JP2002514439A - How to detect microorganisms in products - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method and a test kit for economically detecting bacteria in pharmaceuticals and cosmetics. The present invention uses specific probes and primers whose replication can be visualized by using a special indicator system (which emits a fluorescent dye).

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for detecting microbial contaminants in non-sterile products, preferably according to GMP guidelines. The invention further relates to test kits for detecting microbial contaminants in products, especially pharmaceuticals and cosmetics (including their starting materials and intermediates), and the use of primer sequences and probe sequences for measuring microbial contaminants. About.

The method has been used for the quantitative identification of microorganisms by detecting specifically amplified DNA sequences, and has been used in related methods in the European Pharmacopoeia, sections 2.6.12-13, 1997 (EP) and It replaces monographs from other countries (eg, USP).

[0003] According to the GMP guidelines, the manufacture of medicines and cosmetics involves chemical, physical and biological tests to ensure quality. In the case of cosmetics, manufacturers must be aware that the end product does not pose a health hazard (EC
Cosmetic Regulation 76, 768 EEC (KOSVO), 6), Amendment Regulation EC KOSVO 93/35 / EEC, 1993,
And the requirements of German national law (LMBG, section 24)).

[0004] In the case of drugs, the microbiological requirements for purity are much more stringent and encompass the KOSVO requirements (EP, sections 2.6.12-13, 1997).

[0005] This requirement involves two groups: (i) counting the total number of live aerobic bacteria and fungi (total population), and (ii) specific microorganisms: Staphylococcus aureus, green Pseudomonas aeruginosa (Pse
udomonas aeruginosa), Escherichia coli, and E. faecalis (Strepto
coccus faecalis), salmonella, and enterobacte
riaceae) (detection of the indicator group).

With regard measurement method of the total number of bacteria counting viable aerobic bacteria using the current state nutrient medium technique (total cell count group), EP is the growth of the microorganism to be detected during a specific nutrient medium or agar plates Conventional microbiological methods are described, including: Many suitable ready-to-use products or their starting materials are commercially available.

The use of the method described in EP for measuring aerobic bacteria (total population) has the following disadvantages:-requires a long time (3-5 days) to obtain the results Therefore, efficiency is poor.

The results are inaccurate. The tolerance limits vary 5-fold; EP, section 2.6.12.

The test method is poor and the degree of automation is small.

[0010] Due to the nature of the nutrient medium, only well-growing microorganisms can be detected, rather than all necessary aerobic microorganisms.

High storage costs for the medium and the incubator.

[0012] For agents with bacteriostatic properties, the EP method gives some unusable results due to the low recovery of added test microorganisms.

There are expensive plastic wastes.

The cost of preparing the medium and autoclaving the generated waste is high.

-Fertility testing of all lots of medium is very expensive, especially because of the short shelf life of the ready-to-use medium.

[0016] An alternative commercial method for determining the total bacterial count is by a device using laser scanning (eg, CHEMSCAN (Chemunex)).

The method is unsuitable for the detection of microorganisms that do not form individual colonies, as in the case of bacteria of the genus Sarcina.

In addition, the method is not suitable for solid and fatty products.

Detection of Specific Microorganisms Due to Differences in Cultural Properties and Specific Metabolites For the determination of specific bacteria (indicator groups), EP is used for rough distinction in specific selective nutrient media or agar. A microbiological method for growing each microorganism in a plate is described. The specific metabolic reaction of each microorganism is then used for fine distinction. Suitable detection systems (eg, APILAB or VITEK) are widely used.

The use of the method described in EP for measuring a specific bacterium (indicator group) has the same disadvantages (described above) as the use of the method required by EP for measuring aerobic bacteria.
That the selectivity of the detection method is limited to metabolic differences and therefore inadequate discrimination,
A further disadvantage.

Detection of Specific Microorganisms by Measuring ATP Content After Preculture As an alternative to the market, a rapid microbiological test based on the detection of survival by measuring ATP after growing the microorganisms in a nutrient medium ( For example, Millipore Company).

Disadvantages: The species cannot be determined, and the measurement results vary greatly depending on the survival conditions (which vary greatly between different bacterial genera).

Detection of Specific Microorganisms After Preculture Using DNA Probes, Primers, and PCR Another commercial alternative is the use of various PCRs, which are described in Chen et al., 1997, J. Am.
Food Microbiol. 35, 239-250, as described in food testing,
Probably will not meet the stringent GMP requirements for testing drug quality.

In general, the use of existing PCR is susceptible to contamination by PCR products, poor in reproducibility and difficult to quantify. In addition, alternative PCR methods typically require several hybridization steps to detect the PCR product and are time consuming.

In addition, these methods usually require the addition of various reagents at various times during use, and are less automatable and prone to problems.

In the methods of US Pat. Nos. 4,800,159 and 4,683,195, the nucleic acid to be amplified, which is single-stranded or made single-stranded, is hybridized under hybridizing conditions.
And in the presence of a substance that induces polymerization and nucleotides, are treated with a molar excess of the two oligonucleotide primers, the primers having the extension product of each primer (which is complementary to the nucleic acid strand) And when the extension product of a primer is separated from its complement, it is selected to be used as a template to synthesize the extension product of another primer. After removing the extension product from the template used to synthesize the extension product, the resulting extension product is used for another reaction with the primer. The repetitive rotation of these steps results in a theoretically exponential growth of the nucleic acid sequence, which is located at the outer hybridization site of the primer.

An improved method for the quantitative detection of microbial DNA using special fluorescent PCR techniques is the method of Gelfand et al., US Pat. No. 5,210,015, in which an oligonucleotide probe construct is used, which comprises The oligonucleotide probe that hybridizes with a part of the nucleic acid strand of the template is selected so as to fit between a pair of primers (forward primer and reverse primer) for amplification of the diagnostic target sequence of each microorganism. The construction and synthesis of the probe is based on TaqMan technology (Holland et al. And Lee et al., 1993, Nucl. Acids Res., Vol. 21, pp. 3761-3766).

The chemical basis of this new method is the 5 ′ nuclease PC first published in 1991.
R-measurement method (Holland et al., 1991, PNAS USA 88, 7276). An essential part of this method is the use of Taq polymerase 5 'nuclease activity and fluorescently labeled sequence-specific gene probes. These gene probes are labeled at the 5 'end with a fluorescein derivative (reporter) and at the 3' end with a rhodamine derivative (quencher). As a result of the close spatial distance of both dyes, the fluorescent emission of the reporter is absorbed by the quencher dye. During the polymerase chain reaction (PCR), the reporter and quencher are spatially separated from each other by the 5 'nuclease activity of Taq polymerase. The fluorescence emission of the reporter is no longer quenched and can be measured and quantified directly. The more probes are cleaved, the greater the fluorescence emission of the reporter molecule. The amount of luminescence emitted is proportional to the amount of PCR product generated, which is proportional to the copy number of the gene used for PCR. From the number of gene copies, the number of organisms present in the analysis sample is calculated. This method is very sensitive because gene amplification and thus signal amplification occur during the PCR reaction. A variety of reporter dyes are commercially available, so that each reaction can include an internal control or internal standard. In addition, samples can be tested simultaneously for the presence of multiple genes / organisms. Currently, three different reporter dyes are commercially available.

PROBLEM AND SOLUTION A central object of the present invention is to develop a method for the detection of microorganisms, which in experience often appear as product contaminants. For the group of indicator bacteria, these are, in particular, Escherichia coli
li), Pseudomonas aeruginosa, Staphylococcus a
ureus), Salmonella type, and bacteria and intestinal bacteria in the total bacterial population.

It is an object of the present invention to make the detection of microbial contaminants, for example, in non-sterile products according to the requirements of the EP easier, more accurate and more efficient, if one seeks to include fewer components compared to the requirements of the EP, for example. The use of reagents, methods and materials.
Another object is to provide sensitive and quantitative detection of the required microorganism.

The above objective is accomplished by a test kit for detecting microbial contaminants in non-sterile products (including cosmetics and foods) (particularly according to GMP guidelines), the test kit comprising the following sequence and spacer: (a) forward primer (sequence: forward primer); (b) probe (sequence: probe); (c) reverse primer (sequence: reverse primer); (d) optionally, a spacer between the forward primer and the probe; e) optionally a spacer between the probe and the reverse primer; (f) optionally a spacer upstream from the forward primer; (g) optionally a spacer downstream from the reverse primer. The sequence [(sequence: forward primer), (sequence: probe), and (sequence) Row: reverse primer)] also comprises variants in which one, two or three nucleotides have been substituted, deleted and / or inserted, said variants essentially comprising the sequence [(sequence: Forward primer), (sequence: probe), and (sequence: reverse primer)], the probe binds to DNA, and the primer binds to DNA,
It has the function of providing an extendable 3 'end for A polymerase, the spacer comprises 0 to 40 nucleotides, and the DNA fragment comprises: (i) SEQ ID NO: as a forward primer for Staphylococcus aureus 6, SEQ ID NO: 7 as a probe and SEQ ID NO: 8 as a reverse primer, (ii) For Pseudomonas aeruginosa, SEQ ID NO: 9 as a forward primer, SEQ ID NO: 10 as a probe, and SEQ ID NO: 11 as a reverse primer, ( iii) For Escherichia coli, SEQ ID NO: 12 as a forward primer, SEQ ID NO: 13 as a probe, and SEQ ID NO: 14 as a reverse primer. (iv) For Salmonella ssp., SEQ ID NO: 15 as a forward primer, probe As SEQ ID NO: 16, and river SEQ ID NO: 17 as a primer, (v) SEQ ID NO: 18 for a bacterium (bacteria), SEQ ID NO: 19 as a probe, and SEQ ID NO: 20 as a reverse primer, (vi) Forward primer for enterobacteriaceae SEQ ID NO: 44, SEQ ID NO: 46 as a probe, and SEQ ID NO: 45 as a reverse primer, (vii) For enterobacteriaceae (16S rRNA), SEQ ID NO: 47 as a forward primer, SEQ ID NO: 48 as a probe, and as a reverse primer SEQ ID NO: 49, or further selected from the group consisting of all the sequences complementary to the above sequences of SEQ ID NOs: 6-49.

Two, more preferably three, and even more preferably four, most preferably five
A combination of one, six or seven complete sequences is advantageous. Kits containing PCR reagents are preferred. A kit containing a PCR reagent and TaqMan is more preferred.

All of the above sequences are provided in Example 24. For the success of TaqManPCR,
The primer and probe sequence (Example 24) should fulfill the following requirements:-The primer must be between 15 and 30 bases in length.

The probe sequence must be between the primer sequences on the DNA to be amplified.

