EP1565576A2

EP1565576A2 - Method for the detection of microorganisms in pharmaceutical products

Info

Publication number: EP1565576A2
Application number: EP03779871A
Authority: EP
Inventors: Cecilia Dazzi; Volker Eck; Luca Cantelli; Adrian Harri; Peter Brodmann; Ralf Seyfarth
Original assignee: Pharmacia Italia SpA
Current assignee: Nerviano Medical Sciences SRL
Priority date: 2002-11-20
Filing date: 2003-11-07
Publication date: 2005-08-24
Also published as: WO2004046375A3; AU2003288018A8; WO2004046375A2; AU2003288018A1

Abstract

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.

Description

Title: Method for the detection of microorganisms in pharmaceutical products

Field of the invention

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

The production of pharmaceuticals and cosmetics requires very strictly defined manufacturing environments and a vigorous control of the products for their quality under chemical, physical and microbiological respects. The rules to be applied in cosmetics are e. g. delineated in the European Guideline 93/35/EEC, and require that no health hazards may be caused by any cosmetic product. The rules governing the pharmaceutical production are much more precise and cover completely the requirements set forth for cosmetics. The rules are precisely elaborated in the European Guide on Good Manufacturing Practices (EU- GMP Guide) and the European Pharmacopoeia (EP chapter 2.6.1, 2.6.12-13), as well as other national and international guidelines, e. g. WHO GMP guidelines, CFR 21 (USA). In general, the requirements to be met for any contamination by microorganisms can be differentiated into the following classes:

• counting the total number of viable cells from bacteria or fungi expressed as colony forming units as well as viruses and their specific quantification method;

• confirming the absence of pathogens, specifically Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus faecalis, Salmonella enterica,

Bacillus subtilis, Candida albicans, Aspergillus niger and Enterobactericea as indicating germs;

• sterility as the complete absence of any viable cell. Several compendial as well as advanced methods are known to detect and identify microbial contaminants. In the first group, are those methods based on media and on metabolites. In the second group are methods based on chemoluminescence as well as on PCR techniques. The said methods are reviewed here below. Compendial methods based on media.

To determine the total viable count of microorganisms, conventional microbiological methods are used as laid out in the EP. Those methods are based on promoting growth of viable organisms in and on well defined media and under strictly defined conditions. The material to be used is commercially available as well as complete kits that can be employed. The methods as described for example in the EP bear the following disadvantages:

• Low efficiency, as the time to obtain a definitive result sums up to several days

• Lack of precision, as the limits of acceptance can vary by a factor of 5 (EP, 2.6.12)

• High degree of manually performed procedures and difficult to automate • Selective growth of only very highly replicating microorganisms on the media employed. Those replicating at lower rates might be overlooked; however, the requirement is that also the latter ones should be detected by the methodology

High costs for storage of media and auxiliary equipment

In drug products with bacteriostatic properties, those methods will not give reliable results as, because of the drug activity, growth is inhibited

Microorganisms that do not form colonies will be overlooked

Products that are formulated as solids, dispersions or ointments cannot be tested without additional sample preparation.

Compendial methods based on metabolites Microbiological methods for the specific determination of germs, described in the

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.

Advanced methods based on chemoluminescence

Another alternative methodology commercially available is for example the use of 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 disadvantage of this method, however, is the high variance of the ATP production in living cells, that is very dependant from the growth phase and the type of microorganism.

Advanced methods based on PCR techniques Other alternative methods commercially available are those directed to detect microbial contamination in food (Chen et al., 1997). They are not applicable to pharmaceutical goods as they are susceptible to contaminations by the PCR kit used and hence are of low reproducibility and yield questionable quantitative results. Moreover, they are very time consuming, as they imply an additional gel electrophoreses step. Finally, the documentation of the result is time and cost intensive.

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. In presence of DNA polymerase 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). As long as both fluorochromes are on the probe, the fluorescence emitted by the reporter is quenched by the quencher, so that no fluorescence can be observed. However, as Taq polymerase extend the primers, 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. There are many molecules that show fluorescence under diverse conditions, which allow to design internal standards that control the success of each replication. In addition, it is possible to test for the presence of different sequences in parallel using different wavelength to detect the resulting fluorescence of each one.

However, all these applications are based on detecting genomic DNA strands. The disadvantage of this is that 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. There is therefore the need of 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.

