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
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • 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 relates to an analytical method to detect and identify microbial contaminations, preferably in pharmaceutical products and environments. Moreover, the invention relates to a test kit comprising nucleic acid primers and probes targeting species- specific sequences, thus allowing for detection and quantification of microbial contaminations in pharmaceutical products, cosmetics, raw materials, starting materials, intermediates and auxiliaries as well as production environment. Background of the invention
• EP for example, are based on the growth of those germs on specific media and subsequent determination of specific metabolic reactions. Methods of that kind are commercially available. However, the application of the specific methods, as for example described by the EP, has substantially the same disadvantages as the method described for the total count of viable cells (see above). In addition to those, the selectivity of the determination is reduced to differences in the metabolism of the microorganisms and therefore allows only for a very rough differentiation.
• microbiological quick tests which are based on the fact that living organisms consume ATP. This fact can be exploited performing a chemical reaction that emits light. The light intensity is correlated to the amount of living organisms present.
• the method itself is based on the presence of DNA.
• the DNA either already exists in single strands or the double stranded DNA (dsDNA) is split into single strands.
• Two oligonucletide primers added in excess, anneal onto a specific part of the DNA not too distant apart from each other.
• the specific segment defined between the two primers is replicated starting from a single strand DNA coil, i.e. from each of two strands. After polymerization two new coils, i.e. four strands, of identical composition are formed. These can then further replicated in another cycle. If these steps are repeated, they lead to an exponential increase of the presence of this specific segment of DNA (amplification).
• An improved methodology is based on creating an oligonucleotide probe that fits between the two primers and sits on the segment to be replicated.
• This methodology is based on the TaqMan ® -Technology. It is based on the 5'-nuclease activity of Taq Polymerase, published in 1991 by Holland et al. (Holland et al., 1991; Holland et al., 1993; Lee et al., 1993; Gelfand et al. US Patent 5,210,015), exploiting the 5'-nuclease activity of the TaqPolymerase and the application of fluorescence marked and sequence specific probes.
• Those probes are marked at the 5 '-end with a fluorescing agent (reporter) and at the 3 '-end with a fluorescence quencher (or dark quencher).
• a fluorescing agent reporter
• a fluorescence quencher or dark quencher
• the fluorescence emitted by the reporter is quenched by the quencher, so that no fluorescence can be observed.
• the 5' nuclease activity of Taq degrades the probe, releasing the reporter. The more reporter molecules are released, the higher the intensity of the fluorescence signal results.
• the quantity of the fluorescence signal is proportional to the amount of sequences replicated and by an analysis of the kinetics, i.e. the number of cycles that were needed to obtain a certain signal, the initial number of copies of that specific sequence can be calculated. This provides a highly reliable indicator of the contaminant organism's presence.
• This method is extremely sensitive as it replicates the sequences present and hence intensifies the signal to be recorded with each cycle.
• genomic DNA is very stable and therefore not indicative for living organisms.
• the presence of genomic DNA does not really indicate that the organism it stems from is alive and therefore they cannot distinguish between living and dead microbial contaminants.
• an analytical method capable of indicating the presence of live microbial contaminants in pharmaceutical and cosmetic preparations.
• the present invention solves this problem by providing a new analytical method and test kit able to qualitatively and quantitatively detect and identify live microbial contamination.
• the present invention discloses a method for the detection and identification of living microbial contaminant cells in pharmaceutical products, pharmaceutical production environments, cosmetics and food.
• the method of the invention is based on the selective amplification by quantitative PCR of specific target cDNA sequences, in particular sequences encoding ribosomal RNA of the microbial contaminant to be detected.
• the invention also provides for a test kit for the detection and identification of living microbial contaminant cells in pharmaceutical products, pharmaceutical production environments, cosmetics and food comprising primers and probes specific for a ribosomal RNA target gene of the microbial contaminant.
• the primary objective of the present invention is to develop a methodology that allows the detection and quantification of living microorganisms that are often found as contaminants in pharmaceutical products (e.g. drug substances, excipients, auxiliary materials), pharmaceutical production environment, as well as in other relevant areas that are to be controlled under the GMP guidelines.
• pharmaceutical products e.g. drug substances, excipients, auxiliary materials
• Microbial contaminants that can be detected by the new methodology includes, for example, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Bacillus subtilis, Candida albicans, Aspergillus niger and, more generally, the total amount of Fungi, Bacteria and Enterobacteriaceae present in the sample or in the environment.
