EP1082465A2 - Procede de detection de micro-organismes presents dans des produits - Google Patents

Procede de detection de micro-organismes presents dans des produits

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Publication number
EP1082465A2
EP1082465A2 EP99934505A EP99934505A EP1082465A2 EP 1082465 A2 EP1082465 A2 EP 1082465A2 EP 99934505 A EP99934505 A EP 99934505A EP 99934505 A EP99934505 A EP 99934505A EP 1082465 A2 EP1082465 A2 EP 1082465A2
Authority
EP
European Patent Office
Prior art keywords
seq
probe
primer
pcr
dna
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.)
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Application number
EP99934505A
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German (de)
English (en)
Inventor
Klaus-Peter Gerbling
Frank-Roman Lauter
Lutz Grohmann
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GENESCAN ANALYTICS GMBH
Original Assignee
Bioinside GmbH
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Filing date
Publication date
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Publication of EP1082465A2 publication Critical patent/EP1082465A2/fr
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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

Definitions

  • the invention comprises methods for the detection of microbial contamination of non-sterile products, preferably according to GMP guidelines. Furthermore, the invention comprises a test kit for the detection of microbial contamination and the use of primer sequences and probe sequences for the determination of microorganisms in products, in particular in pharmaceuticals and cosmetics including their starting materials and intermediates.
  • the method is used for the quantitative identification of microorganisms by detection of specifically amplified DNA sequences and is to be used as a replacement for corresponding methods in the European Pharmacopoeia, Section 2.6.12-13.1997 (EP) and other national monographs such as USP.
  • the requirements include two groups: (i) the count of the total viable aerobic bacteria and fungi (group total bacterial count) and (ii) the absence proof of certain microorganisms: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus faecalis, Salmonella and Enterobactriaceae (group lead germs) .
  • test methods are poor and can only be automated to a small extent. • Due to the nutrient media properties only well growing ones can
  • Microorganisms but not, as required, all aerobic microorganisms are detected.
  • the storage costs are high for media and incubators.
  • Waste is high. • The fertility check of all media batches is very complex, especially because of the short shelf life of finished media.
  • This method is unsuitable for the detection of microorganisms which, like the bacterial genus Sarcina, do not form individual colonies.
  • the EP describes microbiological techniques which contain the growth of the respective microorganisms in certain selective nutrient media or on agar plates for rough differentiation. Subsequently, specific metabolic reactions of the respective microorganisms were used for fine differentiation. Appropriate detection systems, such as APILAB, or VITEK, are common.
  • Microbiological rapid tests based on a vital evidence by ATP determination e.g. Millipore company
  • ATP determination e.g. Millipore company
  • the existing PCR applications are generally susceptible to contamination from PCR products, are not very reproducible and difficult to quantify. In addition, they are time-consuming, since the alternative PCR methods generally require several hybridization steps to detect the PCR product.
  • nucleic acid to be amplified which is single-stranded or made single-stranded, is added with a molar excess of two oligonucleotide primers
  • primers are selected such that an extension product of the relevant primer complementary to the nucleic acid strand is synthesized for each strand and that an extension product of a
  • Primer when separated from its complement, as a template for the synthesis of a
  • Extension product of the other primer can serve. After disconnecting the
  • Extension products from the matrices on which they were synthesized can the extension products formed are used for renewed reaction with the primers.
  • the cyclic repetition of the steps results in a theoretically exponential increase in a nucleic acid sequence that lies within the outer hybridization positions of the primers.
  • a more refined method is the method according to Gelfand et al. US Pat. No. 5,210,015.
  • An oligonucleotide probe construction is used which hybridizes with a part of the nucleic acid strand of the template, the oligonucleotide probe being selected so that it fits between the primer pairs (forward and backward primer) for the amplification of the diagnostic target sequence of the respective microorganism.
  • the probe construction and synthesis is based on TaqMan technology (Holland et al. 1993 and Lee et al. 1993, Nucl. Acids. Res, Vol 21, p 3761 - 3766).
  • the chemical basis of this new method is the 5'-nuclease PCR assay, first published in 1991 (Holland et al. 1991, PNAS USA 88: 7276).
  • the core of this method is the 5'-nucleotide activity of Taq polymerase and the use of fluorescence-labeled, sequence-specific gene probes. These gene probes are labeled with a fluorescein derivative (reporter) at the 5 'end and with a rhodamine derivative (quencher) at the 3' end. Due to the spatial proximity of the two dyes, the fluorescent radiation of the reporter is absorbed by the quencher dye.