Summary of the invention

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.

Detailed description of preferred embodiments

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.

This goal is reached through a new and inventive application of PCR techniques, in particular of the TaqMan^® PCR, as will be better explained below.

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

PCR techniques, in particular of the TaqMan^® PCR, for the amplification of specific RNA sequences. The term RNA includes 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.

In the method of the invention, 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 Mg²⁺ 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.

In particular, 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. In the previously employed molecular biology methods, based on PCR amplification of genomic DNA, it was not possible to discriminate between living and dead contaminants as DNA, in contrast to RNA, is a much more stable nucleic acid that can be found undegraded in pharmaceutical preparation even when the contaminant microorganism is dead.

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. Below is a list of the advantages provided for by the presently disclosed methodology. • Spores and pathogen microorganisms difficult to cultivate can be easily detected.

• Non-proliferating microorganisms, containing toxins difficult to detect, can be detected as well.

• Because only live microorganisms are detected, the number of false positive signals will be decreased. This is a clear advantage for the producer of the products that have to be controlled.

• The results are available in a much shorter time compared to the cultivation-based methods. The products can be released in a much faster time than using the conventional method. This lowers the costs in development and production. These factors may lead to cheaper products. • The application has ecological advantages as the reduction of the time of analysis and reduction of analytical material decreases the costs for energy and waste management.

• There are no special safety requirements as no component of the method underlies a safety regulation. As stated above, 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. Generally the PCR sensitivity allowed by the TaqMan^® PCR probe (in particular the minor groove binder probe) is less than the sensitivity achievable using the SyberGreen (SYBR) technology. This is because the SYBR system uses a probe that binds to double stranded DNA, allowing, consequently, a higher sensitivity. Unexpectedly, 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.

The selection of 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. In this connection, 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.

In general, the following guidelines are followed in the design of cDNA amplification: 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.

Notwithstanding the general guidelines for the design of primers and probes (Livak et al. 1995), the optimal combination of primer and probe of each TaqMan^® PCR application has to be experimentally determined. In contrast to the expectations, it could be shown in a series of experiments that the development of an optimal TaqMan^® PCR system wasn't possible although all the above mentioned guidelines were followed. In contrast to the general expectations, it is sometimes necessary to choose primer and probe sequences that do not fulfil the above mentioned guidelines for the design of the systems because of the characteristics of the target sequence of the corresponding organisms (e.g. high GC- content, highly repetitive elements or conserved regions of sequences). 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:

Detected organisms Target gene

Escherichia coli GadA/B (SEQ ID NO:l), 5s rRNA (SEQ JD NO:5) and 23S rRNA (SEQ ID NO:9)

Pseudomonas aeruginosa OprL (SEQ ID NO: 13) and 16s rRNA (SEQ ID

NO: 17)

Bacillus subtilis GyrA (SEQ ID NO:21) and 16s rRNA (SEQ ID

NO:25)

Salmonella enterica InvA (SEQ ID NO:29), 5s rRNA (SEQ ID NO:33) and 23S rRNA (SEQ ID NO:37)

Staphylococcus aureus Nuc (SEQ ID NO:41) and 16s rRNA (SEQ ID

NO:45)

Aspergillus niger 28S rRNA (SEQ ID NO:49)

Candida albicans 26S rRNA (SEQ ID NO:53)

Bacteria 16S rRNA (SEQ ID NO: 57)

Fungi 18S rRNA (SEQ ID NO:61)

The sequences of the primers, probes and target genes (amplicons) selected for each contaminant species are reported in the experimental section of the present application and listed in the Sequence Listing section. The results obtained using as target gene the ribosomal RNAs, allow the detection of as few as 1-5 contaminant microbial cells, as can be appreciated in the experimental section. These results provide a considerable improvement in the sensitivity of the test compared to the teachings of the prior art.

The skilled in the art will recognize that the 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.

Moreover, the expert in the art will be able to select target sequences, within the selected target genes, different from those specifically exemplified herewith. The use of such alternative target sequences will of course fall within the scope of the present invention, as they just constitute an alternative embodiment of the same inventive concept. Indeed, the present invention provides for several possible target sequences, that fulfil the sensitivity requirements set forth above.