• These organisms represent the general group of microbial contaminants, often referred to and quantified as colony forming units: the bacteria and the fungi.
• This methodology can also be applied to the detection, identification and quantification of viral contamination in the products and environments cited above.
• the characterizing feature of the new methodology consists of the application of
• RNA sequences contain messenger RNA, transfer RNA and ribosomal RNA and fragments thereof.
• the RNA sequences contain the transcripts of the genes selected as diagnostic target sequences for specific determination of the species, genus, family or class of contaminant microorganisms, as better described in the Examples section.
• RNA sequences are converted into cDNA via a reverse transcription step and subsequently used in a PCR, in particular TaqMan ® PCR, test.
• the specificity and sensitivity of a TaqMan ® PCR test are determined, besides the sequence of primer and probe, by the following parameters: denaturation temperature of the first PCR cycle; annealing temperature during the amplification phase; number of PCR cycles; use of PCR additives as, e.g., glycerine and / or formamide; use of 7-deaza-2- deoxy-GTP besides GTP in genes with a high G/C content; concentration of Mg 2+ ions in the PCR buffer; concentration of primer and probe; units of Taq DNA-polymerase; distance of the cis-oriented primers to the probe. All these parameters were considered during the design and setting of the TaqMan ® -PCR test of the invention.
• the invention provides a suitable and optimised combination of defined primer/probe pairs to be used in the PCR step of the inventive method, as well as an optimised RNA-extraction and reverse transcription procedure.
• the unique combination of these features enables the detection of the contaminant microorganism with high sensitivity and specificity, fulfilling the requirements for GMP provisions according to, for example, the European and US pharmacopoeias.
• PCR techniques employed in the present invention are those disclosed in US patents US 4,800,159, US 4,683,195, US 5,210,015.
• the method of the invention is superior under many respects to the methodologies described, e.g., in the European Pharmacopoeia (EP) ⁇ 2.6.12-13> or other national pharmacopoeias as the United States Pharmacopoeia (USP) or the Japanese Pharmacopoeia (JP) in their current versions and it has the potential to replace these methods completely after a complete validation of the procedure to be applied in a pharmaceutical production environment or for a particular product. Indeed, for the first time it is possible to detect all and only the contaminating gene-expressing bacteria and fungi, i.e. living microorganisms, without the need for prior cultivation of the microbial cell.
• EP European Pharmacopoeia
• USP United States Pharmacopoeia
• JP Japanese Pharmacopoeia
• the sensitivity of the new method allows the detection and identification of 1 or more bacterial or fungal cells in the sample of the product investigated, thus providing an enhanced safety of the product for the consumer.
• Non-proliferating microorganisms containing toxins difficult to detect, can be detected as well.
• the sensitivity of the present method depends upon the peculiar combination of target gene, primers and probes sequences, as well as cDNA amplification step.
• the present inventors have therefore compared the sensitivity of alternative PCR- based amplification systems, namely the TaqMan ® PCR and the SyberGreen (SYBR) technology. Both technologies make use of labelled probes.
• the PCR sensitivity allowed by the TaqMan ® PCR probe is less than the sensitivity achievable using the SyberGreen (SYBR) technology.
• the SYBR system uses a probe that binds to double stranded DNA, allowing, consequently, a higher sensitivity.
• the inventors found that, in the experimental condition of the invention, the SYBR system is less sensitive than the TaqMan ® system.
• PCR-based amplification of cDNA requires a careful selection of target sequences, suitable primers and probes.
• a suitable target sequence plays an important role in the successful application of the method of the invention, in particular for the amplification step via the TaqMan ® PCR procedure.
• cDNA fragments as short as possible have to be chosen. This improves and enlarges the possibilities of choosing primers and probes on the target sequence. Amplification of small fragments allows an easier determination of specific systems. Therefore, the use of minor groove binder probes (with dark quencher), is preferred.
• primer have to be between 10 to 30 base pairs long; the sequence of the probe has to fit between the primers sequences on the cDNA fragment to be amplified; the minor groove binder probes have to be between 14 and 18 base pairs long; the probe should have a content of G and C bases ranging from 40% to 60%; the melting temperature of the probe should be 8 to 12 °C above that of the primer; there should be no G base at the 5'-end of the probe; the sequence of the probe shouldn't contain more than 3 times the same base in a row; there should be no complementary sequence between primer and probe or within the primers and no hindering secondary structure for all primers and probe.
