscope slides with VectashieldTM. Hybridised cells were viewed under blue light excitation using an Olympus BX40 fluorescence microscope using epi-illumination.
  • a probe complementary to a universally conserved 16S rRNA bacterial sequence (Eub338) labelled with HRP served as a positive control, whereas a probe labelled with HRP and complementary to the sequence of probe Eub 338 (i.e. probe NonEub338) served as a negative control.
  • Hybridization occurred at 37° C for 16 h. After hybridization, slides were washed in hybridization buffer for 20 min at 37°C and mounted in Vectashield (Vector Labs, Ca, USA)
  • Example 5 Measurement of activity of single cells via total mRNA quantification by using a poly-T oligonucleotide probe. The activity of individual cells was measured by whole cell in situ hybridization of Escherichia coli strain JM101 .
  • Hybri disati on Hybridisation was carried out by mixing 10 ⁇ l of the ethanolic fixed cell suspensions with 90 ⁇ l of hybridisation solution (20 mM Tris-HCl, pH 7.2; 0.9 M NaCl; 0.1% SDS; 0.25 ng/ml FITC-labelled oligonucleotide probe) . Cells were hybridized for 16 h at 45 °C with a FITC-labelled poly-T oligonucleotide probe (20-mer, 5 ' -FITC) , which was commercially derived.
  • hybridisation buffer hybridisation solution without probe
  • mixture A see permeabilization formula of example 1
  • mixture B 60 ⁇ l pronase 100 U/ml , 40 ⁇ l proteinase K 20 mg/ml
  • a volume of 30 microlitres of the permeabilized cell suspension is mixed with 1 ml of PBS (see example 1) and cells are pelleted by centrifugation at 14.000 rpm. The pellet is resuspended in 400 ⁇ l of PBS/Ethanol (1:1) and dehydrated at -20°C for 1 hr.
  • Cells are pelleted by centrifugation at 14.000 rpm, the pellet is resuspended in 200 ⁇ l of hybridisation mixture (see example 5) and cells are hybridized overnight. A volume of 5 microlitres of the cell suspension is filtered over a 0.2 ⁇ m polycarbonate filter, washed with hybridization buffer and viewed under the microscope using the appropriate filtersets. Fluorescence of cells is scored visually.
  • Example 7 Conventional FISH versus FISH with HRP-labeled oligonucleotide probes in the flow cytometric measurements of cells stained by 16S rRNA-FISH.
  • Faecal, bacterial samples are prepared by suspending 0.5 grams of fresh faecal material in 4.5 ml of PBS by using a dozen sterile glass beads (3 mm diameter) and a vortex. The suspension is centrifuged at low speed (1,000 rpm) for 1 min and the supernatant - which contains the cells - is transferred to a clean microcentrifuge tube. The cell suspension is then fixed in 4% p-formaldehyde at 4°C for 4 hrs. Cells are washed twice in PBS and resuspended in a mixture of PBS/Ethanol or another organic solvent (1:1) for dehydration and/or partial degradation. Cells are dehydrated for at least 1 hr at -20°C.
  • Dehydrated cells are suspended one in ten in hybridization buffer and hybridized as described above without permeabilization. Hybridization was performed with probes NonEub, Eub, Bif and Uni labeled with FITC and NonEub, Eub and Bif labeled with Cy5. Probe concentrations of 2 ng/ ⁇ l were used. Washing was performed both in hybridization buffer from which SDS was ommitted and in ice-cold PBS. In the case of PBS washings, a volume of 1 ml was added to 500 ⁇ l of hybridized cells. The cell suspension was incubated on ice for 5 min and centrifuged at 14,000 rpm for 5 min.
  • the resulting cell pellet was resuspended in 35 ⁇ l of ice-cold PBS and stored on ice. Just prior to flow cytomerty, 5 ⁇ l of cell suspension was diluted into 500 ⁇ l of ice-cold PBS and cellular fluorescence was acquired by flow cytometry. In the case of hybridization buffer washings, a volume of 1 ml of pre-warmed (45°C) hybridization buffer without SDS was added to the hybridized cells and incubated for 30 min at 45°C. Cells were pelleted by centrifugation at 14,000 rpm for 5 min and resuspended in 1 ml of pre-warmed (45 °C) hybridization buffer. The results of the FISH procedure are displayed in table 5 and in figs. 4 and 5.
  • the cell pellet was resuspended in 100 ⁇ l of hybridization mixture consisting of 10 ⁇ l of non-purified HRP-labeled oligonucleotide (1 in 10 diluted in PEN buffer, see example 2) , 8 ⁇ l of 6M ureum and 82 ⁇ l of hybridization buffer as used above, and incubated for 16 hrs at 37°C. After hybridization, cells were washed for 5 min in 1 ml of hybridization buffer with 0.48 M ureum and pelleted.