  • the reporter and quencher are spatially separated from one another by the 5'-nuclease activity of the Taq polymerase.
  • the reporter's fluorescence radiation is no longer quenched and can be measured and quantified directly.
  • the more probes that are cleaved the higher the fluorescence emission of the reporter molecules.
  • the amount of emission released is proportional to the amount of the resulting PCR products and this in turn is proportional to the number of copies of the genes used in the PCR.
  • the number of organisms present in the analysis sample can be calculated from the gene copy number.
  • the method is extremely sensitive since gene amplification and signal amplification take place during the PCR reaction. Since various reporter dyes are available on the market, internal controls and standards can be carried out with every reaction. In addition, a sample can be examined for the presence of several genes / organisms at the same time. There are currently three different reporter dyes available on the market. Task and solution
  • the main focus of the present invention is the development of detection methods for microorganisms, which experience has shown to occur frequently as product contaminants. These are especially in relation to the group of the leading germs: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonelia species, in relation to the group of total germ count: the bacteria and the Enterobacteriaceae.
  • the object of the present invention is to provide reagents, methods and the use of substances which make the detection of microbial contaminants of non-sterile products simpler, more precise and more efficient, for example in accordance with EP requirements. It should contain fewer components than, for example, according to EP requirements. Another task is to provide very sensitive and quantitative evidence for the required microorganisms.
  • test kit for the detection of microbial contamination of non-sterile products in particular according to GMP guidelines, also cosmetics and food, comprising at least one DNA fragment which comprises the following SEQ ID and spacers:
  • the SEQ ID [(SEQ ID forward primer); (SEQ ID probe); and (SEQ ID reverse primer)] also comprise variants in which one, two or three nucleotides are substituted, deleted and / or inserted, the variant having essentially the same function as the sequence of the SEQ ID [(SEQ ID Forward- Primer); (SEQ ID probe); and (SEQ ID reverse primer)], the function of binding to DNA for probes and the function of binding to DNA for primers and the provision of a 3 'extendable end for the DNA polymerase; the spacers comprising 0-40 nucleotides,
  • SEQ. ID. NO. 8 as reverse primer (ii) for Pseudomonas aeruginosa
  • SEQ. ID. NO. 9 as a forward primer
  • SEQ. ID. NO. 10 as a probe
  • SEQ. ID. NO. 12 as a forward primer
  • SEQ. ID. NO. 13 as a probe and SEQ. ID. NO. 14 as a reverse primer
  • SEQ. ID. NO. 15 as a forward primer
  • SEQ. ID. NO. 18 as a forward primer
  • SEQ. ID. NO. 20 as reverse primer (vi) for Enterobacteriaceae SEQ. ID. NO. 44 as a forward primer
  • SEQ. ID. NO. 45 as reverse primer (vii) for Enterobacteriaceae (16S rRNA)
  • SEQ. ID. NO. 47 as a forward primer
  • SEQ. ID. NO. 48 as a probe
  • SEQ. ID. NO. 49 as a reverse primer or further all the sequences which are complementary to the previous sequences SEQ ID NO 6 to 49.
  • a combination of two, more preferably three, more preferably four and most preferably five, six or seven total sequences is advantageous.
  • a kit with PCR reagents is preferred.
  • a kit with PCR reagents and TaqMan is more preferred. All the sequences mentioned are listed in Example 24. For a successful TaqMan - PCR, the following requirements are placed on the primer and probe sequences (example 24):
  • Probe sequence must be between primer sequences on the DNA to be amplified.
  • Probe should be between 18-30 bases long if necessary. • The probe should have a GC content of 40 - 60%.
  • the Tm of the probe (melting point) should be 5 - 10 C ° above the Tm of the
  • the same base should never follow more than 3 times in a row.
  • Probe sequences (a - c) determined by the following parameters: (i) High denaturation temperature in the first PCR cycles
  • the nucleic acids that can be used to use the amphication method and detection method for the above-mentioned target organisms are understood to mean, in particular, genomic nucleic acids.
  • Genomic nucleic acid sequences also contain, among other things, the genes or gene fragments that are suitable for a specific type, type, family, or type of microorganism -Department are characteristic.
  • the nucleic acid sequences can be used in a PCR test as diagnostic target sequences for a specific detection of this type, genus, family or department
  • a primer is a molecule that has a number of nucleotides on a polymeric backbone.