Also included within the scope of the invention is the combination of two or more target sequences in the detection method. 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. Especially, 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. Therefore, 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. As 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. In a more detailed description, 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:

(i) for Escherichia coli: SEQ ID NO: 10 as forward primer SEQ ID NO: 11 as probe and 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; (iii) for Bacillus subtilis:

SEQ ID NO:26 as forward primer SEQ ID NO:27 as probe and SEQ J-D NO:28 as reverse primer

(iv) for Salmonella enterica:

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 and SEQ ID NO:48 as reverse primer (vi) fox Aspergillus niger: 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 and

SEQ ID NO: 56 as reverse primer (viii) for Bacteria:

SEQ ID NO:58 as forward primer SEQ ID NO:59 as probe and SEQ ID NO:60 as reverse primer (ix) for Fungi:

SEQ ID NO:62 as forward primer SEQ ID NO:63 as probe and 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; and quantifying the emitted fluorescence signal as a measure of the number of the living microbial contaminants present in the sample.

Furthermore, 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 and

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;

(iii) for Bacillus subtilis:

SEQ ID NO:26 as forward primer SEQ ID NO:27 as probe and SEQ ID NO:28 as reverse primer (iv) for Salmonella enterica:

SEQ U) NO:38 as forward primer SEQ ID NO:39 as probe and SEQ J-D NO:40 as reverse primer (v) for Staphylococcus aureus: SEQ ID NO:46 as forward primer SEQ JJD NO:47 as probe and SEQ ID NO:48 as reverse primer (vi) for Aspergillus niger:

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 and SEQ ID NO:56 as reverse primer

(viii) for Bacteria:

SEQ ID NO:58 as forward primer SEQ ID NO:59 as probe and SEQ ID NO: 60 as reverse primer (ix) for Fungi:

SEQ ID NO:62 as forward primer SEQ ID NO:63 as probe and SEQ ID NO:64 as reverse primer; Examples In this section, the inventors firstly provide a general method for the isolation and cleaning of Ribonucleic acid (RNA) of bacteria and fungi. Secondly, under Examples 1 to 58, the application of the inventive quantitative PCR detection method for the identification of microbial contaminants is presented. In particular, Examples 1 to 9 refer to the identification of Escerichia coli; Examples 10 to 17 refer to the identification of Pseudomonas aeruginosa; Examples 18 to 25 refer to the identification of Bacillus subtilis; Examples 26 to 34 refer to the identification of Salmonella enterica; Examples 35 to 42 refer to the identification of Staphylococcus aureus; Examples 43 to 46 refer to the identification of Aspergillus niger; Examples 47 to 50 refer to the identification of Candida albicans; Examples 51 to 54 refer to the identification of all Bacteria; Examples 55 to 58 refer to the identification of all Fungi. Finally, in 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.

Isolation and cleaning of RNA from Bacteria and Fungi

A. Preparation of the product sample The procedure requires the following steps:

• Filter the sample using an appropriate filter, for example a 0,2μm pore size filter using a vacuum pump. In this way all the microbial contaminants are collected on the filter.

• 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 μl of lysis buffer for 30 min at room temperature.

• The lysate is transfered in an appropriate apparatus for further automatic processing or applying equivalent manual procedures.

B. Preparation of the standard

To quantify exactly the amount of viable cells present, the following procedure has to be applied:

• 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).

• Depending on the cell concentration a specific volume of the cell suspension is transferred into a sterile tube.

• 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 μl 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:

Escherichia coli strain ATCC 8739 Pseudomonas aeruginosa strain ATCC 9027 Bacillus subtilis strain ATCC 6633 Salmonella enterica strain NCTC 6017 • Staphylococcus aureus strain ATCC 6538

Aspergillus niger strain ATCC 16404 Candida albicans strain ATCC 10231

C. RNA extraction and cDNA synthesis

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.

This process of 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.

The following description may serve as an example of a procedure:

^■ Basically chemicals for analytical and molecular biology purpose should be used.

^■ The solutions operations should be carried out in a sterile area.

■ The use of aerosol-protected pipette tips serves as a protection against contamination.

