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EP1472370A1 - Procede de detection specifique rapide de bacteries pathogenes presentes dans des denrees alimentaires - Google Patents

Procede de detection specifique rapide de bacteries pathogenes presentes dans des denrees alimentaires

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Publication number
EP1472370A1
EP1472370A1 EP03704530A EP03704530A EP1472370A1 EP 1472370 A1 EP1472370 A1 EP 1472370A1 EP 03704530 A EP03704530 A EP 03704530A EP 03704530 A EP03704530 A EP 03704530A EP 1472370 A1 EP1472370 A1 EP 1472370A1
Authority
EP
European Patent Office
Prior art keywords
seq
bacteria
species
oligonucleotide
tct
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03704530A
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German (de)
English (en)
Inventor
Jiri Snaidr
Claudia Beimfohr
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vermicon AG
Original Assignee
Vermicon AG
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Filing date
Publication date
Application filed by Vermicon AG filed Critical Vermicon AG
Publication of EP1472370A1 publication Critical patent/EP1472370A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a method for the detection of pathogenic food-relevant bacteria, in particular a method for the simultaneous specific detection of bacteria of the genus Listeria and the species Listeria monocytogenes by in situ hybridization and a method for the specific detection of bacteria of the species Staphylococcus aureus by in situ hybridization and a method for the specific detection of bacteria of the genus Campylobacter and the species C. coli and C. jejuni by in situ hybridization, as well as corresponding oligonucleotide probes and kits with which the methods according to the invention can be carried out.
  • Listeria are gram-positive, short, movable sticks.
  • the genus Listeria (L.) includes six species: L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L. welshimeri.
  • L. grayi L. grayi
  • L. innocua L. innocua
  • L. ivanovii L. monocytogenes
  • L. seeligeri L. welshimeri.
  • the worldwide distribution of these ubiquitous bacteria extends to aquatic areas as well as to the soil and vegetation.
  • listeriosis which has a very variable incubation period of a few days to two months, is caused by the species L. monocytogenes, although L. ivanovii, L. seeligeri and L. welshimeri have also been detected in some diseases.
  • Listeria infection can manifest itself in serious clinical pictures such as sepsis, meningitis or encephalitis. Especially in newborns who can be infected via the placenta or during birth, and in the elderly, there is a high health risk in the case of listeriosis. The mortality rate in the case of newborn listeriosis is up to 50%.
  • the fetus may also abort.
  • the occurrence of listeriosis in elderly or otherwise defenseless people can be fatal in up to 30% of those affected.
  • the transmission usually takes place through the consumption of contaminated food.
  • Dairy products in particular are a common source of infection. But almost all other foods are potential sources of Listeria infections. So in the past, in addition to milk and various
  • Dairy products such as cheese, butter or ice cream also identify other foods as the source of listeriosis. These include various products such as coleslaw, mussels, pork, chicken, fish, corn flour or rice salad. In many cases, outbreaks of listeriosis caused by consumption of the foods mentioned have been accompanied by deaths.
  • Listeria are still capable of reproduction at 4 ° C (in milk even at -0.3 ° C). This means that despite refrigerated storage of food, listeria can multiply and accumulate in it. Even after cooking, roasting or smoking, listeria can accumulate in the food in question as a result of inadequate treatment or secondary contamination.
  • Staphylococcal intoxications are among the most common bacterial and foodborne diseases worldwide. These are triggered in particular by strains of Staphylococcus (S.) aureus.
  • S. aureus is a gram-positive, immobile coagulase-positive bacterium and occurs on the skin, the mucous membrane of the nasopharynx, in stool, feces, abscesses and pustules.
  • S. aureus is also widespread in the healthy population: Half of all healthy people have S. aureus in the nasopharynx.
  • S. aureus is usually also transmitted through the consumption of contaminated food, although the spectrum of possible sources of infection is quite broad.
  • the following foods were involved in diseases: ready-made meat dishes, pies, cooked ham, raw ham, milk and milk products, egg-containing preparations, salads, creams, cake fillings, ice cream, pasta. Routine detection nowadays mostly takes place via the culture and confirmation tests of suspect colonies, since the detection of enterotoxins is quite difficult to carry out. If culture is detected, the sample to be examined is first incubated for 48 h on a suitable selective medium (eg Baird) at 37 ° C. If this first cultivation step was carried out in liquid medium, a second one closes
  • a suitable selective medium eg Baird
  • Campylobacter Only for over 20 years has a previously underestimated germ played a greater role than food poisoner: Campylobacter. Unlike salmonella, for example, it rarely reproduces in food, but a few hundred bacterial cells are sufficient for infection with this pathogen.