  • the sequence of the nucleobases is chosen such that they are more than 80% complementary to successive bases of the nucleotide sequence to be amplified.
  • This molecule has at least one extendable end. Extension is understood in particular to mean the enzyme-catalyzed coupling of base units using mononucleoside triphosphate units or oligonucleotides.
  • a DNA polymerase is preferably used as the enzyme.
  • the nucleic acid which contains nucleotide sequences to be amplified, serves as a template for the specific incorporation of bases. The sequence of the template determines the sequence of the bases attached to the primer.
  • Molecules with 15-30 bases are used as primers.
  • the 3 'end is preferably used as the extendable end.
  • Primers which are completely homologous to a partial sequence of the target nucleotide sequences SEQ are particularly preferred. ID. NO.1-5 are (Example 24).
  • Probe definition (including variations): A probe is understood to mean a molecule that, like the primers, has a number of nucleotides on a polymeric backbone.
  • a probe construction method according to US Pat. No. 5,210,015, which has already been described above, is used.
  • the nucleic acid probes of the present invention are 18-30 nucleobases in length. Specific sequences are obtained by selecting an at least 18 base long sequence from the respective matrices (SEQ. ID. NO. 1-5, Example 24). According to the invention, probes are therefore preferred which are at least 90% homologous to part of the respective matrices (SEQ. ID. NO. 1-5). Probes with strict homology are particularly preferred.
  • the invention relates to nucleotide sequences which are at least 80%, preferably 90%, most preferably 95% complementary to the target nucleotide sequences SEQ. ID. NO. 1 to 5 and 46 and 48.
  • the homology results from the number of identical purine or pyrimidine bases in a given nucleotide sequence.
  • Hybridization occurs when the following process steps are present, preferably the following conditions.
  • the primers and probes according to the invention bind to complementary bases, preferably to complementary nucleotide sequences in the genetic material of the target organisms the group total bacterial count and complementary nucleotide sequences in the genetic material of the target organisms from the group lead germs
  • nucleic acid sequences that are specific for other microorganisms
  • microorganism This term primarily includes organisms that can cause diseases in human and animal bodies and are only perceptible under the microscope.They are usually single-celled or occur in loose groups of cells of the same type and are called yours because of their simple cellular organization morphological and cultural-biochemical characteristics, as well as their chemical composition, antigen properties and genetic characteristics are well documented in the literature, eg in microbiological diagnostics, Burkhardt, 1992
  • PCR reagents are substances that are necessary for a PCR reaction with maximum sensitivity and specificity, in particular DNA
  • Mg 2+ ions such as MgCl2
  • potassium salts such as KCI
  • additives such as
  • Glycenn or DMSO or formamide primers and probes, deoxynucleotides, buffer substance such as Tris-Base and optional additives in the form of passive fluorescence reference compounds such as the fluorescent dye derivative ROX and e.g. 7-Deaza-2-deoxy-GTP as Replacement of dGTP
  • complementary Complementary structures correspond to or complement each other.
  • the polynucleotide strands of the natural DNA double helix are complementary. They form two complementary strands based on the specific base pairing (AT or GC). This means that the nucleotide sequence is in the other strand clearly defined, not identical, but complementary
  • cDNA has a structure complementary to an mRNA Complementary structure in which (aa) the sequence of the forward primer and the sequence of the probe or (bb) the sequence of the probe and the reverse primer of a previously mentioned group (i) to (vii) both complementary to the defined sequences are. More preferred is a complementary structure in which the sequence of the forward primer, the sequence of the probe and the reverse primer of a previously mentioned group (i) to (vii) are all three complementary to the defined sequences.
  • the invention further comprises a method for the detection of microorganisms in products, in particular pharmaceuticals or cosmetics, which method comprises the following steps: a) insertion of primers and fluorescence-labeled probes with the corresponding sequences and their variations, (i) for Staphylococus aureus
  • SEQ. ID. NO. 6 as a forward primer
  • SEQ. ID. NO. 7 as a probe
  • SEQ. ID. NO. 8 as reverse primer
  • ii for Pseudomonas aeruginosa
  • SEQ. ID. NO. 10 as a probe and SEQ. ID. NO. 1 1 as reverse primer (iii) for Escherichia coli
  • SEQ. ID. NO. 12 as a forward primer
  • SEQ. ID. NO. 13 as a probe
  • SEQ. ID. NO. 14 as reverse primer (iv) for Salmonella ssp.