■ If DNA is then inserted in the real-time PCR, free of powder gloves have to be used. he material to be used is:

Gloves, free of powder

Aerosol-protected pipette tips

20 ml glas tubes

Plastic loops

Different types of reaction tubes and racks

Different pipettes at different volumes

Microcentrifuge (at least 12,000 x g)

Vortex

Spectophotometer

ABI Prism 6700

The following products from Applied Biosystems may be used

4305673 TOTAL RNA PURIFICATION TRAY (10 PKG)

4311758 SPLASH GUARDS (20 PKG)

4308456 ABI PRISM 6700 SYSTEM FLUID 4.00 L

4306737 MICROAMP OPTICAL 96 WELL PLATE (20 PKG)

4311971 ABI PRISM OPTICAL ADHESIVE (10 PKG)

4304831 REAGENTS RESERVOIRS 120 ml (32 PKG)

4306377 TIPS 1000 μL (24 RACKS/PKG)

4306375 TIPS 200 μL (24 RACKS/PKG)

■ deionized sterile water

■ the following products from Applied Biosystems may be used

4308456 ABI PRISM 6700 SYSTEM FLUID 4.00 L

322171 High capacity cDNA Archive Kit

4305545 Abolute RNA Wash solution

4305895 LYSIS SOLUTION 250 ml

4305893 ELUTION SOLUTION 1.00 L 4305891 WASH SOLUTION 1 1 L

4305890 WASH SOLUTION II 1 L

■ When handling samples avoid contamination from single-use material (spatule, etc.) and solutions for decontamination (e.g. 14% Javelle- water), hydrochloric acid (pH 2), DNA-Away™", etc.).

^■ Transfer the lysates, prepared as previously described, into 96 well Falcon plate and cover it with lid.

^■ Transfer plate into the automated sample manipulation instrument, e. g. ABI Prism 6700. ^■ Control if all reagents and disposables are in the workstation. (Wash solution 1, Wash solution 2, AbsoluteRNA Wash Solution, Elution Solution, 200 and 1000 μl tips, splash guard, Total RNA Purification Tray, Microamp Optical 96 Well plate)

■ Activate following programs: RNA/DNA Archive and cDNA Archive.

■ Activate under RNA DNA Archive the program to fix the remaining RNA and get rid of the lysase the DNA lysis products

^■ Activate under cDNA Archive the transcription from RNA to cDNA

■ Run workstation. ^■ After finishing of the run, cover plate with adhesive covers and store at -80° C.

Example 1

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 γ-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.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following GadA/B DNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence:

5' CCT GCG CCG AAA AAT GG 3' [SEQ ID NO:2]

Probe:

5' - FAM - CAG GCC GTT GGC AC - MGB - 3' [SEQ ID NO:3]

Reverse primer sequence: 5' GCC TCG GAA GAA CCA ATG GT [SEQ ED NO:4]

The probe was manufactured by the company Applied Biosystems, Weiterstadt, Germany. The probe is a single stranded oligonucleotide which was labelled at the 5' end with fluorescence derivate (FAM = 6-carbooxyfluorescein) 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 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 2

PCR conditions for the detection of E. coli

After variation of primer and probe concentration following conditions aroused as optimal:

Total 20

Real-Time-PCR profile for specific systems for the detection of E. coli

Incubation of the PCR-plate (Microamp® Optical/96-well reaction plate) in the TaqMan using the following temperature-time-programme:

* systems to avoid "carry-over"-contamination. Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).

Example 3

Selectivity of the E. coli PCR detection test To evaluate the selectivity of the PCR test specific for E. coli, 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:

List of the tested cDNA samples:

Example 4

Sensitivity of the E. coli test

To determine the sensitivity of E. coli PCR test, cDNA was prepared and deployed in the PCR experiments.

Different amounts of cDNA of E. coli 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 result shows that 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 5

In addition we are strongly convinced that the claimed primer and probe derived form the 5 s rRNA of E. coli will fulfil the desired attributes to detect E. coli exclusively.

Due to cDNA sequence comparison and use of different primer and probe combinations following 5 s rRNA sequences were determined hypothetically as the optimal primer and probe combination:

Forward primer sequence:

5' GGA ACT GCC AGG CAT CAA AT 3' [SEQ ID NO: 6]

Probe:

5 ' - FAM AGC GTG CTG ATA TGG - MGB - 3 ' [SEQ ID NO: 7]

Reverse primer sequence: 5' GGG TGC GCT CTA CCA ACT GA 3' [SEQ ID NO: 8]

Example 6

Escherichia coli can be detected targetting the sequence of the 23 S rRNA gene.