• Consequence of the limitation to the guidelines is that, for the achievement of the necessary specificity and sensitivity of a TaqMan ® PCR test, the choice of the diagnostic target sequence in the genome of the microorganisms to be determined, as well as the experimental determination of optimal primer- and probe sequences, are essential.
• the whole determination procedure is depending on a steady gene expression of the target sequence as the primary target of the tests is an RNA molecule. Therefore, the gene expression of the target sequences has to be checked carefully. It has been shown in many experiments (Abee and Wouters 1999, Int. J. Food. Microbiol. 15: 65-91; Penalva and Arst. 2002, Microbiol. Mol. Biol. Rev. 66:426-246), that even genes that were thought to be expressed during the whole life cycle of a microorganism are often either not expressed or expressed at a too low level to be detectable with the necessary sensitivity.
• the expression rate of single copy genes has been shown to satisfy the needs of the invention only in a few examples. Therefore, ribosomal RNAs expressed at high level during all stages of the life cycle of the mictoorganism, has been shown to solve this problem.
• the following target genes were chosen for the detection of steady gene expression:
• Escherichia coli GadA/B SEQ ID NO:l
• 5s rRNA SEQ JD NO:5
• 23S rRNA SEQ ID NO:9
• Pseudomonas aeruginosa OprL SEQ ID NO: 13
• 16s rRNA SEQ ID NO: 16
• Bacillus subtilis GyrA (SEQ ID NO:21) and 16s rRNA (SEQ ID NO:22)
• SEQ ID NO:21 Bacillus subtilis GyrA
• 16s rRNA SEQ ID NO:16
• Salmonella enterica InvA SEQ ID NO:29
• 5s rRNA SEQ ID NO:33
• 23S rRNA SEQ ID NO:37
• Candida albicans 26S rRNA (SEQ ID NO:53)
• Bacteria 16S rRNA (SEQ ID NO: 57)
• sequences of forward primers, reverse primers and probes also include variants of the described ones where one, two or three nucleotides are substituted, deleted and/or inserted, provided, however, that such variants essentially fulfil the identical function as the sequence of the forward primer, reverse primer and probe from which they are derived.
• the method of the invention can also be used to detect viruses.
• the invention includes the detection of all kind of viruses where the procedure described can be applied.
• RNA viruses are a target of the methods described. These viruses can be detected during their infectious stage.
• the method is especially interesting for the detection of viruses as the systems does detect only active viruses which have infected a host cell.
• the procedure can also be used to test the sterility in pharmaceutical products.
• the invention includes the detection of all kinds of microorganisms that may form colonies. According to the definition of sterility, no viable microorganism of any kind should be present in any sample.
• the test looks for colony forming contaminants, that over a certain period of time (10 to 14 days) under most favourable conditions will form colonies that then can be visually detected by rendering the test solution opalescent.
• the invention has the ability to generally not only test for bacteria and fungi but also for those microorganisms, that do not form colonies or do not spontaneously proliferate, the application of the test procedure, reagents, and substances will be superior to the actual compendial testing methods.
• the invention provides for a method for the detection and identification of 1 or more living microbial contaminant cells in pharmaceutical products, pharmaceutical production environments, cosmetics and food, comprising the following steps: extracting total RNA from the microbial contaminant present in the sample; retro-transcribing the RNA into cDNA; subjecting the cDNA thus obtained to TaqMan ® PCR amplification using the following primers and probes specific for a ribosomal RNA target gene of the microbial contaminant:
• SEQ ID NO:26 as forward primer
• SEQ ID NO:27 as probe
• SEQ J-D NO:28 as reverse primer
• SEQ ID NO:38 as forward primer SEQ ID NO:39 as probe and SEQ ID NO:40 as reverse primer (v) for Staphylococcus aureus:
• SEQ ID NO:46 as forward primer
• SEQ ID NO:47 as probe
• SEQ ID NO:48 as reverse primer
• SEQ ID NO:50 as forward primer
• SEQ ID NO:51 as probe and SEQ ID NO:52 as reverse primer (vii) for Candida albicans:
• SEQ ID NO:54 as forward primer
• SEQ ID NO:55 as probe
• SEQ ID NO: 56 as reverse primer (viii) for Bacteria:
• SEQ ID NO:58 as forward primer
• SEQ ID NO:59 as probe
• SEQ ID NO:60 as reverse primer (ix) for Fungi:
• SEQ ID NO:62 as forward primer
• SEQ ID NO:63 as probe
• SEQ ID NO: 64 as reverse primer
• subjecting the amplified cDNA to a source of light of specific wavelength that excitate the fluorescing label present on the probe
• quantifying the emitted fluorescence signal as a measure of the number of the living microbial contaminants present in the sample.