  • Campylobacter (C.) genus comprises 20 species and subspecies. These bacteria, which have so far been difficult to cultivate, are gram-negative, slim, curved to spirally wound rods that require microaerophilic conditions for their growth.
  • the species C. jejuni, C. coli and C. laris are medically relevant. They colonize the small and large intestines and cause acute gastroenteritis accompanied by the following symptoms: diarrhea, abdominal pain, fever, nausea, vomiting. These symptoms are difficult to distinguish from those of a stomach ulcer. A careful differential diagnosis is therefore essential. Routine detection is currently carried out via multi-stage cultivation, starting with an 18-hour enrichment in selective liquid medium (Campylobacter-selective medium according to Preston), followed by twice 48 h on two different fixed media (Karmali agar followed by Columbia blood agar). These five-day cultivations are followed by biochemical or serological identification.
  • nucleic acid-based detection methods are therefore available.
  • PCR the polymerase chain reaction
  • a characteristic piece of the respective bacterial genome is amplified with specific primers. If the primer finds its destination, a piece of the genetic material multiplies millions of times.
  • a qualitative assessment can take place. In the simplest case, this leads to the statement that the target sites for the primers used were present in the examined sample. No further statements are possible; these target sites can originate from a living bacterium as well as from a dead bacterium or from naked DNA. A differentiation is not possible here. Since the PCR reaction is positive even in the presence of a dead bacterium or naked DNA, false positive results often occur.
  • a further development of this technique is quantitative PCR, in which an attempt is made to establish a correlation between the amount of bacteria present and the amount of amplified DNA.
  • the advantages of PCR lie in its high specificity, ease of use and in a short amount of time.
  • Significant disadvantages are their high susceptibility to contamination and thus false positive results as well as the already mentioned lack Possibility to differentiate between living and dead cells or naked DNA.
  • FISH fluorescence in situ hybridization method
  • the FISH technique is based on the fact that there are certain molecules in bacterial cells that, due to their vital function, have undergone only little mutation in the course of evolution: the 16S and the 23S ribosomal ribonucleic acid (rRNA). Both are components of the ribosomes, the sites of protein biosynthesis, and due to their ubiquitous distribution, their size, and their structural and functional constancy, they can serve as specific markers (Woese, CR, 1987. Bacterial evolution. Microbiol. Rev. 51, p. 221 -271). Based on a comparative sequence analysis, phylogenetic relationships can be established based solely on this data. To do this, this sequence data must be aligned. In the alignment, which is based on knowledge of the secondary structure and tertiary structure of these macromolecules, the homologous positions of the ribosomal nucleic acids are reconciled.
  • rRNA ribosomal ribonucleic acid
  • phylogenetic calculations can be carried out.
  • the use of the latest computer technology makes it possible to carry out large-scale calculations quickly and effectively, and to create large databases that contain the alignment sequences of the 16S rRNA and 23S rRNA. Thanks to the quick access to this data material newly obtained sequences can be analyzed phylogenetically in a short time.
  • These rRNA databases can be used to construct species- and genus-specific gene probes. Here, all available rRNA sequences are compared with each other and probes designed for specific sequence sites that specifically detect a bacterial species, genus or group.
  • these gene probes which are complementary to a specific region on the ribosomal target sequence, are introduced into the cell.
  • the gene probes are usually small, 16-20 base long, single-stranded deoxyribonucleic acid pieces and are directed against a target region, which is typical for a type or group of bacteria. If the fluorescence-labeled gene probe finds its target sequence in a bacterial cell, it binds to it and the cells can be detected in the fluorescence microscope due to their fluorescence.
  • the FISH analysis is fundamentally carried out on a slide, and the evaluation visualizes the bacteria by irradiating them with high-energy light, that is, makes them visible.
  • This is one of the disadvantages of the classic FISH analysis: since only relatively small volumes can naturally be analyzed on a slide, the sensitivity of the method can be unsatisfactory and not sufficient for a reliable analysis.
  • the present invention therefore combines the advantages of classic FISH analysis with those of cultivation.
  • a comparatively short cultivation step ensures that the bacteria to be detected are present in sufficient numbers before the bacteria are detected using specific FISH.