-Optionally, the probe must be between 18 and 30 bases in length.

The probe must have a GC content of 40-60%.

The Tm (melting point) of the probe must be 5-10 ° C. higher than the Tm of the primer.

[0038] There must be no G at the 5 'end of the probe.

-No more than three identical bases must be present consecutively in the probe sequence.

There must be no complementarity between or within the probe and primer and no significant secondary structure within the probe and primer.

These general guidelines for designing primers and probes (Livak
Despite the 1995, guidelines for designing TaqMan fluorescent probes for 5 'nuclease assays, Perkin Elmer Research News), each TaqMan PCR
For use, the optimal primer and probe combination must be determined empirically. As demonstrated in many examples (Example 25), even if the above guidelines were adhered to, no optimal TaqMan PCR system could be developed. on the other hand,
The sequence characteristics of the diagnostic target sequence of each organism (eg, high GC content, highly repetitive sequences or highly conserved sequence regions) may require the selection of primer and probe sequences that do not meet the above design guidelines. As a result of these limitations of the guidelines, the selection of diagnostic target sequences from the genome of the microorganism to be detected and the experimental determination of optimal primer and probe sequences achieve the required specificity and sensitivity of the TaqMan PCR test Required to do.

PCR reaction conditions including TaqMan buffer: Apart from the primer and probe sequences (ac), the specificity and sensitivity of the TaqMan PCR test is determined by the following parameters: (i) the initial PCR cycle Level of denaturation temperature (ii) level of annealing temperature during the amplification phase during PCR (iii) number of PCR cycles (iv) use of PCR additives such as glycerol and / or formamide (v) genes with high G / C content Use of 7-deaza-2-deoxy-GTP in addition to GTP to (vi) Mg ²⁺ ion concentration level in PCR buffer (vii) Primer and probe concentrations (viii) Amount of Taq DNA polymerase (ix) Distance of cis-Oriented Primer from Probe In developing the TaqMan PCR test disclosed herein, all these parameters are considered experimentally (data not shown).

Description of Nucleic Acids Used as Diagnostic Target Sequences In particular, the nucleic acids used in the methods for amplifying and detecting the target organisms are understood to be genomic nucleic acids. In particular, genomic nucleic acid sequences also include genes or gene fragments characteristic of a particular species, genus, family, or subclass of the microorganism. The nucleic acid sequence can be used in a PCR test as a diagnostic target sequence for the specific detection of such a species, genus, family, or subclass.

To detect the target organism, the following target sequences are selected: The gene for which the diagnostic target sequence was selected will be described in detail in Examples.

Definitions Definition of primers (including variants thereof) : A primer is understood to be a molecule having many nucleotides on the polymer backbone. The sequence of the nucleobases is selected to have greater than 80% complementarity to contiguous bases of the nucleotide sequence to be amplified. Each of these molecules has at least one extendable end. In particular, extension is understood to be the enzymatically catalyzed attachment of base units using mononucleotide triphosphate units or oligonucleotides. DNA polymerase is preferably used as an enzyme.
The nucleic acid containing the nucleotide sequence to be amplified is used as a template for specific incorporation of a base. The sequence of the template determines the sequence of the base bound to the primer. Molecules with 15-30 bases are used as primers. In the case of a DNA polymerase, the 3 'end preferably acts as an extendable end. Primers that are completely homologous to the partial sequence of the target nucleotide sequence SEQ ID NOS: 1-5 (Example 24) are particularly preferred.

Definition of probe (including its variants) : A probe, like a primer, is understood to be a molecule having many nucleotides on the polymer backbone. Here, the probe preparation method described in the aforementioned US Pat. No. 5,210,015 is used. The nucleic acid probes of the present invention are 18 to 30 nucleobases long. By selecting one sequence of at least 18 bases in length from each template (SEQ ID NOS: 1-5, Example 24), a specific sequence is obtained. Therefore, in the present invention, a probe having at least 90% homology to a part of each template (SEQ ID NOS: 1 to 5) is preferable. Probes with exact homology are particularly preferred.

Definition of homology : The present invention relates to nucleotide sequences that are at least 80%, preferably 90%, more preferably 95% complementary to the target nucleotide sequence SEQ ID NOS: 1-5, 46 and 48. . Homology (in%) results from the number of identical purine and pyrimidine bases in a given nucleotide sequence.

Definitions of hybridization : The following processing steps and preferably the following conditions: The primers and probes of the present invention are complementary bases, preferably complementary to one another in the genotype of the target organism from the total bacterial population. Binds to nucleotide sequences and complementary nucleotide sequences in the genotype of the target organism from the indicator organism group, and furthermore that they do not bind to nucleic acid sequences specific to other microorganisms. , Hybridization is present.

Definition of drugs : These substances are active substances, ingredients, adjuvants and preparations described in the EP monograph and intended for use as human and veterinary medicine.

Definitions of cosmetics : These substances are not listed in the pharmacopoeia monographs, but KOSVO and LMBG
Including ingredients, adjuvants, and formulations intended for use in humans and animals as directed.

Definition of microorganism : This term mainly includes organisms that cause disease in the human and animal body and is only visible by microscopic means. Generally these are unicellular and appear as loosely bound like cells and are called protists because of their simple cellular composition.
Its morphological and culture-biochemical characteristics, as well as its chemical composition, antigenicity and genetic characteristics, are described in the literature (eg, Mikrobiologische Diagnostik, Burkhardt, 1992
).

Definition of PCR reagent: PCR reagent is a substance required for a PCR reaction having maximum sensitivity and specificity.
DNA polymerase, Mg ²⁺ ions, such as is in MgCl _2, potassium salts such as KCl,
Additives such as glycerol or DMSO or formamide, primers and probes, buffer substances such as deoxynucleotides, Tris bases, and passive fluorescent standard compounds (such as the fluorochrome derivative ROX, and 7 as an alternative to dGTP, for example) -Deaza-2-deoxy-GTP).

Definition of complementarity: Complementary structures correspond to each other or complement each other. For example, the natural DN
The polynucleotide strand of an A double helix is complementary and forms two complementary strands due to its specific base pairing (AT and GC, respectively). As a result, the nucleotide sequence in the other strand is clearly determined (not identical but complementary).
This is also true for DNA-RNA hybrids (with AU pairs instead of AT pairs). cDNA has a structure complementary to mRNA. The sequence of the (aa) forward primer and the probe of the group already described in (i) to (vii), or both the sequence of the (bb) probe and the reverse primer of the group are complementary to the specified sequence. , A complementary structure is preferred. The sequence of the forward primer, the sequence of the probe, and the sequence of the reverse primer (ie, all three above) of the group already described in (i) to (vii) have a structure complementary to the sequence in a defined manner. More preferred.

[0054] The present invention product, relates in particular detection method of microorganisms in medicine and cosmetics, the method comprising the following steps: a) the use of primers and fluorescently labeled probes having the appropriate sequence and its variants, (i) SEQ ID NO: 6 as a forward primer for Staphylococcus aureus, SEQ ID NO: 7 as a probe, and SEQ ID NO: 8 as a reverse primer. (ii) SEQ ID NO: 9 probe as a forward primer for Pseudomonas aeruginosa (Iii) For Escherichia coli, SEQ ID NO: 12 as a forward primer SEQ ID NO: 13 as a probe, and SEQ ID NO: 14 as a reverse primer (iv) For Salmonella species Is the forward primer SEQ ID NO: 15 as a probe, and SEQ ID NO: 17 as a reverse primer (v) For bacteria SEQ ID NO: 18 as a forward primer SEQ ID 19 as a probe, and SEQ ID NO as a reverse primer SEQ ID NO: 20 (vi) Enterobacteria SEQ ID NO: 44 as a forward primer SEQ ID NO: 46 as a probe, and SEQ ID NO: 45 as a reverse primer (vii) For enterobacteria (16S rRNA) SEQ ID NO: 47 as a forward primer SEQ ID NO: 48 as a probe, and SEQ ID NO: 49 as a reverse primer Alternatively, all sequences complementary to the above sequences of SEQ ID NOs: 6-49; b) amplifying the DNA using PCR; and c) irradiating at a specific wavelength to excite the fluorescent dye; d) Measuring and quantifying the emission of the excited fluorescent dye.

The invention includes the preparation of a probe based on the method of the invention, the TaqMan detection method.

Essence of the Invention The essence of the invention is a specific selected probe / primer pair combination that can detect microorganisms in a satisfactory manner. Probe / Primer pairs and EP, 2.6.12-13: Microbial contamination of products that do not need to be compatible with tests for sterilization (
It is also essential to optimize PCR conditions for the sensitivity and suitability of GMP compliant product testing according to 1997). The PCR methods of U.S. Pat.Nos. 4,800,159 and 4,683,195 are used,
In particular, the TaqMan method described in U.S. Pat. No. 5,210,015 issued May 11, 1993 is used.

The method of the present invention or the test kit of the present invention comprises a fluorescent PCR for the above-mentioned target microorganism.
It is a special embodiment of the law (TaqMan).

Advantages In many respects, the methods and test kits of the present invention are far superior to the analytical methods defined in EP (for cosmetics, it is not necessary to define the method yet), Once the efficacy has been proven for each product to be tested, it is intended to completely replace the EP method. The EP (General) clearly acknowledges the possibility of using other analytical methods if they achieve the same result as the prescribed method.

In particular, the method of the present invention has the following advantages.

(A) Kits and Methods for Detecting Microorganisms from Total Populations Using the kits and methods, their sequences are described in the NIH database (USA, as of November 1997). Analytical measurement of all contaminating bacteria
This is only possible without prior cultivation, where live bacteria that cannot grow can be detected very accurately and with the sensitivity of one to three bacteria in the product to be tested.
One consequence of such use is improved product safety for the consumer. The reason is as follows.

[0061] The detection of spores and microorganisms that are difficult to cultivate, which can be dangerous to health.

[0062] Non-proliferable microorganisms containing difficult-to-detect toxins can be detected.

-RDNA technology must now be proven to be absent in biological agents and products (EP, 1997 and USP 1995). Contaminated DNA of bacterial origin is present in all products to be tested. , Can be detected easily and efficiently.

In addition, there is no need for specific safety instructions since none of the components of the kit are regulated for hazardous materials.

(B) All claimed kits and methods: The previous methods take a few extra days and often require clearance.
) Such use has economic advantages for consumers and manufacturers because it is the rate-limiting step in the analysis. The rapid results on the microbiological safety of the biologically sensitive products to be tested lower the costs of development and production (eg, storage costs), speed up the response to various commercial demands, In addition, the production cost is reduced as a whole, and the product price is reduced.