• the invention provides for a test kit for the detection and identification of 1 or more living microbial contaminant cells in pharmaceutical products, pharmaceutical production environments, cosmetics and food, comprising the following primers and probes specific for a ribosomal RNA target gene of the microbial contaminant: (i) for Escherichia coli:
• SEQ ID NO: 10 as forward primer
• SEQ J_D NO: 11 as probe
• SEQ ID NO: 12 as reverse primer (ii) for Pseudomonas aeruginosa: SEQ ID NO: 18 as forward primer SEQ ID NO: 19 as probe and SEQ ID NO:20 as reverse primer;
• SEQ ID NO:26 as forward primer
• SEQ ID NO:27 as probe
• SEQ ID NO:28 as reverse primer
• SEQ U) NO:38 as forward primer SEQ ID NO:39 as probe and SEQ J-D NO:40 as reverse primer
• SEQ ID NO:46 as forward primer
• SEQ JJD NO:47 as probe
• SEQ ID NO:48 as reverse primer
• Aspergillus niger
• SEQ ID NO:50 as forward primer
• SEQ ID NO:51 as probe
• SEQ ID NO: 52 as reverse primer (vii) for Candida albicans:
• SEQ ID NO: 54 as forward primer
• SEQ ID NO:55 as probe
• SEQ ID NO:56 as reverse primer
• SEQ ID NO:58 as forward primer
• SEQ ID NO:59 as probe
• SEQ ID NO: 60 as reverse primer (ix) for Fungi:
• SEQ ID NO:62 as forward primer
• SEQ ID NO:63 as probe
• SEQ ID NO:64 as reverse primer
• the inventors firstly provide a general method for the isolation and cleaning of Ribonucleic acid (RNA) of bacteria and fungi.
• RNA Ribonucleic acid
• Examples 1 to 58 the application of the inventive quantitative PCR detection method for the identification of microbial contaminants is presented.
• Examples 59 to 62 the application of the method of the invention for testing water, surfaces, pharmaceutical packaging material and pharmaceutical products, respectively, is presented.
• the filter is transferred in a sterile tube, for example a 1.5 ml tube.
• the filter is treated with lysis buffer in order to lyse the microorganisms present in the filter, for example the filters are treated with 300 ⁇ l of lysis buffer for 30 min at room temperature.
• the testing material is present on solid growing medium. • The proliferated cells are resuspended in sterile water.
• the number of cells present has to be measured for example using the reading of OD (optical density) at 600 nm on a spectrophotometer and using solid growing medium to count the colony forming units (CFU).
• OD optical density
• CFU colony forming units
• the bacteria and fungi present in the samples are harvested by centrifugation at 130,000 x g for 10 min. Decant the supernatant, and carefully remove all remaining media by aspiration. • The cells are treated with 300 ⁇ l of lysis buffer for 30 min at room temperature buffer in order to lyse the microorganisms.
• the lysate is transferred in an appropriate apparatus for further automatic procession or applying equivalent manual procedures.
• the standard microbial strains are the following:
• RNA preparation Prior to PCR analysis, the lysates have to be treated in order to obtain a clean RNA preparation. This includes purification from genomic DNA fragments. The purified RNA is then reverse-transcribed to the cDNA.
• RNA purification and cDNA synthesis can be performed using for example an automatic equipment or an equivalent manual procedures.
• the resulting cDNA can be used for further determinination of the cell number in the starting sample with quantitative PCR.
• RNA/DNA Archive RNA/DNA Archive and cDNA Archive.
• Escherichia coli can be detected targetting the sequence of the GadA/B gene. Specific areas of the GadA/B gene served as diagnostical target for the development of a rapid detection kit for the detection of E. coli.
• the GadA/B gene encodes for the - decarboxylation of L-glutamatic acid to yield ⁇ -aminobutic acid and carbon dioxide. It has been reported that the enzyme is limited to E. coli (Mc Daniels et al. 1996 Appl. Environ. Microbiol. 62, 3350-3354; Smith et al. 1992 J. Bacteriol. 174, 5820-5826). Therefore it was chosen to serve as a genetic marker to detect the enterobacteria specie E. coli.