  • cultivation is understood to mean the multiplication of the bacteria contained in the sample in a suitable cultivation medium.
  • the cultivation can be carried out, for example, in V_ Fraser broth for 24 h at 30 ° C.
  • S Aureus can be cultivated, for example, as a blood culture (for example BACTEC 9240, Becton Dickinson Instruments) for 8 h to 48 h at 35 ° C.
  • cultivation can be carried out, for example, in Preston selective medium for 24 h at 42 ° C.
  • the person skilled in the art can in any case find the suitable cultivation methods from the prior art.
  • fixing the bacteria is understood to mean a treatment with which the bacterial envelope is made permeable to nucleic acid probes. Ethanol is usually used for fixing. If the cell wall cannot be penetrated by the nucleic acid probes with these measures, the person skilled in the art will know Sufficient other measures are known which lead to the same result, for example methanol, mixtures of alcohols, a low-percentage paraformaldehyde solution or a dilute formaldehyde solution, enzymatic treatments or the like.
  • the fixed bacteria are incubated for the “hybridization” with fluorescence-labeled nucleic acid probes.
  • nucleic acid probes which consist of an oligonucleotide and a marker attached to it, can then penetrate the cell envelope and adhere to the target sequence corresponding to the nucleic acid probe inside the cell
  • the binding is to be understood as the formation of hydrogen bonds between complementary pieces of nucleic acid.
  • the nucleic acid probe can be complementary to a chromosomal or episomal DNA, but also to an mRNA or rRNA of the microorganism to be detected. It is advantageous to choose a nucleic acid probe that is complementary to an area that is present in the number of copies of more than 1 in the microorganism to be detected.
  • the sequence to be detected is preferably 500-100,000 times per cell, particularly preferably 1,000-50,000 times.
  • the rRNA is preferably used as the target site, since the ribosomes in the cell as sites of protein biosynthesis are present thousands of times in each active cell.
  • the nucleic acid probe in the sense of the invention is a DNA or RNA probe, which will generally comprise between 12 and 1000 nucleotides, preferably between 12 and 500, more preferably between 12 and 200, particularly preferably between 12 and 50 and between 15 and 40, and most preferably between 17 and 25 nucleotides.
  • the nucleic acid probes are selected on the basis of whether a complementary sequence is present in the microorganism to be detected. By selecting a defined sequence, a bacterial species, a bacterial genus or an entire bacterial group can be recorded. Complementarity should exist for a probe of 15 nucleotides over 100% of the sequence. With oligonucleotides with more than 15 nucleotides, one or more mismatching sites are allowed.
  • the nucleic acid probe molecules according to the invention have the following lengths and sequences:
  • the detection method for Listeria and L. monocytogenes is carried out, for example, as follows:
  • the oligonucleotide SEQ ID No. 1 is specifically labeled, for example with a green fluorescent dye, and is used for the specific detection of all bacteria of the genus Listeria.
  • the oligonucleotide SEQ ID No. 2 remains unlabeled and, as a competitor, prevents the labeled oligonucleotide SEQ ID No. 1 from binding to bacteria that do not belong to the Listeria genus.
  • the oligonucleotide of SEQ ID No. 3 is also specific, but unlike the oligonucleotide SEQ ID No.
  • the ohgonucleotides SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 again remain unlabeled and, as competitors, prevent the labeled oligonucleotide SEQ ID No. 3 from binding to bacteria that do not belong to the species L. monocytogenes. In this way, the simultaneous and highly specific detection of bacteria possible belonging to the genus Listeria or to the species L. monocytogenes.
  • the different markers for example a green fluorescent dye on the one hand and a red fluorescent dye on the other hand, are easily distinguishable from one another, for example by using different filters in fluorescence microscopy.
  • nucleic acid probe molecules according to the invention have the following lengths and sequences:
  • the detection method for S. aureus is carried out as follows, for example:
  • the ohgonucleotides SEQ ID No. 7 and SEQ ID No. 10 are specifically labeled, for example with a red fluorescent dye, and are used for the specific detection of all bacteria of the species Staphylococcus aureus.
  • the ohgonucleotides SEQ ID No. 8 and 9 and SEQ ID No. 11, 12 and 13, on the other hand, remain unmarked and prevent competitors from binding the labeled ohgonucleotides to bacteria that do not belong to the species S. aureus. In this way, highly specific detection of bacteria belonging to the species S. aureus is possible.