Such use has the advantage of being environmentally friendly, since the reduction in analysis time and materials (plastics and culture media) significantly reduces the considerable energy costs.

EXAMPLES The following examples demonstrate the use of target microorganisms (including all sequence variants and target sequences).
The following describes a rapid PCR test developed to detect E. coli: (i) Staphylococcus aureus (Examples 1-5) (ii) Pseudomonas aeruginosa (Examples 6-9) (iii) E. coli (Example 10) 13) (iv) Salmonella species (Examples 14-17) (iv) Bacteria (Examples 18-23) (vi) Target, probe and primer sequences (Example 24) (vii) Sequence variants (Example 25) (Viii) Developed sequences of probes and primers with unsatisfactory test specificity / sensitivity Example 26 Example 1 DNA release after initial accumulation Dissolve 100 μl aliquots of each microbial culture each time DNA was released (M
akino et al., Applied Environ. Microbiol. 3745-3747, 1995). The DNA was purified to remove proteins and other PCR inhibitors and then used for PCR amplification experiments.

Example 2 Detection of Staphylococcus aureus Detection of S. aureus was performed by species-specific amplification of the cap-8 gene sequence of the present invention (SEQ ID NO: 1, Example). 24). The cap-8 gene cluster encodes a protein involved in capsular biosynthesis of S. aureus. The capsule covers the surface of these bacteria while presenting a defense mechanism against the defense mechanisms of the host organism. The molecular composition of the capsule is specific for S. aureus and shows a so-called molecular fingerprint of this Staphylococcus species. ORF-O (reading frame O) of cap-8 gene cluster is Staphylococcus aureus (
Conserved in various serotypes of S. aureus (Sau and Lee 1996, J. Bacteri
178, 2118-2126). The DNA sequence from ORF-O of the cap-8 gene cluster (SEQ ID NO: 1) was selected as a diagnostic DNA sequence for synthesizing species-specific DNA primers and probes.

As a result of comparing DNA sequence databases using various primer / probe combinations and a practical optimization procedure, the following cap-8-specific DNA sequences were determined as the optimal primer / probe combinations.

1. PCR probe 20 mer 5'-TAMRA-CCT GGT CCA GGA GTA GGC GG 3'-FAM (probe cap-8 # 15460 *, used as reverse complement) [SEQ ID NO: 7] The probe is PE Applied Biosystems Division, Weiterstadt (Germany). These are 5 'fluorescent derivatives (FAM = 6-carboxyfluorescein)
It is a single-stranded oligonucleotide modified at the terminus and modified at the 3 'terminus with a rhodamine derivative (TAMRA = 6-carboxytetramethylrhodamine). Synthesis and purification
Performed according to the instructions of PE Applied Biosystems.

2. PCR primer 24mer: 5'-AGA TGC ACG TAC TGC TGA AAT GAG-3 '(primer cap-8 forward # 15297 *) [SEQ ID NO: 6] 26mer: 5'-GTT TAG CTG TTG ATC CGT ACT TTA TT-3 '(primer cap-8 reverse # 15485 *, used as reverse complement) [SEQ ID NO: 8] * Position is ca, published by Sau and Lee (1996, J. Bacteriol. 178, 2118-2126)
In the p-8 DNA sequence. Synthesis and purification of PCR primer oligonucleotides were performed by PE Applied Biosystems according to its protocol.

Example 3 PCR conditions for detection of Staphylococcus aureus After changing the primer and probe concentrations and the MgCl ₂ concentration, the following conditions were found to be optimal.

All components were purchased from PE Applied Biosystems, Weiterstadt (Germany). Preparation of the TaqMan PCR reaction mixture, performance of the PCR reaction, and operation of the PCR heating table and fluorescence detector (PE 30 ABD Model 7700 or Model LS50B) were performed according to the manufacturer's instructions (User's Manual, ABI Prism 7700). Sequence detection system, PE Applied Biosystems 1997, and user's manual, PE ABI LS5
0B).

The following components were mixed in a PCR reaction vessel (PE Applied Biosystems, order no. N8010580).

[0075] In order to optimize the reproducibility of the results, care must be taken in each PCR cycle to premix as many components of the PCR mix as possible in the so-called master mix. Under standard conditions, only the DNA substance to be tested (0-15.25 μl)
Is separately added as a component to each PCR reaction vessel.

The PCR reaction is performed on a PCR heating table of ABI Sequence Detector 7700. Functionally equivalent are PCR heating tables with comparable heating and heat transfer, such as the PE ABI instrument models 7200, 9700, 9600, and 2400.

[0077] For a detailed description of the PCR cycle profile, see Prism 7700 Sequ
Please refer to the user's manual of the ence detection system (PE Applied Biosystems 1997).

Example 4 Selectivity of S. aureus PCR Rapid Test 4.1 Electrophoretic Analysis Genomic DNA from various organisms was isolated and used in PCR tests to estimate the selectivity of the PCR test. (FIG. 1, Sambrock et al., 1993). The generated PCR product was analyzed by electrophoresis. The size of the PCR product was 213 base pairs. Control sequencing of the PCR products confirmed that they were cap8-0 DNA (not shown).

DNA (10 ng per lane, 2-14) of all S. aureus strains used (lanes 2-5) was detected with cap8-0 primers (# 15297 and # 15485). In contrast, the closely related Staphylococcus species, ie, S. epidermidis (lane 6)
And DNA of other bacterial genera (lanes 7 to 11) were not detected. Fungal, fish and human DNA (lanes 12-14) were used as controls, but there was no detected signal. NTC (= no template control) was a water control and did not use DNA.

4.2 Fluorescence Analysis In addition to electrophoretic analysis, the selectivity of diagnostic PCR was measured as a TaqMan fluorescence test using the above primers and fluorescent probe. The results are shown as Ct values (threshold cycles).

Ct value: Hydrolysis of the fluorescent probe that occurs during TaqMan PCR increases the reporter fluorescence emission from one PCR cycle to the next. The number of cycles in which the reporter fluorescence emission first rises above the system background emission (NTC) and rises linearly is called the "threshold cycle" (Ct). (Background radiation (NTC
) Is the reporter fluorescence emission of a PCR control reaction without template DNA. ) The amount of reporter radiation emitted and the "threshold cycle" (Ct threshold number of the cycle)
The amount of gene copy (number of bacteria) used is proportional to the amount of PCR product formed and therefore
Is proportional to

The more gene copies used, the lower the Ct value obtained. For a PCR system with 100% efficiency, the Ct value is reduced by one cycle for every doubling of the starting number of gene copies. For example, in a PCR reaction containing 40 cycles in which no PCR product was generated, the Ct value would be 40 by definition.

For the specificity test, 10 ng of template DNA is used in each PCR reaction. Reaction conditions are specified in Example 3.

[0084] After about 17 cycles, a linear increase in FAM fluorescence above the FAM background emission of the fluorescent probe was detected for the first time when using S. aureus genomic DNA in fluorescent PCR. Staphylococcus epidermidis (S. aureu in the genus Staphylococcus)
s), which is a closely related species), no significant increase in FAM reporter fluorescence was detected.

The results of PCR analysis using DNA from various bacterial genera (Staphylococcus sp. And Staphylococcus aureus strains) show the results of the developed S. aureus test. Prove the specificity. Only S. aureus DNA is detected by cap-8 primers and probes.

Example 5 Sensitivity of S. aureus Detection Method To determine the sensitivity of the S. aureus PCR test, genomic DNA of S. aureus was prepared. Used in PCR experiments.

10 fg of genomic S. aureus DNA corresponds to three genomes (
Strauss and Falkow 1997, Science 276, 707-712).

10 fg = 3 gfu 10 pg = 3,000 gfu 10 ng = 3,000,000 gfu Various amounts of S. aureus DNA (1 fg-100 ng) were used in fluorescent PCR (FIG. 2). The data shown are the average of six independent experiments. The amount of emitted fluorescence, and thus the amount of PCR product generated, is indicated as the Ct value.

The results show that DNA from three bacterial cells can be detected by fluorescent PCR.
PCR rapid tests span 5 log levels (i.e. between 3 and 300,000 gfu (1 ng DNA
)), Allowing for linear quantification of the S. aureus genome used.

Example 6 Detection of Pseudomonas aeruginosa Pseudomonas aeruginosa was obtained by species-specific amplification of the algQ gene sequence of the present invention.
nosa) (see Example 24 for sequence). The algQ gene encodes an element of the defense mechanism that evolved from Pseudomonas aeruginosa during evolution, which mechanism is specific to this bacterial species.

[0091] Alginate production is a unique pathogenicity of Pseudomonas aeruginosa. Alginate is a polymer of mannuronic acid and guluronic acid (1,4-
Glycoside linkage). This polymer forms a viscous gel on the surface of the bacteria. The production of this biogel is subject to sensitive controls. The ability to synthesize alginate is
Present in all Pseudomonas aeruginosa strains and is characteristic of this bacterial species. Alginate synthesis is an energy consuming process and is therefore controlled. The gene that controls alginate synthesis is the algQ gene (Konyec
sni and Deretic 1990, J. Bacteriol. 172, 2511-2520). It encodes a sensory component of the signaling system (Roychoudhury et al., 1993, PNAS USA 90, 965-969).
). Since the algQ gene is involved in the regulation of specific defense mechanisms, it presents a diagnostic genetic marker in the identification of Pseudomonas aeruginosa species.

As a result of a comparison of the DNA sequence databases and a practical optimization procedure using various primer / probe combinations, the following algQ-specific DNA sequences were determined as the optimal primer / probe combinations.

1. PCR probe: 26 mer: 5'-FAM-CCA ACG CCG AAG AAC TCC AGC ATT TC-TAMRA (probe algQ # 911): [SEQ ID NO: 10] The probe was a PE Applied Biosystems Division, Weiterstadt ( Germany). These are the fluorescent derivatives (FAM = 6-carboxyfluorescein)
Single-stranded oligonucleotide modified at the 'end and modified at the 3' end with a rhodamine derivative (TAMRA = 6-carboxytetramethylrhodamine). Synthesis and purification were performed according to the instructions of PE Applied Biosystems.

2. PCR primer: 23 mer: 5'-CTT CGA TGC CCT GAG CGG TAT TC-3 '(primer algQ forward # 876 *) [SEQ ID NO: 9] Reverse primer sequence (# 1147): 23 mer : 5'-CTG AAG GTC CTG CGG CAA CAG TT-3 '(primer algQ reverse # 1147 *, used as reverse complement) SEQ ID NO: 11 Means the DNA sequence published by.

The synthesis and purification of the PCR primer oligonucleotides were performed by PE Applied Biosystems according to its protocol.