Specific areas of the 23 S rRNA gene served as diagnostical target for the development of a rapid detection kit for the detection of E. coli.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following 23 S rRNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence:

5' GGGTGACAGCCCCGTACA 3' [SEQ ID NO: 10]

Probe:

5' - FAM - ATGCACATGCTGTGAGCTC - MGB - 3' [SEQ ID NO: 11] Reverse primer sequence:

5' CGTGTCCCGCCCTACTCAT 3' [SEQ ID NO: 12]

Example 7

PCR conditions for the detection of E. coli

Total 20 Real-Time-PCR profile for specific systems for the detection of E. coli using 23 S rRNA target gene

Example 8

Selectivity of the E. coli PCR detection test using 23 S rRNA target gene.

To evaluate the selectivity of the PCR test specific for E. coli using 23 S rRNA target gene, 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:

List of the tested cDNA samples:

Example 9

Sensitivity of the E. coli test

The result shows that 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.

Example 10

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). As 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.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following oprL DNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence: 5' CGT GCG ATC ACC TTC TA 3' [SEQ ID NO: 14]

Probe:

5' - FAM - TTC GAG TAC GAC AGC TC - MGB - 3' [SEQ ID NO: 15]

Reverse primer sequence:

5' CAT GGC TTC CGG CTT CAG 3' [SEQ ID NO: 16]

Example 11

PCR conditions for the detection of P. aeruginosa After variation of primer and probe concentration following conditions aroused as optimal:

Total 20

Real-Time-PCR profile for specific systems for the detection of P. aerusinosa

Example 12

Selectivity of the P. aerusinosa PCR detection test

To evaluate the selectivity of the PCR test specific for P. aeruginosa, 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:

List of the tested cDNA samples:

Example 13

Sensitivity of the P. aeruginosa test

To determine the sensitivity of P. aeruginosa PCR test, cDNA was prepared and deployed in the PCR experiments.

Different amounts of cDNA of P. aeruginosa 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 result shows that 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.

Example 14

Pseudomonas aeruginosa can be detected targetting thesequence of the 16S rRNA gene.

Specific areas of the 16S rRNA gene served as diagnostical target for the development of a rapid detection kit for the detection of P. aeruginosa. Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following 16S rRNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence: 5 ' CGC GTA GGT GGT TCA GCA A 3 ' [SEQ ID NO: 18]

Probe:

5' - FAM - TTG GAT GTG AAA TCC CCG G - MGB - 3' [SEQ ID NO: 19]

Reverse primer sequence:

5' GGA TGC AGT TCC CAG GTT GA 3' [SEQ ID NO: 20]

Example 15

PCR conditions for the detection of P. aerusinosa

Total 20

Real-Time-PCR profile for specific systems for the detection of P. aeruginosa using

16S rRNA target gene

* systems to avoid "carry-over"-contamination. Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG). Example 16

Selectivity of the P. aei-usinosa PCR detection test using 16S rRNA target gene.

To evaluate the selectivity of the PCR test specific for P. aeruginosa using 16S rRNA target gene, 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:

List of the tested cDNA samples:

Example 17 Sensitivity of the P. aerusinosa test

Example 18

Bacillus subtilis can be detected targetting the sequence of the gyrA gene.

Specific areas of the gyrA gene served as diagnostical target for the development of a rapid detection kit for the detection of B. subtilis. It was shown from comparative sequence analysis that the gyrA sequences provide a firm framework for accurate classification and identification of Bacillus subtilis (Chun and Bae, 2000, Antonie Van

Leeuwenhoek 78, 123-127).

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following gyrA DNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence:

5' AAC CTG CTT GCG CTT GTT G 3' [SEQ ID NO: 22]

Probe:

5' - FAM - TGG CCA GCC GAA AG - MGB - 3 ' [SEQ ID NO: 23] Reverse primer sequence:

5' TCC AGG CAT TGC TTA AGA GTT AAA 3 ' [SEQ ID NO: 24]

Example 19

PCR conditions for the detection of B. subtilis

Total 20

Real-Time-PCR profile for specific systems for the detection of B. subtilis Incubation of the PCR-plate (Microamp® Optical/96-well reaction plate) in the

TaqMan using the following temperature-time-programme:

• systems to avoid "carry-over"-contamination. Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).