  • SEQ. ID. NO. 15 as a forward primer
  • SEQ. ID. NO. 16 as a probe
  • SEQ. ID. NO. 17 as a reverse primer
  • SEQ. ID. NO. 18 as a forward primer
  • SEQ. ID. NO. 19 as a probe
  • SEQ. ID. NO. 20 as reverse primer
  • SEQ. ID. NO. 44 as a forward primer
  • SEQ. ID. NO. 46 as a probe
  • SEQ. ID. NO. 45 as a reverse primer (vii) for Enterobacteriaceae (16S rRNA)
  • SEQ. ID. NO. 49 as reverse primer or further all the sequences which are complementary to the previous sequences SEQ ID NO 6 to 49, b) amplification of the DNA with PCR; and c) irradiation with specific wavelengths that excite the fluorescent dye, d) measurement and quantification of the emission of the excited fluorescent dye.
  • the invention comprises a method according to the invention, the production of the probes being based on the TaqMan detection technology.
  • the core of the invention is the combination of certain selected probe / primer pairs which can detect microorganisms satisfactorily.
  • the optimization of the probes / primer pairs and the PCR reaction conditions for sensitivity and suitability for GMP-compliant product testing according to EP, 2.6.12-13: Microbial contamination of products not required to comply with the test for sterility (1997) is also essential.
  • a PCR technology according to US Pat. Nos. 4,800,159 and 4,683,195 is used.
  • TaqMan technology is used, which is described in US Pat. No. 5,210,015, which was issued on May 11, 1993 as a patent.
  • the method according to the invention or the test kit according to the invention is a special embodiment of the fluorescence PCR technology (TaqMan) for the above-mentioned target microorganisms.
  • the methods according to the invention and the test kits are in many respects far superior to the analytical methods prescribed in the EP (no prescribed method is currently required for cosmetics) and should completely replace them after the method has been validated with the respective test product.
  • the possibility of using other analytical methods is explicitly permitted in the EP (General Notices) if they produce the same results as the prescribed methods.
  • the method according to the invention has the following advantages:
  • (A) Kit and method for the detection of microorganisms of the group total bacterial count For the first time, all contaminating bacteria, the sequence of which is described in the NIH database, USA, status 11.1997, can be determined analytically using this kit and method without prior cultivation. Living and non-reproductive bacteria are detected quantitatively and very precisely with a sensitivity of 1-3 bacteria in the test product. The consequence of the application is a significantly increased product safety for the consumer, because:
  • Non-reproducible microorganisms that contain toxins that are difficult to detect can also be detected, • Contaminating DNA of bacterial origin, the absence of which must currently be shown in biologicals and products from rDNA technology (EP, 1997 and USP 1995) in all Test products can be proven easily and efficiently.
  • there are no special safety requirements for the application since no components of the kit are subject to a hazardous substances regulation.
  • the application has economic advantages for consumers and manufacturers, since the previous methods are several days more time-consuming and often represent the time-determining step in the release analysis. Rapid results on the microbiological safety of a biologically susceptible test product lead to lower costs in development and production, e.g. lower storage costs or faster response to variable market inquiries and thus overall to lower the production costs, which result in cheaper products.
  • Example 2 Detection of Staphylococcus aureus The detection of Sureus was carried out by the species-specific amplification according to the invention of cap-8 gene sequences (SEQ ID NO 1, see Example 24).
  • the cap-8 gene cluster encodes proteins which are involved in the biosynthesis of the capsule of Sureus
  • the capsule envelops the surface of these bacteria and represents a protective mechanism against the defense mechanisms of the host organisms.
  • the molecular composition of the capsule is specific for S aureus and represents, so to speak, a molecular fingerprint of this staphylococcal species
  • the (open reading frame O) ORF-0 of the cap -8 gene cluster is conserved in the different serotypes of S aureus (Sau and Lee 1996, J Bactenol 178, 2118-2126).