Example 7 PCR conditions for detection of P. aeruginosa The following conditions were found to be optimal by changing the primer / probe concentration and the MgCl ₂ concentration.

[0097] In order to optimize the reproducibility of the results, care must be taken in each PCR cycle to premix as many components of the PCR mix as possible in the so-called master mix. Under standard conditions, only the DNA substance to be tested (0-15.25 μl)
Is separately added as a component to each PCR reaction vessel.

[0098] The PCR reaction is performed on a PCR heating table of the ABI Sequence Detector 7700. Functionally equivalent are PCR heating tables with comparable heating and heat transfer, such as the PE ABI instrument models 7200, 9700, 9600, and 2400.

The profile of the PCR cycle of Pseudomonas aeruginosa PCR is as follows.

[0100] See Example 3 for details of the PCR conditions.

EXAMPLE 8 Selectivity of Pseudomonas aeruginosa PCR Rapid Test To estimate the selectivity of the PCR test, genomic DNA of various organisms was isolated and fluorescent PC
Used in R test. The amount of PCR product generated is indicated as _Ct value (for threshold cycle, _Ct value see definition in Example 4).

[0102] In a rapid PCR test, only Pseudomonas aeruginosa gave a positive result. After 19 cycles (C _t = 19), a linear increase in fluorescence was measured only when using 10 ng of P. aeruginosa DNA. This PCR test was very specific. Even the closely related Pseudomonas putida and Pseudomonas fluoreszenz did not give a fluorescent signal in the PCR rapid test.

As a positive control, the same bacterial DNA as analyzed in the algQ PCR test was tested using a generic 16S rRNA PCR system (see Example 19). All bacterial DNA
Gave a positive signal with the 16S rRNA PCR system. That is, 16S rRNA PCR
Amplified all DNA, but only P. aeruginosa DNA was algQ
PCR amplification was enabled. The algQ system is specific for Pseudomonas aeruginosa.

Further, the generated PCR product was analyzed by electrophoresis (see Example 3). The size of the PCR product was 294 base pairs (not shown). Control sequencing of the PCR product confirmed that this was algQ DNA (not shown).

EXAMPLE 9 To determine the sensitivity of the rapid P. aeruginosa PCR test and the sensitivity of the linear P. aeruginosa PCR test, genomic P. aeruginosa DNA Was prepared and used in PCR experiments (FIG. 3). Pseudomonas aeruginosa (P
aeruginosa) genomic copy was used in fluorescent PCR (FIG. 3). The data shown are the mean and standard deviation of four independent experiments. The amount of emitted fluorescence, and thus the amount of PCR product generated, is indicated as the _Ct value. The PCR reaction was performed for 45 cycles or more.
C _t values of the water control (no NTC = template control) is 45.

The results show that DNA from three bacterial cells can be detected by fluorescent PCR.
The PCR rapid test allows for linear quantification of the P. aeruginosa genome used over 4 log levels (ie between 3 and 30,000 gfu).

Example 10 Detection of Escherichia coli Escherichia coli was detected by species-specific amplification of the murA gene sequence.

The specific region of the murA gene was analyzed by PCR rapid test (PCR qui
ck test) was used as a diagnostic target for development. Why did you choose this gene as a diagnostic target? The murA gene encodes the UDP-N-acetylglucosamine enolpyruvyl transferase enzyme and is an important structural gene in E. coli (Marquardt et al. 1992, J. Bacteriol. 174, 5748-5752). This enzyme catalyzes the first step in the synthesis of peptidoglycan (murein in E. coli, which represents an essential component of the bacterial cell wall). The composition of the cell wall is considered characteristic of the bacterial species. The murA nucleotide sequence of Escherichia coli was used to enter the closely related enterobacteriaceae species Enterobacter cloacye (En
terobacter cloacae). Because of the sequence differences identified, the murA gene was selected as a diagnostically diagnostic genetic marker to identify Enterobacteriaceae, an Enterobacteriaceae species.

As a result of the DNA sequence database comparison and the practical optimization procedure using various primer / probe combinations, the following murA-specific DNA sequences were
Determined as probe combination: Forward primer sequence (# 767 *): 5 'GTT CTG TGC ATA TTG ATG CCC GCG 3' [SEQ ID NO: 12] Probe (# 802): 5'-FAM-TCT GCG CAC CTT ACG ATC TGG TT-TAMRA 3 '[SEQ ID NO. 13] Reverse primer sequence (# 884): 5' GCA AGT TTC ACT ACC TGG CGG TTG 3 '(used as reverse complement) [ SEQ ID NO. 14] * This position refers to the DNA sequence published in Marquardt et al. 1992, J. Bacteriol. 174, 5748-5752 (Genebank: M92358).

The probes were manufactured by PE Applied Biosystems Division, Weiterstadt, Germany. They are fluorescent derivatives (FAM = 6-carboxyfluorescein)
Is a single-stranded oligonucleotide modified at its 5 ′ end and at its 3 ′ end with a rhodamine derivative (TAMRA = 6-carboxytetramethylrhodamine). Synthesis and purification were performed according to the instructions of PE Applied Biosystems.

Example 11 PCR conditions for detection of Escherichia coli The following conditions were found to be optimal by changing the concentrations of primers and probes, the concentrations of MgCl ₂ and glycerol, and the nucleotide composition.

[Table 1] PCR cycle profile for E. coli PCR:

[Table 2] See Example 3 for details.

Example 12 Selectivity of Escherichia coli PCR Rapid Test In order to evaluate the selectivity of the PCR test, genomic DNAs from various organisms were isolated and subjected to a fluorescent PCR test. The amount of PCR product generated was obtained as the _Ct value (threshold cycle; table).

List of DNA isolation products tested (10 ng of genomic DNA analyzed each hour)

[Table 3] Only the E. coli strain gave a positive result in the PCR rapid test. 16 PCR cycles (
After C _t = 16), a linear increase in fluorescence could only be measured using 10 ng of E. coli DNA. The PCR test was very specific. Even the closely related enterobacterial species, Enterobacter cloisy, did not give a fluorescent signal in the PCR rapid test (Table).

As a positive control, the same bacterial DNA analyzed in the murA PCR test (Table) was tested using the universal 16S rRNA PCR system (see Example 19). All bacterial DNA gave a positive signal by the 16S rRNA system (ie, all D
NA could be propagated by 16S rRNA PCR), but only E. coli DNA was capable of murA PCR amplification. The murA system is specific for E. coli.

Furthermore, the produced PCR product was analyzed by electrophoresis and Staphylococcus aureus (S.
taphylococcus aureus). The PCR product had a size of 142 base pairs (not shown). The control sequence of the PCR product confirmed that this was murA DNA (not shown).

Example 13 Selectivity of E. coli test In order to measure the selectivity of the E. coli PCR test, genomic E. coli DNA was prepared and used in PCR experiments (FIG. 4). The amount of E. coli genome copy used in fluorescent PCR was changed (
Figure 4). The data shown represent the mean and standard deviation of four independent experiments. The emitted fluorescence, ie the amount of PCR product produced, is indicated by the _Ct value. PCR reactions were performed for over 40 cycles. Water as a control: C _t value (NTC no template control) is 40
Met.

The results show that DNA from three bacterial cells can be detected by fluorescent PCR. PCR rapid tests exceed 6 log levels, ie, 3-3,000,
Allows linear quantification of the E. coli genome used between 000 gfu.

Example 14 Detection of Salmonella ssp. (Subspecies) Detection of Salmonella enterica (Salmonella enterica) species was performed using the specific amplification of the invA gene sequence according to the present invention.

The specific region of the invA gene was used as a diagnostic target to develop a rapid PCR test for Salmonella ssp. Why did you select this gene as a diagnostic target? The invA gene encodes a virulence factor unique to Salmonella. Various studies on numerous Salmonella have demonstrated that these bacterial species bind to epithelial cells. In this process, the actin system of the host cell is affected by the bacteria. In response, the host cell encloses the bacterial cell.
After complete containment, the bacteria are present in vesicles in the cytoplasm of the host cell. The so-called inv gene of Salmonella (invA-H) is involved in this invasion process. invA
Mutants of the gene bind to the host cell but are no longer incorporated as well. The inv gene sequence is highly conserved among Salmonella subspecies (Salye
rs and Whitt 1994, Salmonella Infection,
in: Bacterial Pathogenesis), published by ASM Press, Washington DC, p. 233).
The Salmonella invA gene was isolated and its nucleotide sequence was elucidated (
Galan and Curtis 1989, PNAS USA 86, 6383-7; Galan and Curtis 1991, Infection and Immunity 59, 2901-2908, and Yards et al. 1992, Mo.
l. Cell. Probes 6, 271-279). The invA gene has been implicated in the expression of Salmonella specific virulence mechanisms and is therefore a diagnostic genetic marker in the identification of Salmonella ssp. (Rahn et al. 1992, Mol. Cell. Prob.
es. 6, 271-279).

As a result of the DNA sequence database comparison and the practical optimization operation using various primer / probe combinations, the following invA-specific DNA sequences
It was determined as a probe combination.

Forward primer sequence (# 269 *): 5 'GTG AAA TTA TCG CCA CGT TCG GGC 3' [SEQ ID NO: 15]; Probe (# 333): 5'-FAM-CTT CTC TAT TGT CAC CGT GGT CCA- TAMRA 3 '[SEQ ID NO: 16]; reverse primer sequence (# 542): 5' GGT TCC TTT GAC GGT GCG ATG AAG 3 '[SEQ ID NO: 17]; (used as reverse complement) , Appl. Environ. Microbiol. 62, 804-808 (Genebank: U43237).

The probe was manufactured by PE Applied Biosystems Division (Weiterstadt, Germany). They are fluorescent derivatives (FAM = 6-carboxyfluorescein)
Is a single-stranded oligonucleotide whose 5 ′ end is modified by a rhodamine derivative (TAMRA = 6-carboxytetramethylrhodamine). Synthesis and purification were performed according to the instructions of PE Applied Biosystems.

Example 15 PCR Conditions for Detection of Salmonella The following conditions were found to be optimal by changing the concentrations of primers and probes and the concentration of MgCl ₂ .

[0124]

[Table 4] PCR cycle profile of Salmonella ssp .:

[Table 5] See Example 3 for details.

Example 16 Selectivity of Salmonella ssp. Rapid PCR Test To evaluate the selectivity of the PCR test, genomic DNA from various organisms was isolated and used in a fluorescent PCR test. The amount of the produced PCR product, indicated by C _t values (threshold cycle for C _t definitions, see Example 4).