  • Figs. 6 demonstrates the shift in fluorescence upon labeling with HRP probes in FISH in graphical display.
  • the intensity of HRP labeling is comparable to the staining with the nucleic acid stain YOPRO-1 at a dilution of 40x (see fig 5, panel B) .
  • These types of intensities are required for flow cytometric detection of FISH in bacterial cells, especially in cells that exist in a low level metabolic state, i.e. in which conventional FISH results in fluorescence levels too low for convenient detection such as soil bacteria or bacteria in natural water bodies.
  • Example 8 Detection of nisin mRNA gene-transcripts in Lactococcus lactis.
  • Example 4 the procedure used in example 4 is used to detect messenger RNA's in whole cells of Lactococcus lactis for the antimicrobial peptide nisin.
  • cells of strain 161-5 of nisin producing Lactococcus lactis were subjected to nisin-inducing growth conditions. Samples were withdrawn every hour for 8 hrs and the cells were treated and hybridized with a HRP-labeled oligonucleotide specific for nisin-mRNA as described in example 4.
  • a non-producing strain (strain 1614) served as a negative control in this experiment. Fluorescence was monitored visually and photographs were taken for later verification.
  • Example 9 Detection of Lactobacillus helveticus by 16S rRNA-targeted fluorescence in si tu hybridization.
  • the cells are dehydrated in a graded ethanol series (50%, 70% and 100% of ethanol) by submersing the entire slides in the various ethanol solutions for 2 min.
  • the slides are dried to air and a volume of 50 ⁇ l of hybridization mixture with FITC labeled probes is applied to the slides.
  • the entire slide is covered by a large cover glass, and incubated in a hybridization chamber for a period of 4 hrs.
  • Upon hybridization, slides are washed in hybridization buffer at 45 °C for 5 min, dried in air and mounted for microscopic examination.
  • NAM N-acetylmuramic sugar / structura acids
  • the pH of the measuring buffer was 7.4 in these experiments.
  • FIG. 1 Illumination series demonstrating the enormous (80x) increase in fluorescence emission from HRP-labelled probe (photograph D: 0.5 sec illumination) compared to conventional FITC-labelled oligonucleotide probes (photographs A, B and C: 0.5, 4 and 10 sec illumination, respectively) .
  • Hybridizations were performed on cheese starter-culture LD-Bos.
  • the HRP-labelled probe used was targeted to the 23S rRNA of Lactococcus lactis subspecies cremoris cells (over 50%) .
  • the FITC-labelled probe was targeted to the 16S rRNA of all bacterial cells present in the sample.
  • the protocol for in situ hybridization with HRP- labelled probes of example 1 was applied.
  • the average fluorescence of cells in photograph D was 181, that of photograph C was 53 (in AU) as determined by image cytometry.
  • Figure 3 Measurement of mRNA via an FITC-labelled poly-T oligonucleotide probe in individual Escherichia coli cells by flow cytometry.
  • Figure 4 Flow cytometer cytograms representing the measurements of human intestinal (faecal) flora with Bifidobacterium genus- specific 16S rRNA targeted in si tu hybridization probes labeled at the 5 ' end with FITC (panel A) and Cy5 (panel B) . Fluorescence intensities (y-axis) are plotted versus Forward Scattering Signal (x-axis) . With both labels, a distinct group of cells with higher fluorescence than the body of the population can be distinguished. Pthe pH of the measuring medium was 10.4 for FITC and 7.5 for Cy5 with TOPRO-3 and YOPRO-1 as the DNA counter stains for FITC and Cy5, respectively.
  • Panel B top: The YOPRO-1 fluorescence (x-axis) versus the cell counts-. YOPRO-1 was applied as a 4Ox diluted commercial preparation. Panel B, bottom; The Cy5 fluorescence derived from these cells (x-axis) versus the cell counts. Panel C, top: The Cyto- 9 fluorescence (x-axis) versus the cell counts. Panel C, bottom; The Cy5 fluorescence derived from these cells (x-axis) versus the cell counts.
  • Panel B 5' end with HRP (panel B) .
  • No nucleic acid-counterstains were used.
  • Panel B, top The FITC fluorescence derived from HRP-labeled probe LLC used in combination with FT-substrate (x-axis) versus the cell counts.
  • Nisin mRNA detection in Lactococcus lactis Panel A: Non nisin-producing strain 1614 after 3 hrs of growth under nisin-inducing conditions. Panel B: Nisin-producing strain 161-5 at various times during 6 hrs of growth under nisin- inducing conditions. See text for details.

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