• Reverse primer sequence 5' GCC TCG GAA GAA CCA ATG GT [SEQ ED NO:4]
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 10 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 5 log steps, i.e. between 10 and 100000 cfu.
• Reverse primer sequence 5' GGG TGC GCT CTA CCA ACT GA 3' [SEQ ID NO: 8]
• Escherichia coli can be detected targetting the sequence of the 23 S rRNA gene.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 1 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 7 log steps, i.e. between 1 and 1000000 cfu.
• Pseudomonas aeruginosa can be detected targetting thesequence of the oprL gene.
• the outer membrane proteins of P. aeruginosa play important roles in the interaction of the bacterium with the environment (Hanock et al. 1990, Mol. Microbiol. 4, 1069-1075).
• the oprL genes are specific outer membrane lipoprotein genes for P. aeruginosa (De Vos et al. 1997, J. Clinic. Microb. 35, 1295-1299).
• the coding genes for the protein have been implicated in efflux transport systems or affect cell permeability, the oprL gene represents a molecular marker with diagnostical power to detect R. aeruginosa.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 1000 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 3 log steps, i.e. between 1000 and 100000 cfu.
• Pseudomonas aeruginosa can be detected targetting thesequence of the 16S rRNA gene.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 1 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 7 log steps, i.e. between 1 and 1000000 cfu.
• Bacillus subtilis can be detected targetting the sequence of the gyrA gene.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 10 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 5 log steps, i.e. between 10 and 100000 cfu.
• Bacillus subtilis can be detected targetting thesequence of the 16S rRNA gene.
• Probe 5' - FAM - CAA TCC CAC AAA TCT GTT CT - MGB - 3' [SEQ ID NO: 27]
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 1 bacterial cell could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 7 log steps, i.e. between 1 and 1000000 cfu.
• Salmonella enterica can be detected targetting thesequence of the invA gene.
• Specific areas of the invA gene served as diagnostical target for the development of a rapid detection kit for the detection of S. enterica.
• the invA gene encodes for a specific Salmonella virulence factor. Different investigations have shown that these bacteria are binding to epithelia cells. The host cells are enclosing the bacterial cells. At this process the invA genes are involved. As the invA gene is involved in a specific virulence mechanism of Salmonella, the gene has the power to serve as a genetic marker to detect Salmonella ssp. (Rahn et al. 1992, Mol. Cell. Probes. 6, 271-279).
• Probe 5' - FAM - CGC CGC CAA ACC - MGB - 3' [SEQ ID NO: 31]
• the probe was manufactured by the company Applied Biosystems, Rothstadt,
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Example 27 PCR conditions for the detection of S. enterica After variation of primer and probe concentration following conditions aroused as optimal:
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA extraction Different amounts of cDNA of S. enterica were deployed in the fluorescence PCR.
• the number of starting cells for RNA extraction and the Ct values are given in the following table.
• the Ct values are mean values of six autonomous replications.
• RNA of 10 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 5 log steps, i.e. between 10 and 100000 cfu.
• Example 30 In addition we are strongly convinced that the claimed primer and probe derived form the 5s rRNA of S. enterica will fulfil the desired attributes to detect S. enterica exclusively.
• Salmonella enterica can be detected targetting thesequence of the 23 S rRNA gene.
• the probe was manufactured by the company Applied Biosystems, Rothstadt,
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA extraction Different amounts of cDNA of S. enterica were deployed in the fluorescence PCR.
• the number of starting cells for RNA extraction and the Ct values are given in the following table.
• the Ct values are mean values of six autonomous replications.
• RNA of 1 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 7 log steps, i.e. between 1 and 1000000 cfu.
• Staphylococcus aureus can be detected targetting thesequence of the nuc gene.
• S. aureus is coagulase positive and produce a thermostable nuclease, which is encoded by the nuc gene. It is a nuclease specific for Staphylococcus aureus. It is an enzyme that degrades nucleic acids of the host (Kuroda et al. 2001, The Lancet. 357, 1225-1240). Therefore it was chosen to serve as an genetic marker to detect the enterobacteria specie S. aureus.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA extraction Different amounts of cDNA of S. aureus were deployed in the fluorescence PCR.