List of DNA isolates tested (10 ng of genomic DNA was analyzed each hour)

[Table 6] Only Salmonella gave a positive result in the PCR rapid test. After 15 PCR cycles (C _t = 15), a linear increase in fluorescence could only be measured when using 10 ng of Salmonella ssp. DNA. The PCR test was very specific. Even closely related E. coli strains did not give a fluorescent signal in the PCR rapid test.

As a positive control, the same bacterial DNA analyzed in the invA PCR test was tested using the universal 16S rRNA PCR system. All bacterial DNA gave a positive signal by the 16S rRNA system. Therefore, all DNAs could be amplified by 16s rRNA PCR, but only Salmonella DNA could be amplified by invA PCR. The invA system is specific for Salmonella.

Furthermore, the PCR products produced were analyzed by electrophoresis. The PCR product had a size (not shown) of 287 base pairs. Depending on the control sequence of the PCR product,
This (not shown) was confirmed to be invA DNA.

Example 17 Selectivity of PCR Rapid Test In order to determine the selectivity of the Salmonella ssp. PCR test , genomic Salmonella typhimurium DNA was prepared and used in PCR experiments (FIG. 5). Various amounts of Salmonella
A copy of the Typhimurium genome was used in fluorescent PCR (FIG. 5). The data shown represent the mean and standard deviation of four independent experiments. The emitted fluorescence, ie the amount of PCR product produced, is indicated as the _Ct value. The PCR reaction was performed for more than 40 cycles. C _t values for the control in which water (control without NTC = template) was 40 der.

[0130] The results show that DNA from three bacterial cells can be detected by fluorescent PCR. The PCR rapid test is performed above 6 log levels, i.e., 3-3,000,000 gfu
Allows linear quantification of the genome of Salmonella typhimurium used between

Example 18 Release of DNA without Preliminary Accumulation in Nutrient Medium DNA from various test microorganisms was extracted and purified according to Boom et al., 1990 to obtain proteins and other PCR inhibitors (Quiagen Column Kit, 1995). It was removed and used for PCR amplification experiments.

Example 19 Detection of bacteria, detection of universal bacteria, was performed by specific amplification of the conserved 16S rRNA gene sequence according to the invention (SEQ ID NO: 5, see Example 24). Specific 16S rRNA-specific D
NA sequences have been conserved during evolution; therefore, they are present in the genomes of all bacteria and can be used as primers and probes in universal detection of bacteria (Relman 1993, Weisburg et al., 1991, J. Bacteriol. 173).

As a result of DNA sequence database comparison and practical optimization using various primer / probe combinations, the following 16S rRNA-specific DNA sequences were determined to be optimal primer / probe combinations. .

1. PCR probe 23 mer: 5′-FAM-TTA AGT CCC GCA ACG AGC GCA AC-TAMRA-3 ′ (probe 16S rRNA # 1090): [SEQ ID NO: 19]; The probe was a PE Applied Biosystems Division ( Weiterstadt, Germany). They are fluorescent derivatives (FAM = 6-carboxyfluorescein)
Is a single-stranded oligonucleotide modified at the 5 ′ end and at the 3 ′ end by a rhodamine derivative (TAMRA = 6-carboxytetramethylrhodamine). Synthesis and purification were performed according to the instructions of PE Applied Biosystems.

2. PCR primer 19 mer: 5′-GCA TGG CTG TCG TCA GCT C-3 ′ (Primer 16S rRNA forward # 1053 *) [SEQ ID NO: 18]; 20 mer: 5′-TGA CGG GCG GTG TGT ACA AG-3 '(Primer 16S rRNA reverse # 1386 *) [SEQ ID NO: 20]; * This position refers to the DNA sequence of the 16S rRNA gene (Weisburg et al., 1991, J.B.
acteriol. 173). Synthesis and purification of PCR primer oligonucleotides were performed according to the protocol of PE Applied Biosystems.

Example 20 PCR Conditions for Universal Detection of Bacteria The concentration of primers and probes and the concentration of MgCl ₂ , the temperature and cycle profile of PCR and the spacing of reporter dyes from quencher dyes were varied. The following conditions were found to be optimal: The following components were mixed in a PCR reaction vessel (PE Applied Biosystems, order no. N8010580):

[Table 7] For optimal reproduction of this result, in the so-called master mix at each PCR cycle, as many components of the PCR mix as possible are
Care must be taken to mix before use. Under standard conditions, only the DNA material to be tested (0-15.25 μl) is individually added as a component to each PCR reaction vessel.

The PCR cycle profile is as follows:

[Table 8] This formulation is a PCR device with a heating stage, for example, GeneAMP from Perkin Elmer.
Compatible with PCR instruments 2400 and 9600, and ABI Prism 7700 sequence detection system. See Example 3 for details.

After completion of the PCR reaction, the sample was transferred to a fluorimeter LS-50B in a Perkin Elmer microtiter plate containing an additional unit for detecting fluorescence. The measurement and quantification of the fluorescence emission is described in the manufacturer's instructions (PE Applied Biosystems, Weite
rstadt, Germany).

Example 21 Universal Bacterial PCR Rapid Test Selectivity To evaluate the selectivity of the PCR test, genomic DNA from various organisms was isolated and used in the universal PCR test (FIG. 6). The amount of PCR product produced is expressed in relative fluorescent units (r
elative fluorescenece units) (Fig. 6).

The developed PCR test selectively detects bacteria.

[0141] Changes in the signal intensity of the bacterial sample reflect changes in the amount of DNA used.

[0142] The PCR products produced were analyzed by electrophoresis. The PCR product had a size of 330 base pairs (not shown). Depending on the control sequence of these PCR products,
This was confirmed (not shown) to be 16S rRNA. The PCR rapid test is 16S rRNA specific.

Example 22 Salmonella DNA was prepared and used in PCR experiments to determine the sensitivity of a rapid test for detecting bacteria and the sensitivity of a linear PCR test. Various dilutions of DNA were made. Each dilution was prepared three times in parallel and used for PCR testing (FIG. 7). The amount of emitted fluorescence is indicated as the so-called RQ value.

The RQ value is calculated based on the fluorescence emission (R ⁺ ) of the reporter (R) in the PCR reaction using the template DNA (genomic Salmonella DNA in this case) and the fluorescence emission of the reporter in the PCR reaction using no DNA. (R ⁻ ). Therefore, R ⁻ corresponds to background radiation. Reporter radiation (R) and quencher radiation (Q
) Is expressed as a ratio. Quencher emission is not subject to changes during the PCR reaction, and thus represents an internal standard used for normalization.

The results show that DNA from 1-3 Salmonella bacteria can be detected by fluorescent PCR. The fluorescent emission produced by the 40 PCR cycles is significantly higher than the background emission.

Fluorescent PCR testing is at least above 4 log levels, ie 1-3 and 30,000 g
It allows for linear quantification of the Salmonella genome used between fu (FIG. 7).

Example 23 Product Testing Using Bacterial Rapid Test The application of the developed PCR rapid test was tested using a spiking experiment. 10 ml of WFI (water for injection, lot # 63022) was spiked with 50 gfu of Salmonella (5 gfu / ml). DNA was prepared from various spiked samples (Boom et al., 1990), purified (Qiagen, 1995) and analyzed in a rapid PCR test (FIG. 8).

It was possible to detect spiked Salmonella in the products to be tested. The amount detected was 90% of the amount of DNA used (FIG. 8). This value reflects the loss of material that occurs during the preparation of DNA from spiked products. Despite such a loss, 1-3 gfu / ml could be detected in the spiked product to be tested. On the other hand, Salmonella bacteria were not detected in the unspiked test product (FIG. 8). The sterility of the test products was proved using membrane filtration according to the EP method (1997).

[0149] Target genes for Examples 24 different organisms / group, primer and probe sequences SEQ ID NO 1: Staphylococcus aureus (Staphylococcus aureus) 5 'AGATGCACGT ACTGCTGAAA TGAG TAAGCT AATGGAAAAC ACATATAGAG ACGTGAATAT TGCTTTAGCT AATGAATTAA CAAAAATTTG CAATAACTTA AATATTAATG TATTAGTTGT GATTGAAATG GCAAACAAAC ATCCGCGTGT TAATATCCAT CAA CCTGGTC CAGGAGTAGG CGG TCATT GT TTAGCTGTTG ATCCGTACTT TATT 3 '(underlined primer and probe sequences); SEQ ID NO: 6 5' AGATGCACGT ACTGCTGAAA TGAG 3 '; SEQ ID NO: 75'-TAMRA-CCTGGTCCAG GAGTAGGCGG-FAM-3 used as complement); SEQ ID NO: 8 5 'GTTTAGCTGT TGATCCGTAC TTTATT 3' ( used as the reverse complement); SEQ ID NO: 2: Pseudomonas aeruginosa (Pseudomonas aeruginosa) 5 'CAGGC CTTCG ATGCCCTGAG CGGTATTC AG GCACCGGCGC CCAACGCCGA AGAACTCCAG CATTTC TGCC AATTGCTGCT GGACTATGTA TCTGCCGGAC ACTTCGAGGT CTACGAGCAA CTGACGGCGG AAGGCAAGGC CTTCGGCGAT CAGCGCGGCC TGGAGCTGGC CAAGCA GATC TTCCCCCGGC TGGAAGCCAT CACCGAATCC GCGCTGAACT TCAACGACCG CTGCGACAAC GGCGATTGCC GTGAAGGAGC CTGCCTCATC GCGGAG CTGA AGGTCCTGCG GCAACAGTT G CACGAACGCT 3 '(Underlined primer and probe sequences); SEQ ID NO: 9 5' - TAMRA - 3 '; SEQ ID NO: 11 5' CTGAAGGTCC TGCGGCAACA GTT 3 ' ( used as the reverse complement); SEQ ID NO: 3: E. (Escherichia coli) 5' AAAGTAGAAC GTAATG GTTC TGTGCATATT GATGCCCGCG ACGTTAATGT AT TCTGCGCA CCTTACGATC TGGTT AAAAC CATGCGTGCT TCTATCTGGG CGCTGGGGCC GCTGGTAGCG CGCTTTGGTC AGGG GCAAGT TTCACTACCT GGCGGTTG TA CGATCGGTGC GCGTCCGGTT GATCTACACA TTTCTGGCCT CGAACAATTA GGCGCGACCACCC TC 3 '(underlined primer and probe sequence); SEQ ID NO: 12 5''; SEQ ID NO: 14 5' GCAAGT TTCACTACCT GGCGGTTG 3 (Used as reverse complement ..); SEQ ID NO: 4: Salmonella · ssp (Salmonella ssp) 5 ' TGATTGAAGC CGATGCCG GT GAAATTATCG CCACGTTCGG GC AATTCGTT ATTGGCGATA GCCTGGCGGT GGGTTTTGTT GT CTTCTCTA TTGTCACCGT GGTCCA GTTT ATCGTTATTA CCAAAGGTTC AGAACGTGTC GCGGAAGTCG CGGCCCGATT TTCTCTGGAT GGTATGCCCG GTAAACAGAT GAGTATTGAT GCCGATTTGA AGGCCGGTAT TATTGATGCG GATGCCGCGC GCGAACGGCG AAGCGTACTG GAAAGGGAAA GCCAGCTTTA C GGTTCCTTT GACGGTGCGA TGAAG TTTAT 3 '(underlined primer and probe sequences); SEQ ID NO: 15 5' GTGAAATTAT CGCCACGTTC GGGC 3 '; SEQ ID NO: 16 5'-FAM-CTTCTCTATT GTCACCGTGG TCCA-TAMRA-3 '; SEQ ID NO: 17 5' GGTTCCTTTG ACGGTGAT GAAG 3 '(used as the reverse complement); SEQ ID NO: 5: bacterial (bacteria) 5' GCATGGCTGT CGTCAGCTC G TGTTGTGAAA TGTTGGG TTA AGTCCCGCAA CGAGCGCAAC CCTTATCCTT TGTTGCCAGC GGTCCGGCCG GGAACTCAAA GGAGACTGCC AGTGATAAAC TGGAGGAAGG TGGGGATGAC GTCAAGTCAT CATGGCCCTT ACGACCAGGG CTACACACGT GCTACAATGG CGCATACAAA GAGAAGCGAC CTCGCGAGAG CAAGCGGACC TCATA AAGTG CGTCGTAGTC CGGATTGGAG TCTGCAACTC GACTCCATGA AGTCGGAATC GCTAGTAATC GTGGATCAGA ATGCCACGGT GAATACGTTC CCGGGC CTTG TACACACCGC CCGTCA 3 ′ (underlined primer and probe sequences);
SEQ ID NO: 18 5 'GCATGGCTGT CGTCAGCTC 3'; SEQ ID NO: 19 5 '-FAM-TTAAGTCCCG CAACGAGCGC AAC-TAMRA-3'; SEQ ID NO: 20 5 'CTTGTACACA CCGCCCGTCA 3' (used as reverse complement); Example 25 primers and probes define detecting a target DNA sequence in variant equivalent specificity of sequences (100%) and comparable sensitivity (> 70%), such as sequences identified in example 24, the primer / probe sequence combinations and variants I do.