  • the intensity of the signals obtained can be increased by using so-called "helper probes".
  • helper probes are unlabeled ohgonucleotides that have the following sequence:
  • helper probes are not mandatory, but optional.
  • the helper probes facilitate the binding of the marked probes to their target sites and thus improve the signal intensity. The detection method works very well even without these helper probes.
  • nucleic acid probe molecules according to the invention have the following lengths and sequences:
  • the detection method for the genus Campylobacter or the species C. coli and C. jejuni is carried out as follows:
  • the ohgonucleotides SEQ ID No. 16 to SEQ ID No. 19 and the ohgonucleotides SEQ ID No. 24 to SEQ ID No. 28 and the oligonucleotide SEQ ID No. 30 and the ohgonucleotides SEQ ID No. 32 to 34 are specifically labeled, for example with a green fluorescent dye, and are used for the specific detection of all bacteria of the genus Campylobacter.
  • the ohgonucleotides SEQ ID No. 20 to 23 and the oligonucleotide SEQ ID No. 29 and the oligonucleotide SEQ ID No. 31 remain unlabeled and prevent as
  • Competitors bind the abovementioned labeled ohgonucleotides specific for the genus Campylobacter to bacteria which do not belong to the genus Campylobacter.
  • the ohgonucleotides SEQ ID Nos. 35 and 36 and the oligonucleotide SEQ ID No. 39 are also specific, but different from the ohgonucleotides SEQ ID Nos. 16 to 19, 24 to 28, 30 and 32 to 34, i.e. distinguishable from them, e.g. marked with a blue fluorescent dye and are used for the specific detection of all bacteria of the species Campylobacter coli.
  • the ohgonucleotides SEQ ID No. 37, 38 and 40 to 42 again remain unlabeled and, as competitors, prevent the labeled, for C. coli specific, ohgonucleotides from binding to bacteria which do not belong to the C. coli species.
  • the ohgonucleotides SEQ ID Nos. 43 and 48 are also specific, but again different from the aforementioned ohgonucleotides, that is to say distinguishable from them again, for example with a red fluorescent dye, and are used for the specific detection of all bacteria of the species Campylobacter jejuni.
  • the ohgonucleotides SEQ ID Nos. 44 to 47 and 49 in turn remain unlabeled and, as competitors, prevent the labeled ohgonucleotides specific for C. jejuni from binding to bacteria which do not belong to the C. jejuni species. In this way, the simultaneous and highly specific detection of bacteria belonging to the genus Campylobacter or to the species C. coli or C. jejuni is possible.
  • the intensity of the signals received can optionally be increased by using so-called helper probes.
  • the helper probes are also unlabeled, but make it easier to bind the labeled probes to their target locations and thus improve the signal intensity. This is only an amplification of the signal intensity, the detection procedure naturally also works without these helper probes.
  • the intensity of the signals obtained with the oligonucleotide SEQ ID No. 24 can be increased by using the following unlabelled ohgonucleotides as helper probes:
  • Competitors in the context of the present invention are understood to mean, in particular, ohgonucleotides which have a higher specificity for genus or species not to be detected than the marked ohgonucleotides which are specific for the genus or species to be detected.
  • the invention also relates to modifications of the above oligonucleotide sequences which, despite the deviations in the sequence and / or length, result in a specific hybridization with target nucleic acid sequences of the particular Show bacterium and are therefore suitable for use in a method according to the invention.
  • sequence region of the nucleic acid molecule in at least 60%, 65%, preferably in at least 70%, 75%, more preferably in at least 80 %, 84%, 87% and particularly preferably in at least 90%, 94%, 96%, of the bases agree (whereby the sequence region of the nucleic acid molecule is to be considered which corresponds to the sequence region of one of the above-mentioned ohgonucleotides (SEQ ID No. 1 to SEQ ID No. 51) corresponds to, and not approximately the entire sequence of a nucleic acid molecule which may be in the
  • Hybridization techniques known hybridization conditions bind only the ribosomal RNA of the target organisms, but not the rRNA of non-target organisms to the oligonucleotide.
  • SEQ ID NO. 51 is complementary, hybridize under stringent conditions.