• the number of starting cells for RNA extraction and the Ct values are given in the following table.
• the Ct values are mean values of six autonomous replications.
• RNA of 10 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 5 log steps, i.e. between 10 and 100000 cfu.
• Stafilococcus aureus can be detected targetting thesequence of the 16S rRNA gene.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA extraction Different amounts of cDNA of S. aureus were deployed in the fluorescence PCR.
• the number of starting cells for RNA extraction and the Ct values are given in the following table.
• the Ct values are mean values of six autonomous replications.
• RNA of 1 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 7 log steps, i.e. between 1 and 1000000 cfu.
• Aspergillus niger can be detected targetting thesequence of the 28S rRNA gene.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• FAM fluorescence derivate
• MGB Minor Groove Binder molecule
• Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 1 fungal cell could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 7 log steps, i.e. between 1 and 1000000 cfu.
• Candida albicans can be detected targetting thesequence of the 26S rRNA gene.
• Probe 5' - FAM - CGG CAG GAT AAT GG - MGB - 3' [SEQ ID NO: 55]
• the probe was manufactured by the company Applied Biosystems, Rothstadt,
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany.
• the primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA was extracted from different organisms and a following Reverse Transcriptase step was performed to obtain cDNA.
• the cDNA was used to perform a fluorescence PCR Test.
• the amount of amplified PCR products was listed as the Ct value (Threshold Cycle) in following table:
• RNA of 1 fungal cell could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 6 log steps, i.e. between 1 and 100000 cfu.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• the probe is a single stranded oligonucleotide which was labelled at the 5' end with fluorescence derivate (VIC) and at the 3' end with a Minor Groove Binder molecule (MGB). Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the probe was VIC labelled to be able to run multiplex real time PCR. Therefore it might be possible to quantify in one PCR reaction the amount of bacterial contamination in addition to the determination of specific pathogens.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany. The primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA of different organisms was extracted and in a subsequent step transcribed to cDNA.
• the cDNA was amplified with the universal bacteria detection test.
• the developed PCR test detects selective bacteria. Following bacteria were tested and were amplified:
• RNA extraction Different amounts of cDNA of a mixture of five different bacteria were deployed in the fluorescence PCR.
• the number of starting cells for RNA extraction and the Ct values are given in the following table.
• the Ct values are mean values of six autonomous replications.
• RNA of 5 bacterial cells could be detected using fluorescence PCR.
• the PCR detection test allows a linear quantification about 6 log steps, i.e. between 5 and 500000 cfu.
• the probe was manufactured by the company Applied Biosystems, Rothstadt, Germany.
• the probe is a single stranded oligonucleotide which was labelled at the 5' end with fluorescence derivate (VIC) and at the 3' end with a Minor Groove Binder molecule (MGB). Manufacturing and purification was performed according to the instructions of Applied Biosystems.
• the probe was VIC labelled to be able to run multiplex real time PCR. Therefore it might be possible to quantify in one PCR reaction the amount of fungal contamination in addition to the determination of specific pathogens.
• the primers were manufactured by the company MWG Biotech, Ebersberg, Germany. The primers are single stranded oligonucleotides which are not modified. Manufacturing and purification was performed according to the instructions of MWG Biotech.
• Example 56 PCR conditions for the detection of all fungi
• Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).
• RNA of different organisms was extracted and in a subsequent step transcribed to cDNA.
• the cDNA was amplified with the universal fungi detection test.
• the developed PCR test detects selective fungi. Following fungi were tested and were amplified:
• RNA extraction Different amounts of cDNA of a mixture of two different fungi were deployed in the fluorescence PCR.
• the number of starting cells for RNA extraction and the Ct values are given in the following table.
• the Ct values are mean values of six autonomous replications.
• the PCR detection test allows a linear quantification about 6 log steps, i.e. between 5 and 200000 cfu.
• Example 59 Application to Testing of Water Human pathogens (mainly enteric bacteria, fungi and viruses) introduced into the water system used as source of manufacturing for example pharmaceutical products, are viable in water and therefore pose significant health risks. So their absence has to be controlled, water and therefore pose significant health risks.
• Traditional techniques used to examine water for the presence of pathogens rely mainly on the culturing of non-pathogenic indicator organisms for detection by inference. The methods used are slow, are unable to distinguish between closely related pernicious or benign strains, and fail to detect viable but non-prolifering bacteria.