Example 20

Selectivity of the B. subtilis PCR detection test

To evaluate the selectivity of the PCR test specific for B. subtilis, 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:

List of the tested cDNA samples:

Example 21 Sensitivity of the B. subtilis test

To determine the sensitivity of B. subtilis PCR test, cDNA was prepared and deployed in the PCR experiments.

Different amounts of cDNA of B. subtilis 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 result shows that 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 22

Bacillus subtilis can be detected targetting thesequence of the 16S rRNA gene.

Specific areas of the 16S rRNA gene served as diagnostical target for the development of a rapid detection kit for the detection of B. subtilis.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following 16S rRNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence:

5' AGC GAA ACC GCG AGG TTA A 3' [SEQ ID NO: 26]

Probe: 5' - FAM - CAA TCC CAC AAA TCT GTT CT - MGB - 3' [SEQ ID NO: 27]

Reverse primer sequence:

5' GCA GAC TGC GAT CCG AAC TG 3' [SEQ ID NO: 28]

Example 23

PCR conditions for the detection of B. subtilis

Total 20 Real-Time-PCR profile for specific systems for the detection of R. subtilis using 16S rRNA target gene

digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).

Example 24

Selectivity of the B. subtilis PCR detection test using 16S rRNA target gene.

To evaluate the selectivity of the PCR test specific for B. subtilis using 16S rRNA target gene, 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:

List of the tested cDNA samples:

Example 25

Sensitivity of the B. subtilis test

The result shows that 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.

Example 26

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).

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following invA DNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence:

5' TTA AAT TCC GTG AAG CAA AAC GTA 3' [SEQ ID NO: 30]

Probe: 5' - FAM - CGC CGC CAA ACC - MGB - 3' [SEQ ID NO: 31]

Reverse primer sequence:

5' TGC TCG CCT TTG CTG GTT 3' [SEQ ID NO: 32]

The probe was manufactured by the company Applied Biosystems, Weiterstadt,

Germany. The probe is a single stranded oligonucleotide which was labelled at the 5' end with fluorescence derivate (FAM = 6-carbooxyfluorescein) 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 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:

Total 20

Real-Time-PCR profile for specific systems for the detection of S. enterica

* systems to avoic 'carry-over"-contamination. Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).

Example 28

Selectivity of the S. enterica PCR detection test

To evaluate the selectivity of the PCR test specific for S. enterica, 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:

List of the tested cDNA samples:

Example 29

Sensitivity of the S. enterica test

To determine the sensitivity of S. enterica PCR test, cDNA was prepared and deployed in the PCR experiments.

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.

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.

Forward primer sequence:

5' CAT GCC GAA CTC AGA AGT GA 3' [SEQ ID NO: 34]

Probe:

5' - FAM - ACG CCG TAG CGC C - MGB - 3' [SEQ ID NO: 35]

Reverse primer sequence:

5' GGG AGA CCC CAC ACT ACC AT 3' [SEQ ID NO: 36]

Example 31

Salmonella enterica can be detected targetting thesequence of the 23 S rRNA gene.

Specific areas of the 23 S rRNA gene served as diagnostical target for the development of a rapid detection kit for the detection of S. enterica. Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following 23 S rRNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence: 5' TTC TCC CCG AAA GCT ATT TAG GT 3' [SEQ J-D NO: 38]

Probe:

5' - FAM - AAG CCG GGA TGG CCC - MGB - 3' [SEQ J-D NO: 39]

Reverse primer sequence:

5' CCC GTG ATA ACA TTC TCC GGT AT 3' [SEQ J-D NO:40]

The probe was manufactured by the company Applied Biosystems, Weiterstadt,

Example 32

PCR conditions for the detection of S. enterica

Total 20

Real-Time-PCR profile for specific systems for the detection of S. enterica using 23 S rRNA target gene

Example 33

Selectivity of the S. enterica PCR detection test using 23 S rRNA target gene.

To evaluate the selectivity of the PCR test specific for S. enterica using 23 S rRNA target gene, 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:

List of the tested cDNA samples:

Example 34

Sensitivity of the S. enterica test

The result shows that 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. Example 35

Staphylococcus aureus can be detected targetting thesequence of the nuc gene.