  • Nuclemic acid molecules that have an ohgonucleotide sequence of SEQ ID No. 1 to SEQ ID No. 51 or the sequence of a nucleic acid molecule according to a) or b) and in addition to the sequences mentioned or their Modifications according to a) or b) have at least one further nucleotide and enable specific hybridization with nucleic acid sequences of target organisms.
  • the degree of sequence identity of a nucleic acid molecule with the probes SEQ ID No. 1 to SEQ ID No. 51 can be determined using conventional algorithms.
  • the program for determining the sequence identity is suitable here, which is available at http://www.ncbi.nlm.nih.gov/BLAST (on this page e.g. the link "Standard nucleotide-nucleotide BLAST [blastn]").
  • the specific oligonucleotide probes preferably correspond to the oligonucleotides SEQ ID No. 1 or SEQ ID No. 3.
  • modifications are also possible, as long as a specific hybridization between the probes is nevertheless possible and target sequence takes place. It may be sufficient that the oligonucleotide probe used in 15, preferably 16 and 17 and particularly preferably 18 and 19 successive nucleotides matches SEQ ID No. 1 or SEQ ID No. 3. The same applies to the ogonucleotides serving as competitors with regard to the sequences SEQ ID No. 2, 4, 5 and 6.
  • the specific oligonucleotide probes preferably have a sequence which in 13 and 14 and preferably 15, 16 or 17 consecutive nucleotides corresponds to those of SEQ ID No. 7 or 10.
  • the specific oligonucleotide probes preferably have a sequence which is in 13 or 14, preferably 15 or 16 and particularly preferably 17 or 18 consecutive Nucleotides with SEQ ID Nos. 16-19, 24-28, 30, 32-36, 39, 43 and 48.
  • the nucleic acid probe molecules according to the invention can be used with various hybridization solutions as part of the detection method.
  • Various organic solvents can be used in concentrations of 0 - 80%.
  • Compliance with stringent hybridization conditions ensures that the nucleic acid probe molecule actually hybridizes with the target sequence.
  • Moderate conditions in the sense of the invention are e.g. 0% formamide in a hybridization buffer as described below.
  • Stringent conditions in the sense of the invention are, for example, 20-80% formamide in the hybridization buffer.
  • a typical hybridization solution contains 0% - 80% formamide, preferably 20% - 60% formamide, particularly preferably 40% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0.5 mol / 1 - 1.0 mol / 1, more preferably 0.7 mol / 1 - 0.9 mol / 1 , particularly preferably of 0.9 mol / 1, the salt preferably being sodium chloride.
  • the hybridization solution usually comprises a detergent, such as sodium dodecyl sulfate (SDS), in a concentration of 0.001% - 0.2%, preferably in a concentration of 0.005 - 0.05%, more preferably 0.01 - 0.03%, particularly preferably in a concentration of 0.01%.
  • a detergent such as sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various compounds such as Tris-HCl, sodium citrate, PIPES or HEPES can be used to buffer the hybridization solution, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.05 mol / 1, in a pH range of 6.0-9.0, preferably 7.0 to 8.0.
  • the particularly preferred invention the execution of the hybridization solution contains 0.02 mol / 1 Tris-HCl, pH 8.0.
  • a typical hybridization solution contains 0% -80% formamide, preferably 20% -60% formamide, particularly preferably 20% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0.7 mol / 1 - 0.9 mol / 1, particularly preferably 0.9 mol / 1, where it is the salt is preferably sodium chloride. Furthermore, the hybridization solution usually comprises a detergent, such as e.g.
  • Various compounds such as Tris-HCl, sodium citrate, PIPES or HEPES can be used to buffer the hybridization solution, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.05 mol / 1, in a pH range of 6.0-9.0, preferably 7.0 to 8.0.
  • the particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / 1 Tris-HCl, pH 8.0.
  • a typical hybridization solution contains 0% -80% formamide, preferably 20% -60% formamide, particularly preferably 20% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0.7 mol / 1 - 0.9 mol / 1, particularly preferably 0.9 mol / 1, where it is the salt preferably around
  • the hybridization solution usually comprises a detergent, such as sodium dodecyl sulfate (SDS), in a concentration of 0.001% - 0.2%, preferably in a concentration of 0.005-0.05%, more preferably 0.01-0.03%, particularly preferably in a concentration of 0.01%.
  • a detergent such as sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various compounds such as tris HC1, sodium citrate, PIPES or HEPES are used, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.05, in a pH range of 6, 0 - 9.0, preferably 7.0 to 8.0.