  • the DNA sequences from the ORF-0 of the cap-8 gene cluster (SEQ ID NO 1) were used as diagnostic DNA - Sequences selected for the synthesis of species-specific DNA panners and probes
  • FAM fluorescence derivative
  • TAMRA 6-carboxytetramethylrhodamine
  • Example 3 PCR conditions for the detection of Staphylococcus aureus After variation of primer and probe concentration and MgCl2 concentration, the following conditions were found to be optimal:
  • PE ABD model 7700 or model LS50B All components were purchased from PE Applied Biosystems, Rothstadt. Production of the TaqMan-PCR reaction mixtures, implementation of the PCR reactions and operation of the PCR heating blocks or the fluorescence detector (PE ABD model 7700 or model LS50B) was carried out according to the instructions of the device manufacturer (User's Manual, ABI Prism 7700 Sequence Detection System, PE Applied Biosystems 1997, or Users Manual, PE ABI LS50 B).
  • PCR reactions are carried out in the PCR heating block of the ABI Sequence Detector 7700. PCR heating blocks with comparable heating and
  • Heat transfer properties such as. B. the PE ABI devices model 7200, 9700, 9600 and
  • the PCR cycle profile is as follows:
  • genomic DNA was isolated from various organisms and used in the PCR test (Fig. 1, Sambrock et al. 1993). The resulting PCR products were analyzed ectrophoretically. The PCR products were 213 base pairs in size. Control sequencing of the PCR products verified that it was cap8-0 DNA (not shown).
  • the DNA (10 ng per lane, 2-14) of all S. aureus strains used (lane 2-5) was detected by the cap8-0 primers (# 15297 and # 15485). In contrast, the DNA of a closely related Staphylococcus species, S. epidermidis (Lane 6) and that of other bacterial genera (Lane 7-11) were not detected. Fungus, fish and human DNA (Lane 12-14) were used as controls and gave no detection signal. NTC ( no template control) is the water control in which no DNA was used.
  • the selectivity of the diagnostic PCR was performed as a TaqMan fluorescence test using the above-mentioned primers and
  • Ct value The hydrolysis of the fluorescent probe that takes place during TaqMan-PCR leads to an increase in reporter fluorescence radiation from one PCR cycle to the next. The number of cycles at which the reporter fluorescence radiation over the
  • NTC Background radiation
  • Ct Background radiation
  • NTC Background radiation
  • Ct threshold value cycle number Both the amount of reporter radiation released and the “threshold cycle” (Ct threshold value cycle number) are proportional to the amount of PCR being generated Products and thus the amount of gene copies used (bacterial count). The more gene copies are used, the lower the resulting Ct value. In a PCR system with 100% efficiency, the Ct value decreases by one cycle with every doubling of the starting gene copy number. In the case of a PCR reaction, e.g. 40 cycles, and no PCR product is produced, the Ct value by definition
  • DSM 1128 (ATCC 9027) 40 DSM 3227 (ATCC 19429) 40 DSM 50071 (ATCC 10145) 40 Salmonella typhimurium DSM 5569 (ATCC 1331 1) 40
  • DSM 2981 (ATCC 14506) 40 (reclassified DSM 2570 (ATCC 29212) 40 as Enterococcus faecalis)
  • DSM 6134 40 Escherichia coli
  • genomic S aureus DNA was prepared and used in PCR experiments
  • the result shows that the DNA of 3 bacterial cells can be detected by means of fluorescence PCR.
  • the rapid PCR test allows a linear quantification of the S aureus genomes used over 5 log steps, i.e. between 3 and 300,000 CFU (Ing DNA)
  • Pseudomonas aeruginosa was detected by the species-specific amplification of a / gQ gene sequences according to the invention (sequences see Example 24).
  • the a / gQ gene encodes elements of a protective mechanism which was developed by Pseudomonas aeruginosa in the course of evolution and which is specific for this type of bacteria
  • alginate is a unique virulence property of Pseudomonas aeruginosa.
  • Alginate is a polymer of mannuronic and guluronic acid (1, 4 glycosidically linked). This polymer forms a viscous gel on the bacterial surface. The production of this organic gel is very sensitively regulated. The ability to synthesize alginate , is present in all Pseudomonas aeruginosa strains. It is characteristic of this type of bacteria. Alginate synthesis is energy-consuming Process and therefore regulated.
  • One gene that regulates alginate synthesis is the algQ gene (Konyecsni and Deretic 1990, J. Bacteriol. 172, 2511-2520).
  • FAM fluorescence derivative
  • TAMRA 6-carboxytetramethylrhodamine
  • PCR primer 23 mer: 5 -CTT CGA TGC CCT GAG CGG TAT TC-3 '
  • Reverse primer sequence (# 1147):
  • Positions refer to those in Konyecsni and Deretic 1990, J. Bacteriol. 172, 2511-2520 published DNA sequence.