Specific areas of the nuc gene served as diagnostical target for the development of a rapid detection kit for the detection of S. aureus. 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.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following nuc DNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence: 5 ' AGC TCA GCA AAT GCA TCA CAA 3 ' [SEQ ID NO: 42]

Probe:

5 ' - FAM - CAG ATA ACG GCG TAA AT - MGB - 3 ' [SEQ ID NO: 43]

Reverse primer sequence:

5' ACT GTT GGA TCT TCA GAA CCA CTT C 3' [SEQ ID NO: 44]

The probe was manufactured by the company Applied Biosystems, Weiterstadt, Germany. The probe is a single stranded oligonucleotide which was labelled at the 5' end with fluorescence derivate (FAM = 6-carbooxyfluorescein) 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 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 36

PCR conditions for the detection of S. aureus

Total 20

Real-Time-PCR profile for specific systems for the detection of S. aureus

Example 37

Selectivity of the S. aureus PCR detection test To evaluate the selectivity of the PCR test specific for S. aureus, 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:

List of the tested cDNA samples:

Example 38

Sensitivity of the S. aureus test

To determine the sensitivity of S. aureus PCR test, cDNA was prepared and deployed in the PCR experiments.

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.

The result shows that 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 39

Stafilococcus aureus can be detected targetting thesequence of the 16S rRNA gene.

Specific areas of the 16S rRNA gene served as diagnostical target for the development of a rapid detection kit for the detection of S. aureus.

Forward primer sequence: 5' CTG GGA TAA CTT CGG GAA ACC 3' [SEQ J-D NO: 46]

Probe:

5' - FAM -CCG GAT AAT ATT TTG AAC CGC AT - MGB - 3' [SEQ ID NO: 47]

Reverse primer sequence:

5' GAC AGC AAG ACC GTC TTT CAC TT 3' [SEQ ID NO: 48]

Example 40

PCR conditions for the detection of S. aureus

Total 20

Real-Time-PCR profile for specific systems for the detection of S. aureus using 16S rRNA target gene

* systems to avoid "carry-over"-contamination. Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG). Example 41

Selectivity of the S. aureus PCR detection test using 16S rRNA target gene.

List of the tested cDNA samples:

Example 42 Sensitivity of the S. aureus test

Example 43

Aspergillus niger can be detected targetting thesequence of the 28S rRNA gene.

Specific areas of the 28S rRNA gene served as diagnostical target for the development of a rapid detection kit for the detection of A. niger. It has been shown in many experiments (Melese et al. Curr. Opin. Cell. Biol. 7, 319-324) 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. Therefore the determination was performed on ribosomal RNAs which are expressed on a high level during all stages of the life cycle of the target organisms. Therefore it was chosen to serve as a genetic marker to detect the fungal specie A. niger.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following 28S rRNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence: 5' GCG GCC GGT CAA AGG 3' [SEQ ID NO: 50]

Probe:

5' - FAM - TGG AAT GTA GTA CCC TCC GG - MGB -3' [SEQ ID NO: 51]

Reverse primer sequence:

5' TTG CAC CCC TGG CTA TAA GG 3' [SEQ ID NO: 52]

Example 44

PCR conditions for the detection of A. niser

Total 20

Real-Time-PCR profile for specific systems for the detection of A. niger Incubation of the PCR-plate (Microamp® Optical/96-well reaction plate) in the TaqMan using the following temperature-time-programme:

Steps

UNG-activity* 2 min./ 50 °C activation of AmpliTaq Gold 10 min./ 95°C amplification (50 cycles) 15 sec./ 95°C 60 sec./ 60°C * systems to avoid "carry-over"-contamination. Contaminating amplicons are digested before PCR by the enzyme Uracil-N-Glykosylase (UNG).

Example 45

Selectivity of the A. niger PCR detection test To evaluate the selectivity of the PCR test specific for A. niger, 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:

List of the tested cDNA samples:

Example 46

Sensitivity of the A. niser test

To determine the sensitivity of A. niger PCR test, cDNA was prepared and deployed in the PCR experiments.

Different amounts of cDNA of A. niger 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 result shows that 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.

Example 47

Candida albicans can be detected targetting thesequence of the 26S rRNA gene.