  • the particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / 1 Tris-HCl, pH 8.0.
  • the person skilled in the art can select the stated concentrations of the constituents of the hybridization buffer in such a way that the desired stringency of the hybridization reaction is achieved.
  • Particularly preferred embodiments reflect stringent to particularly stringent hybridization conditions. Using these stringent conditions, the person skilled in the art can determine whether a specific nucleic acid molecule enables specific detection of nucleic acid sequences of target organisms and thus was used reliably in the context of the invention. The person skilled in the art is able, by changing the parameters of the hybridization buffer, to increase or decrease the stringency if necessary or depending on the probe or target organism.
  • the concentration of the nucleic acid probe in the hybridization buffer depends on the type of its labeling and the number of target structures. In order to enable rapid and efficient hybridization, the number of nucleic acid probe molecules should exceed the number of target structures by several orders of magnitude. However, with fluorescence in situ hybridization (FISH) care must be taken to ensure that an excessively large amount of fluorescence-labeled nucleic acid probe molecules leads to increased background fluorescence.
  • the concentration of the nucleic acid probe molecules should therefore be in a range between 0.5-500 ng / ⁇ l, preferably between 1.0-100 ng / ⁇ l and particularly preferably between 1.0-50 ng / ⁇ l.
  • the preferred concentration in the context of the method according to the invention is 1-10 ng of each nucleic acid probe molecule used per ⁇ l of hybridization solution.
  • the volume of the hybridization solution used should be between 8 ⁇ l and 100 ml, in a particularly preferred embodiment of the method according to the invention it is 30 ⁇ l.
  • the duration of the hybridization is usually between 10 minutes and 12 hours; hybridization is preferably carried out for about 1.5 hours.
  • the hybridization temperature is preferably between 44 ° C. and 48 ° C., particularly preferably 46 ° C., the parameter of the hybridization temperature, as well as the concentration of salts and detergents in the hybridization solution, depending on the nucleic acid probes, in particular their lengths and the degree of complementarity can be optimized for the target sequence in the cell to be detected.
  • the person skilled in the art is familiar with the relevant calculations here.
  • this washing solution can contain 0.001-0.1% of a detergent such as SDS, preferably 0.005-0.05%, particularly preferably 0.01%, as well as Tris-HCl in a concentration of 0.001-0.1 mol / 1. preferably 0.01 - 0.05 mol / 1, particularly preferably 0.02 mol / 1, the pH of Tris-HCl in the range from 6.0 to 9.0, preferably at 7.0 - 8, 0, particularly preferably 8.0.
  • a detergent may be included, but is not essential.
  • the washing solution usually also contains NaCl, the concentration depending on the stringency required being from 0.003 mol / 1 to 0.9 mol / 1, preferably from 0.01 mol / 1 to 0.9 mol / 1.
  • a NaCl concentration of 0.07 mol / l (method for the simultaneous specific detection of bacteria of the genus Listeria and the species L. monocytogenes) or 0.215 mol / 1 (method for the specific detection of bacteria of the species S) is particularly preferred. aureus) or from 0.215 mol / 1 (process for simultaneous specific Detection of bacteria of the genus Campylobacter and the species C. coli and C. jejuni).
  • the washing solution can contain EDTA, the concentration preferably being 0.005 mol / 1.
  • the washing solution can also contain preservatives known to the person skilled in the art in suitable amounts.
  • buffer solutions are used in the washing step, which in principle may look very similar to hybridization buffers (buffered sodium chloride solution), except that the washing step is usually carried out in a buffer with a lower salt concentration or at a higher temperature.
  • hybridization buffers buffered sodium chloride solution
  • Td dissociation temperature in ° C
  • the "washing off" of the unbound nuclear acid probe molecules usually takes place at a temperature in the range from 44 ° C. to 52 ° C., preferably from 44 ° C to 50 ° C and particularly preferably at 46 ° C for a period of 10 to 40 minutes, preferably for 15 minutes.
  • the nucleic acid molecules according to the invention are used in the so-called Fast FISH method for the specific detection of the specified target organisms.
  • the Fast FISH method is known to the person skilled in the art and e.g. described in patent applications DE 199 36 875 and WO 99/18234. Reference is hereby expressly made to these documents with regard to their disclosure in order to carry out the detection methods described there.
  • the specifically hybridized nucleic acid probe molecules can then be detected in the respective cells.