  • PCR reactions are carried out in the PCR heating block of the ABI Sequence Detector 7700.
  • Functionally equivalent are PCR heating blocks with comparable heating and heat transfer properties, e.g. B. the PE ABI devices model 7200, 9700, 9600 and 2400.
  • the PCR cycle profile for the Pseudomonas aeruginosa PCR is as follows:
  • the a / gQ system is specific to Pseudomonas aeruginosa.
  • PCR products were analyzed electrophoretically (see Example 3).
  • the PCR products were 294 base pairs in size (not shown).
  • E. coli was detected by the species-specific amplification of murA gene sequences according to the invention
  • the murA gene encodes the enzyme UDP-N-acetylglucosamine enolpyruvyltransferase, an important structural gene of E. coli (Marquardt et al. 1992, J. Bacteriol. 174, 5748-5752). This enzyme catalyzes the first step of peptidoglycan synthesis, in the case of E. coli des mureins, which is an essential part of the bacterial cell wall.
  • the cell wall composition can be seen as a characteristic feature of bacterial species.
  • the murA gene was selected as a genetic marker with diagnostic potency for identifying the Enterobacteriaceae species Escherichia coli.
  • Positions refer to those in Marquardt et al. 1992, J. Bacteriol. 174, 5748-5752 published DNA sequence (Genbank: M92358).
  • FAM fluorescence derivative
  • TAMRA 6-carboxytetramethylrhodamine
  • Baciilus subtiiis 40 Salmonella typhimurium ATCC 13311 40 Pseudomonas mirabelis DSM 788 40 Staphylococcus aureus DSM 6538P 40 Streptococcus faecalis DSM 2981 40 Klebsiella pneumonia ATCC 10031 40 Citrobacter freundii DSM 30040 40
  • Neurospora crassa 40 Arabidopsis thaliana 40
  • the / 77i / r> 4 system is specific for Escherichia coli.
  • the PCR products were 142 in size
  • Example 13 Sensitivity of the E. coli test
  • the PCR rapid test allows a linear quantification of the Escherichia co // ' genome used over 6 log levels, ie between 3 and 3,000,000 CFU.
  • Salmonella enterica The detection of Salmonella spp. of the species Salmonella enterica was carried out by specific amplification according to the invention of / wA gene sequences
  • the invA gene encodes a Salmonella-specific virulence factor.
  • Salmonella-specific virulence factor Various studies on a number of Salmonella have shown that these types of bacteria bind to epithelial cells. In this process, the actin system of the host cells is influenced by the bacteria. In response, the host cells enclose the bacterial cells. After complete confinement, the bacteria exist in vesicles in the cytoplasm of the host cells. The so-called inv genes (InvA-H) of Salmonella are involved in this inclusion process.
  • invA gene Since the invA gene is involved in the expression of a specific virulence mechanism of Salmonella, it is a genetic marker with diagnostic potency for the identification of Salmonella ssp. (Rahn et al. 1992, Mol. Cell. Probes. 6: 271-279).
  • Reverse Primer Sequence (# 542): 5 'GGT TCC TTT GAC GGT GCG ATG AAG 3' (use as reverse complement) [SEQ. ID. NO. 17]
  • FAM fluorescence derivative
  • TAMRA 6-carboxytetramethylrhodamine
  • the / nv system is specific to Salmonella.
  • PCR products were analyzed electrophoretically.
  • the PCR products were 287 base pairs in size (not shown). Control sequencing of the PCR products verified that it was invA DNA (not shown).
  • Example 17 Sensitivity of the PCR rapid test In order to determine the sensitivity of the Salmonella ssp. Determining PCR tests has been genomic
  • the result shows that the DNA of 3 bacterial cells can be detected using fluorescence PCR.
  • the PCR rapid test allows a linear quantification of the Salmonella typhimurium genomes used over 6 log steps, i. H. between 3 and 3,000,000 CFU.
  • DNA from various test microorganisms was extracted according to Boom et al., 1990, purified from proteins and other PCR inhibitors (Quiagen rampulen Kit, 1995) and used in PCR amplification experiments.
  • Example 19 Detection of bacteria universal
  • the detection of bacteria was carried out by specific amplification according to the invention of conserved 16S rRNA gene sequences (SEQ. ID. NO. 5, see Example 24).