• this project focuses on developing a rapid molecular method using the real-time polymerase chain reaction (PCR) to test water for the presence and quantification of specific pathogens and unspecific contaminations by bacteria, fungi, and viruses.
• PCR polymerase chain reaction
• a real-time PCR protocol was developed to detect Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enterica, Staphylococcus aureus, Aspergillus niger, Candida albicans and two different systems to detect all possible fungal and bacterial contaminants based on unique RNA sequences (see above), and will be used to examine questions regarding relationships between survival/occurrence of indicator organisms and pathogens in water.
• Human pathogens mainly enteric bacteria, fungi and viruses introduced into the production system used for manufacturing pharmaceutical products can pose significant health risks.
• Traditional techniques used to examine surfaces for the presence of pathogens rely mainly on the culturing of non-pathogenic indicator organisms for detection by inference. The methods used are slow, are unable to distinguish between closely related pernicious or benign strains, and fail to detect viable but non-prolifering bacteria.
• this project focuses on developing a rapid molecular method using the real-time polymerase chain reaction (PCR) to test surfaces for the presence and quantification of specific pathogens and unspecific contaminations by bacteria, fungi, and viruses.
• PCR real-time polymerase chain reaction
• the new protocol would obviate the need to culture organisms for detection, and could remedy shortcomings of traditional techniques by allowing rapid, sensitive, and specific identification of the pathogens of concern rather than indicator organisms.
• a real-time PCR protocol was developed to detect Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enterica, Staphylococcus aureus, Aspergillus niger, Candida albicans and two different systems to detect all possible fungal and bacterial contaminants based on unique RNA sequences (see above), and will be used to examine questions regarding relationships between survival/occurrence of indicator organisms and pathogens on surfaces of the production plant.
• Human pathogens mainly enteric bacteria, fungi and viruses introduced into Pharmaceutical Packaging Material pose significant health risks.
• Traditional techniques used to examine pharmaceutical packaging material for the presence of pathogens rely mainly on the culturing of non-pathogenic indicator organisms for detection by inference. The methods used are slow, are unable to distinguish between closely related pernicious or benign strains, and fail to detect viable but non-prolifering bacteria.
• this project focuses on developing a rapid molecular method using the real-time polymerase chain reaction (PCR) to test pharmaceutical packaging material for the presence and quantification of specific pathogens and unspecific contaminations by bacteria, fungi, and viruses.
• PCR real-time polymerase chain reaction
• the new protocol would obviate the need to culture organisms for detection, and could remedy shortcomings of traditional techniques by allowing rapid, sensitive, and specific identification of the pathogens of concern rather than indicator organisms.
• a real-time PCR protocol was developed to detect Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enterica, Staphylococcus aureus, Aspergillus niger, Candida albicans and two different systems to detect all possible fungal and bacterial contaminants based on unique RNA sequences (see above), and will be used to examine questions regarding relationships between survival/occurrence of indicator organisms and pathogens in pharmaceutical packaging material.
• Human pathogens mainly enteric bacteria, fungi and viruses introduced into pharmaceutical products can pose significant health risks.
• Traditional techniques used to examine pharmaceutical products for the presence of pathogens rely mainly on the culturing of non-pathogenic indicator organisms for detection by inference. The methods used are slow, are unable to distinguish between closely related pernicious or benign strains, and fail to detect viable but non-prolifering bacteria.
• this project focuses on developing a rapid molecular method using the real-time polymerase chain reaction (PCR) to test pharmaceutical products for the presence and quantification of specific pathogens and unspecific contaminations by bacteria, fungi, and viruses.
• PCR real-time polymerase chain reaction
• the new protocol would obviate the need to culture organisms for detection, and could remedy shortcomings of traditional techniques by allowing rapid, sensitive, and specific identification of the pathogens of concern rather than indicator organisms.
• a real-time PCR protocol was developed to detect Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enterica, Staphylococcus aureus, Aspergillus niger, Candida albicans and two different systems to detect all possible fungal and bacterial contaminants based on unique RNA sequences (see above), and will be used to examine questions regarding relationships between survival/occurrence of indicator organisms and pathogens in pharmaceutical products. This will include testing for absence of indicator germs, quantitative microbial contamination and sterility.
• Penalva MA Arst HN Jr.,2002 Regulation of gene expression by ambient pH in filamentous fungi and yeasts. Microbiol. Mol. Biol. Rev.; 66:426-246

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