  • the prerequisite for this is that the nucleic acid probe molecule is detectable, e.g. in that the nucleic acid probe molecule is linked to a marker by covalent binding.
  • detectable markers e.g. fluorescent groups such as CY2 (available from Amersham Life Sciences, Inc., Arlington Heights, USA), CY3 (also available from Amersham Life Sciences), CY5 (also available from Amersham Life Sciences), FITC (Molecular Probes Inc., Eugene, USA), FLUOS (available from Röche Diagnostics GmbH, Mannheim, Germany), TRITC (available from
  • nucleic acid probe molecules so that at their 5 'or 3' end there is another nucleic acid suitable for hybridization. sequence is present.
  • This nucleic acid sequence in turn comprises approximately 15 to 1,000, preferably 15-50 nucleotides.
  • This second nucleic acid region can in turn be recognized by a nucleic acid probe molecule which can be detected by one of the means mentioned above.
  • Another possibility is to couple the detectable nucleic acid probe molecules with a hapten, which can then be brought into contact with an antibody that recognizes the hapten.
  • Digoxigenin can be cited as an example of such a hapten. The skilled worker is also well known about the examples given.
  • the final evaluation is possible depending on the type of marking of the probe used with a light microscope, epifluorescence microscope, chemiluminometer, fluorometer etc.
  • Another advantage is the simultaneous detection of bacteria of the genus Listeria and the species L. monocytogenes. So far, only bacteria of the species L. monocytogenes have been detected with more or less reliability. However, epidemiological studies have shown that in addition to L. monocytogenes, other species of the genus Listeria also can trigger dangerous listeriosis. The sole evidence of
  • Another advantage is the ability to distinguish between bacteria of the genus Listeria and those of the species L. monocytogenes. This is easily and reliably possible through the use of different labels for the respective genus or species-specific nucleic acid probe molecules.
  • the nucleic acid probe molecules used can be used to specifically detect and visualize all species of the genus Listeria, but also highly specifically only the species L. monocytogenes.
  • the species S. aureus is just as reliable and all species of the genus Campylobacter, but also highly specifically only the species C. coli and C. jejuni are detected.
  • Another advantage is the ability to differentiate between bacteria of the genus Campylobacter and those of the species C. coli or C. jejuni. This is due to the use of different markings for the respective genus or. Species-specific nucleic acid probe molecules easily and reliably possible.
  • Another advantage of the method according to the invention is that it is easy to handle.
  • the method can be used to easily test large quantities of samples for the presence of the bacteria mentioned.
  • the methods according to the invention can be used in a variety of ways.
  • food samples e.g. poultry, fresh meat, milk, cheese, vegetables, fruits, fish, etc.
  • environmental samples can also be examined for the presence of the bacteria to be detected. These samples can e.g. taken from the ground or also be parts of plants.
  • the method according to the invention can also be used for the investigation of wastewater samples or silage samples.
  • the method according to the invention can also be used to examine medical samples, e.g. of stool samples, blood cultures, sputum, tissue samples (including sections), wound material, urine, samples from the respiratory tract, implants and catheter surfaces are used.
  • medical samples e.g. of stool samples, blood cultures, sputum, tissue samples (including sections), wound material, urine, samples from the respiratory tract, implants and catheter surfaces are used.
  • the food samples are taken from milk or milk products (yoghurt, cheese, curd cheese, butter, buttermilk), drinking water, beverages (lemonades, beer, juices), baked goods or meat products.
  • Another area of application for the method according to the invention is the examination of pharmaceutical and cosmetic products, e.g. Ointments, creams, tinctures, juices, solutions, drops etc.
  • pharmaceutical and cosmetic products e.g. Ointments, creams, tinctures, juices, solutions, drops etc.
  • kits for carrying out the corresponding methods are also made available.
  • the hybridization arrangement contained in these kits is described, for example, in German patent application 100 61 655.0. On the disclosure in this document regarding the in situ hybridization arrangement is hereby expressly incorporated by reference.
  • kits comprise, as the most important constituent, the respective hybridization solution with the nucleic acid probe molecules (VIT solution) which are described above for the microorganisms to be detected.
  • VIT solution the nucleic acid probe molecules
  • the corresponding hybridization buffer (Solution C) and a concentrate of the corresponding washing solution (Solution D) are also included.
  • Fixation solutions (Solution A (50% ethanol) and Solution B are also included
  • a sample is appropriately cultured for 20-44 hours.