Forward primer Probe reverse primer <br/> Staphylococcus aureus (same PCR conditions as in Example 3) [SEQ ID NO: 6] AGATGCACGT ACTGCTGAAA TGAG / [SEQ ID NO: 7] TAMRA-CCTGGTCCAG GAGT
AGGCGG-FAM / [SEQ ID NO: 8] GTTTAGCTGT TGATCCGTAC TTTATT; [SEQ ID NO: 6] AGATGCACGT ACTGCTGAAA TGAG / [SEQ ID NO: 7] TAMRA-CCTGGTCCAG GA
GTAGGCGG-FAM / [SEQ ID NO: 23] CATTGTTTAGCTGT TGATCCGTAC T; [SEQ ID NO: 24] GCACGT ACTGCTGAAA TGAGTAAG / [SEQ ID NO: 7] TAMRA-CCTGGTCCAG GAG
TAGGCGG-FAM / [SEQ ID NO: 8] GTTTAGCTGT TGATCCGTAC TTTATT; Pseudomonas aeruginosa (same PCR conditions as in Example 7) [SEQ ID NO: 9] CTTCGATGCC CTGAGCGGTA TTC / [SEQ ID NO: 10] FAM-CCAACGCCGA AGAACT
CCAG CATTTC-TAMRA / [SEQ ID NO: 11] CTGAAGGTCC TGCGGCAACA GTT; [SEQ ID NO: 25] CAGGCCTTCG ATGCCCTGA GC / [SEQ ID NO: 10] FAM-CCAACGCCGA AGAACT
CCAG CATTTC-TAMRA / [SEQ ID NO: 11] CTGAAGGTCC TGCGGCAACA GTT; [SEQ ID NO: 9] CTTCGATGCC CTGAGCGGTA TTC / [SEQ ID NO: 10] FAM-CCAACGCCGA AGAACT
CCAG CATTTC-TAMRA / [SEQ ID NO: 26] GCTGAAGGTCC TGCGGCAACA G; Escherichia coli (PCR conditions are the same as in Example 11) [SEQ ID NO: 12] GTTCTGTGCA TATTGATGCC CGCG / [SEQ ID NO: 13] FAM-TCTGCGCACC TTAC
GATCTG GTT-TAMRA / [SEQ ID NO: 14] GCAAGTTTCA CTACCTGGCG GTTG; [SEQ ID NO: 27] TAGAACGTAA TGGTTCTGTGC AT / [SEQ ID NO: 13] FAM-TCTGCGCACC TTACG
ATCTG GTT-TAMRA / [SEQ ID NO: 14] GCAAGTTTCA CTACCTGGCG GTTG; [SEQ ID NO: 12] GTTCTGTGCA TATTGATGCC CGCG / [SEQ ID NO: 13] FAM-TCTGCGCACC TTA
CGATCTG GTT-TAMRA / [SEQ ID NO: 28] CTGGCCTCGA ACAATTAGGC GCG; [SEQ ID NO: 27] TAGAACGTAA TGGTTCTGTGC AT / [SEQ ID NO: 13] FAM-TCTGCGCACC TTAC
GATCTG GTT-TAMRA / [SEQ ID NO: 28] CTGGCCTCGA ACAATTAGGC GCG; Salmonella ssp. (Salmonella ssp) (Same PCR conditions as in Example 15) [SEQ ID NO: 15] GTGAAATTAT CGCCACGTTC GGGC / [SEQ ID NO: 16] FAM-CTTCTCTATTGTCAC
CGTGG TCCA-TAMRA / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG; [SEQ ID NO: 15] GTGAAATTAT CGCCACGTTC GGGC / [SEQ ID NO: 21] FAM-TT (T / C) GTTAT
TGGCGATAGCCTGGC-TAMRA / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG;
[SEQ ID NO: 15] GTGAAATTAT CGCCACGTTC GGGC / [SEQ ID NO: 22] TAMRA-TTCTCTGGATGG
TATGCCCGGTA-FAM / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG; Bacteria Bacteria (same PCR conditions as in Example 20) [SEQ ID NO: 18] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 19] FAM-TTAAGTCCCG CAACGAGC
GC AAC-TAMRA / [SEQ ID NO: 20] CTTGTACACA CCGCCCGTCA; [SEQ ID NO: 29] TGCATGGCTGT CGTCAGCTC / [SEQ ID NO: 19] FAM-TTAAGTCCCG CAACGAG
CGC AAC-TAMRA / [SEQ ID NO: 20] CTTGTACACA CCGCCCGTCA; [SEQ ID NO: 18] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 30] FAM-TTGGGTTAAGTCCCG CAA
CGAGC-TAMRA / [SEQ ID NO: 20] CTTGTACACA CCGCCCGTCA; Enterobacteriaceae (same PCR conditions as in Example 30) Mutant of primer and probe sequences: [SEQ ID NO: 44] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 46] FAM -TTAAGTCCCG CAACGAGC
GC AAC-TAMRA / [SEQ ID NO: 45] TTTATGAGGT CCGCTTGCTC; [SEQ ID NO: 50] GTGCTGCATG GCTGTCGTC / [SEQ ID NO: 46] FAM-TTAAGTCCCG CAACGAGC
GC AAC-TAMRA / [SEQ ID NO: 45] TTTATGAGGT CCGCTTGCTC; [SEQ ID NO: 44] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 51] FAM-AGTCCCGCAA CGAGCGCA
AC CC-TAMRA / [SEQ ID NO: 45] TTTATGAGGT CCGCTTGCTC Example 26 Failure of primer and probe sequences Failure Detects target DNA sequence with insufficient specificity (<100%) and sensitivity (<70%) A primer / probe sequence combination, such as the sequence specified in Example 24, is defined as a failed mutant. See diagram containing primers and probes.