  • Certain 16S rRNA-specific DNA sequences have been preserved in the course of evolution, are therefore present in the genome of all bacteria and can be used as primers and probes for the universal detection of bacteria (Relman 1993, Weisburg et al. 1991, J. Bacteriol. 173).
  • Positions refer to the DNA sequence of the 16S rRNA gene (E. coli in Weisburg et al. 1991, J. Bacteriol. 173)
  • PCR primer oligonucleotides The synthesis and purification of the PCR primer oligonucleotides was carried out by the company PE Applied Biosystems and according to their protocols.
  • the temperature and cycle profile of the PCR and the distance between the reporter dye and the quencher dye within the probe resulted in the following conditions being optimal:
  • the following components were mixed in a PCR reaction vessel (PE Applied Biosystems order no. N8010580):
  • the PCR cycle profile is as follows:
  • the samples were transferred to the fluorimeter LS-50B, with an additive for the detection of fluorescence in microtiter plates from Perkin Elmer.
  • the fluorescence radiation is measured and quantified according to the manufacturer's instructions (PE Applied Biosystems, Rothstadt, Germany).
  • genomic DNA was isolated from various organisms and used in the universal PCR test (Fig. 6).
  • the amount of PCR products formed is given in relative fluorescence units (Fig. 6)
  • the developed PCR test selectively detects bacteria.
  • the different signal intensities of the bacterial samples reflected the variable amounts of DNA used.
  • the resulting PCR products were analyzed electrophoretically.
  • Salmonella DNA was prepared and used in PCR experiments. Various DNA dilutions were made. Each dilution was made three times in parallel and used in the PCR test (Fig. 7). The amount of fluorescence released is called the RQ
  • the RQ value is the difference between the reporter (R) fluorescence radiation in one
  • Reporter radiation is related to the quencher position (Q).
  • the quencher radiation does not change during the PCR reaction and thus represents an internal standard against which norms are made.
  • the result shows that the DNA of 1-3 Salmonella bacteria can be identified
  • Fluorescence PCR was detected.
  • the fluorescence radiation that arises after 40 PCR cycles is significantly above the background radiation.
  • the fluorescence PCR test allows the linear quantification of the Salmonella genomes used over at least 4 log steps d. H. between 1-3 and 30,000 CFU (Fig. 7).
  • Example 23 Product Testing Using the Rapid Bacterial Test The use of the developed rapid PCR test was investigated by spiking experiments. 10 ml WFI (water for injections, lot no. 63022) was spiked with 50 CFU Salmonella (5 CFU / ml). DNA was prepared from the various spiked samples (Boom et al. 1990), purified (Qiagen 1995) and analyzed in the PCR rapid test (Fig. 8).
  • the spiked salmonella was found in the test product.
  • the detection amount was 90% of the amount of DNA used (Fig. 8). This value reflects the material losses that arise from the spiked products during DNA preparation. Despite these losses, 1-3 CFU / ml could be detected in the spiked test product. On the other hand, no Salmonella germs were detectable in the non-spiked test product (Fig. 8). The sterility of the test product was verified by membrane filtration according to the methods in EP (1997).
  • Example 24 Target gene, primer and probe sequences for the different organisms / groups
  • SEQ. ID. NO. 10 5 '- FAM - CCAACGCCGA AGAACTCCAG CATTTC - TAMRA - 3'
  • SEQ. ID. NO. 11 5 'CTGAAGGTCC TGCGGCAACA GTT 3' (use as reverse complement)
  • SEQ. ID. NO. 3 Escherichia coli
  • SEQ. ID. NO. 18 5 'GCATGGCTGT CGTCAGCTC 3'
  • the variants of the primer / probe sequence combinations are defined which detect the target DNA sequences with the same specificity (100%) and comparable sensitivity (> 70%) as the sequences given in Example 24.