  • cultivation can be carried out, for example, in V_ Fraser broth for 24 h at 30 ° C.
  • S. aureus the cultivation can be carried out, for example, as a blood culture (for example BACTEC 9240, Becton Dickinson Instruments) for 8 h to 48 h at 35 ° C respectively.
  • cultivation can be carried out, for example, in Preston selective medium for 24 h at 42 ° C.
  • a suitable aliquot of the fixed cells (preferably 40 ⁇ l) is applied to a slide and dried (46 ° C., 30 min or until completely dry).
  • the cells can also be applied to other carrier materials (e.g. a microtite latte or a filter).
  • the dried cells are then completely dehydrated by adding the fixing solution again (Solution B, preferably 40 ⁇ l).
  • the slide is dried again (room temperature, 3 min or until completely dry).
  • the hybridization solution (VIT solution) with the nucleic acid probe molecules described above for the microorganisms to be detected is then applied to the fixed, dehydrated cells.
  • the preferred volume is 40 ul.
  • the slide is then incubated in a chamber moistened with hybridization buffer (Solution C, corresponds to the hybridization solution without probe molecules), preferably the VIT reactor (46 ° C., 90 min).
  • the slide is then removed from the chamber, the chamber is filled with washing solution (Solution D, 1:10 diluted in distilled water) and the slide is incubated in it (46 ° C, 15 min).
  • the chamber is then filled with distilled water, the slide is briefly immersed and then air-dried in the lateral position (46 ° C, 30 min or until completely dry).
  • the slide is then embedded in a suitable medium (finisher).

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Abstract

La présente invention concerne un procédé pour détecter des bactéries pathogènes présentes dans des denrées alimentaires, notamment un procédé de détection spécifique simultanée de bactéries de l'espèce Listeria et de l'espèce Listeria monocytogenes, par hybridation in situ, un procédé de détection spécifique de bactéries de l'espèce Staphylococcus aureus, par hybridation in situ, ainsi qu'un procédé de détection spécifique simultanée de bactéries de l'espèce Campylobacter et de l'espèce C. coli et/ou C. jejuni, par hybridation in situ, ainsi que des sondes oligonucléotidiques et kits correspondants, permettant la mise en oeuvre des procédés de l'invention.
EP03704530A 2002-02-04 2003-02-04 Procede de detection specifique rapide de bacteries pathogenes presentes dans des denrees alimentaires Withdrawn EP1472370A1 (fr)

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DE10204447 2002-02-04
PCT/EP2003/001092 WO2003066893A1 (fr) 2002-02-04 2003-02-04 Procede de detection specifique rapide de bacteries pathogenes presentes dans des denrees alimentaires

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CN100342033C (zh) * 2004-08-13 2007-10-10 汕头大学 一种检测空肠弯曲菌的方法
JP2010022336A (ja) * 2008-07-24 2010-02-04 Hokkaido Univ 培養併用ｉｎｓｉｔｕハイブリダイゼーション法により生きているカンピロバクター菌を迅速かつ特異的に計数するための遺伝子プローブ及びその方法
JP2010057390A (ja) * 2008-09-02 2010-03-18 Nikken Seibutsu Igaku Kenkyusho:Kk 検査用具
KR102008708B1 (ko) 2010-06-23 2019-08-08 큐알엔에이, 인크. 전압 작동 나트륨 통로, 알파 소단위(scna)에 대한 자연 안티센스 전사체의 저해에 의한 전압 작동 나트륨 통로, 알파 소단위(scna) 관련된 질환의 치료

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US6028187A (en) * 1991-08-01 2000-02-22 Gen-Probe Incorporated Nucleic acid probes to Listeria monocytogenes
EP1098006A1 (fr) * 1994-06-24 2001-05-09 Innogenetics N.V. Detection, identification et differentiation simultanees de taxa d'eubacteriales a l'aide d'une technique d'hybridation
AU5062500A (en) * 1999-04-26 2000-11-10 Amersham Pharmacia Biotech Ab Primers for identifying typing or classifying nucleic acids
DE10012540B4 (de) * 2000-03-15 2004-09-23 Vermicon Ag Oligonukleotide und Verfahren zum spezifischen Nachweis von Mikroorganismen durch Polymerase-Kettenreaktion

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JP2005516627A (ja) 2005-06-09

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