Forward primer Probe reverse primer Staphylococcus aureus (PCR conditions same as in Example 3) [SEQ ID NO: 31] ATGCACGTAC TGCTGAAATG AG / [SEQ ID NO: 32] FAM-AACACATATA GAG
ACGTGAA TATTGC- TAMRA / [SEQ ID NO: 33] GTTTAGCTGT TGATCCGTAC TT; [SEQ ID NO: 6] AGATGCACGT ACTGCTGAAA TGAG / [SEQ ID NO: 32] FAM-AACACATATA GAG
ACGTGAA TATTGC-TAMRA / [SEQ ID NO: 23] CATTGTTTAGCTGT GATCCGTAC T; [SEQ ID NO: 24] GCACGT ACTGCTGAAA TGAGTAAG / [SEQ ID NO: 32] FAM-AACACATATA G
AGACGTGAA TATTGC-TAMRA / [SEQ ID NO: 8] GTTTAGCTGT TGATCCGTAC TTTATT; Pseudomonas aeruginosa (same PCR conditions as in Example 7) [SEQ ID NO: 9] CTTCGATGCC CTGAGCGGTA TTC / [SEQ ID NO: 34] FAM-CAATTGCTGC
TGGACTATGT ATCTG- TAMRA / [SEQ ID NO: 1] CTGAAGGTCC TGCGGCAACA GTT; [SEQ ID NO: 35] CAACGCCGA AGAACTCCAG CATTTC / [SEQ ID NO: 34] FAM-CAATTGCTGC T
GGACTATGT ATCTG-TAMRA / [SEQ ID NO: 11] CTGAAGGTCC TGCGGCAACA GTT; [SEQ ID NO: 9] CTTCGATGCC CTGAGCGGTA TTC / [SEQ ID NO: 36] FAM-AACGCCGA AGAACT
CCAG CATTTCTGC-TAMRA / [SEQ ID NO: 26] GCTGAAGGTCC TGCGGCAACA G; [SEQ ID NO: 9] CTTCGATGCC CTGAGCGGTA TTC / [SEQ ID NO: 36] FAM-AACGCCGA AGAACT
CCAG CATTTCTGC-TAMRA / [SEQ ID NO: 11] CTGAAGGTCC TGCGGCAACA GTT; Escherichia coli (same PCR conditions as in Example 11) [SEQ ID NO: 12] GTTCTGTGCA TATTGATGCC CGCG / [SEQ ID NO: 13] FAM-TCTGCGCACC
TTACGATCTG GTT-TAMRA / [SEQ ID NO: 37] CATTTCTGGC CTCGAACAAT TA; [SEQ ID NO: 27] TAGAACGTAA TGGTTCTGTGC AT / [SEQ ID NO: 38] FAM-CCGCTGGTAG CGC
G (T / C) TTTGG TCA-TAMRA / [SEQ ID NO: 14] GCAAGTTTCA CTACCTGGCG GTTG; [SEQ ID NO: 12] GTTCTGTGCA TATTGATGCC CGCG / [SEQ ID NO: 38] FAM-CCGCTGGTAG CG
CG (T / C) TTTGG TCA-TAMRA / [SEQ ID NO: 37] CATTTCTGGC CTCGAACAAT TA; [SEQ ID NO: 39] ATGAAGCTGC TAAGCCAGCT GGG / [SEQ ID NO: 13] FAM-TCTGCGCACC T
TACGATCTG GTT-TAMRA / [SEQ ID NO: 28] CTGGCCTCGA ACAATTAGGC GCG; [SEQ ID NO: 39] ATGAAGCTGC TAAGCCAGCT GGG / [SEQ ID NO: 38] FAM-CCGCTGGTAG CGC
G (T / C) TTTGG TCA-TAMRA / [SEQ ID NO: 28] CTGGCCTCGA ACAATTAGGC GCG; [SEQ ID NO: 39] ATGAAGCTGC TAAGCCAGCT GGG / [SEQ ID NO: 38] FAM-CCGCTGGTAG CGC
G (T / C) TTTGG TCA-TAMRA / [SEQ ID NO: 37] CATTTCTGGC CTCGAACAAT TA; [SEQ ID NO: 39] ATGAAGCTGC TAAGCCAGCT GGG / [SEQ ID NO: 38] FAM-CCGCTGGTAG CGC
G (T / C) TTTGG TCA-TAMRA / [SEQ ID NO: 14] GCAAGTTTCA CTACCTGGCG GTTG; Salmonella ssp. (Salmonella ssp.) (Same PCR conditions as in Example 15) [SEQ ID NO: 40] TTGAAGCCGA TGCCGGTGAA ATTAT / [SEQ ID NO: 16] FAM-CTTCTCTATTGT
CACCGTGG TCCA-TAMRA / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG; [SEQ ID NO: 40] TTGAAGCCGA TGCCGGTGAA ATTAT / [SEQ ID NO: 21] FAM-TT (T / C) GTTAT
TGGCGATAGCCTGGC-TAMRA / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG; [SEQ ID NO: 40] TTGAAGCCGA TGCCGGTGAA ATTAT / [SEQ ID NO: 22] TAMRA-TTCTCTGGAT
GGTATGCCCGGTA-FAM / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG; [SEQ ID NO: 40] TTGAAGCCGA TGCCGGTGAA ATTAT / [SEQ ID NO: 41] FAM-TTTGTTGTCT T
CTCTATTGT CACC-TAMRA / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG; [SEQ ID NO: 15] GTGAAATTAT CGCCACGTTC GGGC / [SEQ ID NO: 41] FAM-TTTGTTGTCT TC
TCTATTGT CACC-TAMRA / [SEQ ID NO: 17] GGTTCCTTTG ACGGTGCGAT GAAG; Bacteria Bacteria (same PCR conditions as in Example 20) [SEQ ID NO: 18] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 19] FAM-TTAAGTCCCG CAACGA
GCGC AAC-TAMRA / [SEQ ID NO: 42] AAGTCGTAAC AAGGTAACCA; [SEQ ID NO: 29] TGCATGGCTG TCGTCAGCTC / [SEQ ID NO: 19] FAM-TTAAGTCCCG CAA
CGAGCGC AAC-TAMRA / [SEQ ID NO: 42] AAGTCGTAAC AAGGTAACCA; [SEQ ID NO: 43] GGATTAGATA CCCTGGTAGTC / [SEQ ID NO: 30] FAM-TTGGGTTAAGTC
CCG CAACGAGC-TAMRA / [SEQ ID NO: 20] CTTGTACACA CCGCCCGTCA; [SEQ ID NO: 43] GGATTAGATA CCCTGGTAGTC / [SEQ ID NO: 30] FAM-TTGGGTTAAGTC
CCG CAACGAGC-TAMRA / [SEQ ID NO: 42] AAGTCGTAAC AAGGTAACCA; Enterobacteriaceae (same PCR conditions as in Example 30) [SEQ ID NO: 44] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 46] FAM-TTAAGTCCCG CAACGA
GCGC AAC-TAMRA / [SEQ ID NO: 45] TTTATGAGGT CCGCTTGCTC; [SEQ ID NO: 44] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 52] FAM-ATGTTGGGTT AAGTCC
CGCA ACG-TAMRA / [SEQ ID NO: 45] TTTATGAGGT CCGCTTGCTC; [SEQ ID NO: 50] GTGCTGCATG GCTGTCGTC / [SEQ ID NO: 52] FAM-ATGTTGGGTT AAGTCC
CGCA ACG-TAMRA / [SEQ ID NO: 45] TTTATGAGGT CCGCTTGCTC; [SEQ ID NO: 53] GCTGTCGTCA GCTCGTGTT / [SEQ ID NO: 46] FAM-TTAAGTCCCG CAACGA
GCGC AAC-TAMRA / [SEQ ID NO: 45] TTTATGAGGT CCGCTTGCTC; [SEQ ID NO: 53] GCTGTCGTCA GCTCGT GTT / [SEQ ID NO: 46] FAM-TTAAGTCCCG CAACG
AGCGC AAC-TAMRA / [SEQ ID NO: 54] AACTTTATGA GGTCCGCTTG C; [SEQ ID NO: 44] GCATGGCTGT CGTCAGCTC / [SEQ ID NO: 46] FAM-TTAAGTCCCG CAACGA
GCGC AAC-TAMRA / [SEQ ID NO: 54] AACTTTATGA GGTCCGCTTG C Development of a PCR rapid test for detection of Enterobacteriaceae The following example describes a developed, rapid test that includes all sequence variations and target sequences. .

(I) Rapid test for detection of enterobacteria including identification of target, probe and primer sequences (Examples 27-31) (II) Unsuccessful mutations in primer and probe sequences (Example 32) Example 27 Detection of Species from the Enterobacteriaceae Family To develop a diagnostic PCR rapid test for Enterobacteriaceae, the genes that must be found, on the other hand, are the detection of multiple species of the Enterobacteriaceae family. It had to have enough conserved regions to allow for, while also containing enough variable regions to exclude the detection of bacteria not belonging to the Enterobacteriaceae family. Bacterial 16S rRNA as target
By selecting the gene, both conditions were met.

The 16S rRNA gene, together with 23S rRNA and 5S rRNA, cooperates with ribosomal proteins to form a translational machinery for protein biosynthesis, the bacterial ribosomal DN.
Code A

As a result of the DNA sequence database comparison and practical optimization using various primer / probe combinations, the following specific DNA sequences were determined as the optimal primer / probe combinations. Following sequence comparison and practical optimization procedures, the following optimal combinations of primers and probes were determined for detection of enteric bacteria.

Forward primer (# 1053) 5'-GCA TGG CTG TCG TCA GCT C-3 '[SEQ ID NO: 44]; Reverse primer (# 1270) 5'-TTT ATG AGG TCC GCT TGC TC-3' [SEQ ID NO: 45]; Probe (# 1090) 5'-Fam-TTA AGT CCC GCA ACG AGC GCA AC-Tamra-3 '[SEQ ID NO: 46] The probe was manufactured by PE Applied Biosystems Division (Weiterstadt, Germany). They are fluorescent derivatives (FAM = 6-carboxyfluorescein)
Is a single-stranded oligonucleotide modified at the 5 ′ end and at the 3 ′ end by a rhodamine derivative (TAMRA = 6-carboxytetramethylrhodamine). Synthesis and purification were performed according to the instructions of PE Applied Biosystems.

The numerical designation of the oligonucleotide indicates the position of the main chain of the sequence of the Escherichia coli 16S rRNA published in 1978 by Brosius et al.

The position of these sequences within the 16S rRNA gene is shown in SEQ ID NO: 24. Primer
The size of the amplicon flanked by 1053 and 1270 is 238 bp.

Target sequence of the 16S rRNA gene, SEQ ID NO: 47 (forward primer # 1053) 5′-GCATGGCTGTCGTCAGCTC-3 ′ (5′-CTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAA 1082 GAAGCCCTTGGCACTCTGTCCACGACGTACCGACAGCATT sequence from SEQ ID NO: 5; primers for 3'-TCGTTCGCCTGGAGTATTT-5'(reverse primer # 1270) enterobacterial specific detection: SEQ ID NO: 49; TTAAGTCCCGCAACGAGCGCAAC-TAMRA-3'( TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCC 1137 ACAACCCAATTCAGGGCGTTGCTCGCGTTGGGAATAGGAAACAACGGTCGCCAGG GGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGAC 1192 CCGGCCCTTGAGTTTCCTCTGACGGTCACTATTTGACCTCCTTCCACCCCTACTG GTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCAT 1247 CAGTTCAGTAGTACCGGGAATGCTGGTCCCGATGTGTGCACGATGTTACCGCGTA ACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTC 1302 from TijitititishitishititishijishitijijieijishijishitishitishijititishijiCCTGGAGTATTTCACGCAGCATCAG) And probe position It shows a section of the sequence encoding the 16S rRNA. The numbers in the right margin of the sequence indicate the position of each of the last nucleotides in the row. The position refers to the sequence published by Brosius et al. (1978). Primers and probes are indicated according to their position in the 16S rRNA gene. FAM: a fluorescent derivative as a reporter, TAMRA: a tetramethylrhodamine derivative as a quencher.

Example 28 PCR Conditions for Enterobacteriaceae Detection Composition and Components of a TaqMan PCR Reaction Batch for Enterobacteria Detection: Column 1 lists the single components of the PCR reaction batch. are doing. The volume used per reaction batch is shown in column 2, while column 3 shows the final concentration of a single component in the reaction batch. Column 4 shows the amount of material for each single component in 50 μl of PCR. UNG
: Uracil N-glycosylase

[Table 9] The following PCR cycle profile was constructed for intestinal bacteria detection.

[0160]

[Table 10] PCR Profile for Enterobacteriace Detection: Column 1 lists the single components of the PCR reaction batch. The volume used per reaction batch is shown in column 2 while column 3 shows the final concentration of a single component in the reaction batch. Column 4 shows the amount of substance for each single component in 50 μl of PCR. UNG: uracil N-glycosylase.