  • Salmonella ssp (PCR conditions as in Example 15) [SEQ.ID.NO 15] GTGAAATTAT CGCCACGTTC GGGC / [SEQ. ID .NO 16] FAM-CTTCTCTATTGTCACCGTGG TCCA-TAMRA / [SEQ. ID .NO 17] GGTTCCTTTG ACGGTGCGAT GAAG
  • TTAAGTCCCG CAACGAGCGC AAC-TAMRA / [SEQ.ID.NO 45] TTTATGAGGT 15 CCGCTTGCTC
  • the primer / probe combinations which detect the target DNA sequences with unsatisfactory specificity ( ⁇ 100%) and sensitivity 25 ( ⁇ 70%), such as the sequences given in Example 24, cf. Figure with primers and probes
  • Salmonella ssp (PCR conditions as in Example 15)
  • the 16S rRNA gene codes for the bacterial ⁇ bosomal DNA, which together with the 23S rRNA and the 5S rRNA in combination with the ⁇ bosomal proteins form the translation apparatus for protein biosynthesis
  • the numerical designations of the oligonucleotides refer to the positions of the leading strand of the sequence published by Brosius et al 1978 for the 16S rRNA from Escherichia coli. The location of these sequences within the 16S rRNA gene is shown in SEQ ID NO 24. The size of the primer 1053 and 1270 limited amplicons are 238 bp
  • Target sequence of the 16S rRNA gene SEQ ID NO 47 (forward primer # 1053) 5 ' -GCATGGCTGTCGTCAGCTC-3 '
  • Seqence Identifier Number 48 (Probe # 1090) 5 ' -Fam-TTAAgTCCCgCAACgAgCgCAAC-Tamra-3 '
  • composition and components of the TaqMan PCR reaction for the detection of Enterobacteriaceae Composition and components of the TaqMan PCR reaction for the detection of Enterobacteriaceae:
  • UNG uracil-N-glycosylase
  • the gram-negative family Enterobacteriaceae belongs to the gamma group of Proteobacteria (Balows et al. 1991, Holt 1989). Proteobacteria also includes members of the Alpha, Beta, Delta, and Epsilon groups as well as Amoebobacter and some unclassified Proteobacteria Figure 9 shows a simplified taxonomic scheme for the classification of Enterobacteriaceae. The similarity of DNA sequences of different species generally increases with increasing degree of relationship.
  • genomic Escherichia co // ' DNA from strain ATCC 8739 was used to represent the other Enterobacteriaceae.
  • the detection range of the developed rapid PCR test for Enterobacteriaceae ranges from less than 5 CFU (corresponds to 25 fg genomic DNA) to over 5000000 CFU (corresponds to 25 ng genomic DNA) Escherichia coli ( Figure 10). No-template controls (without Enterobacteriaceae DNA) show no reaction with the developed rapid PCR test even after 40 cycles.
  • Example 31 Product analysis Sterile water for injections (WFI, lot 63022) was examined. The result of the investigation showed the absence of Enterobacteriaceae DNA.
  • Example 32 Error Variants in the Primer and Probe Sequences Define variants are defined as the primer / probe combinations which detect the target DNA sequences with unsatisfactory specificity ( ⁇ 100%) and sensitivity ( ⁇ 70%), as those given in Example 27 Sequences.
  • the DNA (10 ng per lane, 2-14) of all S aureus strains used (Lane 2 - 5) was detected by the cap8-0 primers (# 15297 and # 15485).
  • the DNA of a closely related Staphylococcus species S- epidermidis (Lane 6) and those of other bacterial genera (Lane 7 - 11) not detected.
  • the DNA (1-10 ng) of all bacteria used (Bacillus subtihs, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa and Streptococcus faecalis) was detected by the 16S rRNA pnmer / probe set.
  • Genomic DNA (10 ng) from fungi was detected Neurospora crassa), plants (Arabodopsis thaliana) or by humans (Human, Perkin Elmer ABI, 401846), the measured fluorescence radiation corresponded to the water control (no DNA control)
  • RQ (R + / Q) - (R- / Q)
  • the Ct values obtained are shown depending on the nucleating units (CFU) Enterobacteriacea used.

Abstract

L'invention concerne un procédé de détection et un nécessaire de test permettant la détection rapide et économique de germes dans des produits pharmaceutiques et cosmétiques. On utilise à cet effet des sondes et des amorces spécifiques dont la réplication est rendue visible par un système indicateur spécial, un colorant à fluorescence étant libéré.
EP99934505A 1998-05-12 1999-05-10 Procede de detection de micro-organismes presents dans des produits Withdrawn EP1082465A2 (fr)

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DE19822108 1998-05-12
DE19822108A DE19822108A1 (de) 1998-05-12 1998-05-12 Verfahren zur Detektion von Mikroorganismen in Produkten, insbesondere in Arzneimitteln und Kosmetika
PCT/DE1999/001471 WO1999058713A2 (fr) 1998-05-12 1999-05-10 Procede de detection de micro-organismes presents dans des produits

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