Example 29 Selectivity of Enterobacteriaceae Detection: The Gram-negative family of enterobacteria belongs to the gamma group of proteobacteria (Ba
lows et al., 1991, Holt, 1989). Proteobacteria also include α, β, δ, and ε
Groups, as well as Amoebobacter, as well as some unclassified proteobacteria. FIG. 9 shows a simple classification diagram for classifying intestinal bacteria.

The similarity of DNA sequences of different species usually increases with increasing degree of association. Thus, the potential for unwanted cross-reactions is greater in closely related species than in less related species. Therefore, the specificity of the PCR rapid test developed for detection of enterobacteria was specifically examined for genomic DNA related to enterobacteria.

Thirty different Enterobacteriaceae and 14 non-Enterobacteriaceae bacterial species were tested.

All the Enterobacteriaceae genera tested were detected by the PCR rapid test developed here. In contrast, bacteria very close to Enterobacteriaceae, particularly members of the γ group, as well as distantly related bacteria, especially members of Firmicutes (gram-positive bacteria), show no response to the system. Was.

List of Enterobacteria tested: 1 ng of genomic DNA of each Enterobacteriaceae species listed in column 1 was used for the specificity test. The stock used can be inferred from column 2. Column 3 shows the results of each test in the PCR rapid test for intestinal bacteria as + (positive reaction) or-(negative reaction).

[0166]

[Table 11] List of test bacterial strains not belonging to Enterobacteriaceae: 2 ng of genomic DNA of each bacterial species listed in column 1 was used for specificity testing. Column 2 shows the species's attribution to a particular upper order. The strain used can be inferred from column 3. Column 4 shows the result of each HDL test in the PCR rapid test for enterobacteria as + (positive reaction) or-(negative reaction).

[0167]

[Table 12] Example 30 Sensitivity of the Rapid PCR Test In experiments to determine the sensitivity of the rapid PCR test for enterobacteria, genomic E. coli DNA of the ATCC 8739 strain was used as a representative of other Enterobacteriaceae. According to these experiments, the detection range of the developed rapid PCR test for enterobacteria ranges from less than 5 gfu (equivalent to 25 fg genomic DNA) to more than 5,000,000 gfu (equivalent to 25 ng genomic DNA) (FIG. 10). .

Even after 40 cycles, the no-template control (no intestinal bacterial DNA) was obtained using the P
No response to the CR rapid test.

Example 31 Analysis of Product Sterile water for injection (WFI, lot no. 63022) was tested. The test results showed that intestinal bacteria
It indicated the absence of DNA.

Example 32 Unsuccessful Variants of Primer and Probe Sequences Detecting target DNA sequences with insufficient specificity (<100%) and sensitivity (<70%), such as those identified in Example 27 , The primer / probe combination is defined as a failed mutant.

References relating to the Examples: Balows, A., Truper, H., Dworkin, M., Harder, W. and Schleifer, K.-H.
(1991), Prokaryotes: A Handbook on Bacterial Biology (The Prokaryotes: AH
andbook on the Biology of Bacteria), 2nd edition, Vol. 1-4, Springer Verlag
(New York, NY).

Brosius, J., Palmer, JL, Kennedy, JP and Noller, HF (1978), Complete nucleotide sequence of 16S ribosomal RNA from E. coli (Complete Nucleotide Se
quence of a 16S Ribosomal RNA Gene from Escherichia coli), Proc. Natl.
Acad. Sci. USA 75, 4801-4805.

Holt, J. (Editor) (1989), Bergey's Manual on Systematic Bacteriology (Bergey's
Manual of Systematic Bacteriology), 1st edition, Vol. 1-4, Williams & Willia
ms (Baltimore, MD) issued.

[Sequence list]

[Brief description of the drawings]

FIG. 1. DNA of all S. aureus strains used (lanes 2-5) (10 ng per lane, 2-14)
Was detected by cap8-0 primers (# 15297 and # 15485). In contrast,
A closely related species of Staphylococcus, namely S. epidermidis
DNA (lane 6) and DNA of other bacterial genera (lanes 7-11) were not detected. Fungal, fish and human DNA (lanes 12-14) were used as controls, showing no detection signal. NTC (= no template control) is a water control without DNA.

FIG. 2 is a function of DNA amount and threshold cycle.

FIG. 3 shows the number of copies and a threshold cycle.

FIG. 4. The amount of E. coli genome copy used in fluorescent PCR was varied.

FIG. 5 To determine the selectivity of the Salmonella ssp. PCR test, genomic Salmonella typhimurium DNA was prepared and used in PCR experiments. Different amounts of Salmonella typhimurium genome copies were used in fluorescent PCR.

FIG. 6: All bacteria used (Bacillus subtilis, Escherichia coli, Staphylococcus aur
eus), Salmonella typhimurium, Klebsiella pneumoniae (K
lebsiella pneumonia) and the DNA of Streptococcus faecalis
(1-10 ng) was detected with a 16S rRNA primer / probe set. Fungi (Neurospora crassa), plants (Arabidopsis thaliana) or humans (Perkin Elmer ABI 40184)
When using genomic DNA (10 ng) from 6), the measured fluorescence emission was
Control without NA).

FIG. 7: Fluorescence emission was used as a function of the amount of Salmonella DNA. In the rapid PCR test, Salmonella DNA was used in amounts isolated from 1-3, 50, 500, etc. bacteria. The amount of emitted fluorescence is indicated by the so-called RQ value. ^{RQ = (R + / Q)} - (R - / Q)

FIG. 8. Water for injection (10 ml assay volume) was analyzed in four independent experiments for the presence of bacteria. 250 fg of genomic Salmonella DNA (Figure 8, far left) was used as a positive control. In parallel, the test products were spiked with 50 gfu / 10 ml of Salmonella and then analyzed (each on the right). Individual results are shown.

FIG. 9: Schematic representation of the taxonomic relationship of the Enterobacteriaceae: Individual genera of the Enterobacteriaceae are the gamma of the proteobacteria classified as eubacteria
Belong to a group. This figure was the basis reflected for specificity testing. To detect the specificity of the developed rapid enterobacterial PCR rapid test, members of the gamma group and some members of other groups of proteobacteria were mainly used.

[10] The sensitivity of PCR rapid test enterobacteria: shows the resulting C _t values, as a function of the bacteria forming units (gfu) of intestinal bacteria used.

[Procedure amendment]

[Submission Date] February 19, 2001 (2001.1.29)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF Term (reference) 4B024 AA05 AA11 BA80 CA09 CA20 HA14 4B063 QA01 QA13 QA18 QQ06 QQ42 QQ52 QR08 QR55 QR62 QS25 QS34

Claims (3)

[Claims] Claims 1. Microbial contaminants in non-sterile products (including cosmetics and foods)
A test kit for detecting GMP guidelines), comprising: (a) forward primer (sequence: forward primer); (b) probe (sequence: probe); (c) reverse primer ( (D) a spacer between the forward primer and the probe; (e) optionally a spacer between the probe and the reverse primer; (f) optionally a spacer upstream from the forward primer; g) optionally comprising at least one DNA fragment comprising a spacer downstream from the reverse primer, and the sequence [(sequence: forward primer), (sequence: probe), and (sequence: reverse primer)] One, two or three nucleotides are substituted, deleted and / or Includes variants which have been input, the variant is essentially sequence [(the sequence: forward primer), (SEQ:
Probe) and (sequence: reverse primer)], with the probe binding to DNA, and the primer binding to DNA
Having the function of providing an extendable 3 'end for NA polymerase, the spacer comprising 0 to 40 nucleotides, and the DNA fragment comprising: (i) as a forward primer for Staphylococcus aureus SEQ ID NO: 6, SEQ ID NO: 7 as a probe and SEQ ID NO: 8 as a reverse primer, (ii) For Pseudomonas aeruginosa, SEQ ID NO: 9 as a forward primer, SEQ ID NO: 10 as a probe, and SEQ ID NO: 11 as a reverse primer, ( iii) For Escherichia coli, SEQ ID NO: 12 as a forward primer, SEQ ID NO: 13 as a probe, and SEQ ID NO: 14 as a reverse primer. (iv) For Salmonella ssp., SEQ ID NO: 15 as a forward primer, probe As SEQ ID NO: 16, and river SEQ ID NO: 17 as a primer, (v) SEQ ID NO: 18 for a bacterium (bacteria), SEQ ID NO: 19 as a probe, and SEQ ID NO: 20 as a reverse primer, (vi) Forward primer for enterobacteriaceae SEQ ID NO: 44, SEQ ID NO: 46 as a probe, and SEQ ID NO: 45 as a reverse primer, (vii) For enterobacteriaceae (16S rRNA), SEQ ID NO: 47 as a forward primer, SEQ ID NO: 48 as a probe, and as a reverse primer The test kit selected from the group consisting of SEQ ID NO: 49, or all the sequences complementary to the above sequences of SEQ ID NOS: 6 to 49. 2. A method for detecting microorganisms in a product, in particular a drug or cosmetic, comprising the following steps: a) the use of primers and fluorescently labeled probes having the appropriate sequence and its variants, (i) Staphylococcus aureus. (Staphylococcus aureus): SEQ ID NO: 6 as forward primer, SEQ ID NO: 7 as probe, and SEQ ID NO: 8 as reverse primer. (Ii) Pseudomonas aeruginosa: SEQ ID NO: 9 as forward primer, SEQ ID NO: as probe 10, and as a reverse primer, SEQ ID NO: 11, (iii) For Escherichia coli, SEQ ID NO: 12, as a forward primer, SEQ ID NO: 13, as a probe, and SEQ ID NO: 14, as a reverse primer, (iv) Salmonella ssp. ) For SEQ ID NO: 15 as forward primer SEQ ID NO: 16 as a probe and SEQ ID NO: 17 as a reverse primer, (v) For bacteria, SEQ ID NO: 18 as a forward primer, SEQ ID NO: 19 as a probe, and SEQ ID NO: 20 as a reverse primer, (vi) Enterobacteria (Enterobacteriaceae) SEQ ID NO: 44 as a forward primer, SEQ ID NO: 46 as a probe, and SEQ ID NO: 45 as a reverse primer. (Vii) Enterobacteria (16S rRNA) as a forward primer, SEQ ID NO: 47. SEQ ID NO: 48, and SEQ ID NO: 49 as reverse primer or all sequences complementary to the above sequences from SEQ ID NO: 6-49 b) Amplify DNA using PCR and c) Excite fluorescent dye Irradiation at a specific wavelength d) Measure and quantitate the emission of the excited fluorescent dye The method comprising: 3. The probe according to claim 2, wherein the probe is prepared based on TaqMan detection technology.
The method described in.