Specific areas of the 26S rRNA gene served as diagnostical target for the development of a rapid detection kit for the detection of C. albicans. It has been shown in many experiments (Melese et al. Curr. Opin. Cell. Biol. 7, 319-324) 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. Therefore the determination was performed on ribosomal RNAs which are expressed on a high level during all stages of the life cycle of the target organisms. Therefore it was chosen to serve as a genetic marker to detect the fungal specie C. albicans.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following 26S rRNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence:

5' GGC CAG CAT CGG TTT GG 3' [SEQ ID NO: 54]

Probe: 5' - FAM - CGG CAG GAT AAT GG - MGB - 3' [SEQ ID NO: 55]

Reverse primer sequence:

5' GAA GCC GTG CCA CAT TCC T 3' [SEQ ID NO: 56]

The probe was manufactured by the company Applied Biosystems, Weiterstadt,

Example 48 PCR conditions for the detection of C. albicans After variation of primer and probe concentration following conditions aroused as optimal:

Total 20

Real-Time-PCR profile for specific systems for the detection of C. albicans

Example 49

Selectivity of the C. albicans PCR detection test To evaluate the selectivity of the PCR test specific for C. albicans, 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:

List of the tested cDNA samples:

Example 50 Sensitivity of the C. albicans test

To determine the C. albicans PCR test, cDNA was prepared and deployed in the PCR experiments.

Different amounts of cDNA of C. albicans 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 result shows that 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.

Example 51

Detection of bacteria universal

The detection of bacteria takes place through the specific amplification of the conserved region of the 16S RNA sequence, which is part of the patent. Certain specific 16S rRNA DNA sequences were conserved through the evolution. Therefore they are present in the genome of all bacteria and thus they can used as primer and probes for the universal detection of bacteria (Relman 1993, J. Infect. Dis. 168, 1-8). Although it is claimed that the ribosomal RNA is conserved, degenerated primers were designed, to be able to detect almost all bacteria.

Forward primer sequence:

5' CAG CTC GTG TYG TGA RAT G 3' [SEQ ID NO: 58]

Probe:

5' - VIC - TGG GTT AAG TCC C - MGB - 3' [SEQ ID NO: 59]

Reverse primer sequence:

5' RAG GGT TGC GGT CGT T 3' [SEQ ID NO: 60] whereas

Y = C or T R = A or G

The probe was manufactured by the company Applied Biosystems, Weiterstadt, 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.

Example 52

PCR conditions for the detection of all bacteria

Total 20 Real-Time-PCR profile for specific systems for the detection of all bacteria

Example 53

To determine the selectivity of the PCR test, 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:

Example 54

Sensitivity of the universal bacteria test

To determine the sensitivity of universal bacteria PCR test, cDNA of five different bacteria was prepared and deployed in the PCR experiments.

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.

The result shows that 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.

Example 55

Detection of fungi universal

The detection of fungi takes place through the specific amplification of the conserved region of the 18S RNA sequence, which is part of the patent. Certain specific 18S rRNA DNA sequences were conserved through the evolution. Therefore they are present in the genome of all fungi and thus they can used as primer and probes for the universal detection of fungi (Reiss et al. 1998, Med. Mycol. 36, 249-57). Although it is claimed that the ribosomal RNA is conserved, degenerated primers were designed, to be able to detect almost all fungi.

Due to cDNA sequence comparison, practical optimisation work and use of different primer and probe combinations following 18S rRNA sequences were determined as the optimal primer and probe combination:

Forward primer sequence:

5' ACG GAA GGG CAC CAC HAG 3' [SEQ ID NO: 62]

Probe:

5' - VIC - TGG AGC CTG CGG CT - MGB - 3' [SEQ ID NO: 63]

Reverse primer sequence:

5' TTC CCC GTG TTG AGT CAA ATT 3' [SEQ ID NO: 64]

whereas H = A or C or T

The probe was manufactured by the company Applied Biosystems, Weiterstadt, 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

Total 20 Real-Time-PCR profile for specific systems for the detection of all fungi

Steps

UNG-activity* 2 min./ 50 °C activation of AmpliTaq Gold 10 min./ 95°C

Amplification (50 cycles) 15 sec./ 95°C 60 sec./ 50°C

Example 57

To determine the selectivity of the PCR test, 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:

Example 58 Sensitivity of the universal fungal test

To determine the sensitivity of universal fungi PCR test, cDNA of two different fungi was prepared and deployed in the PCR experiments.

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 result shows that RNA of 5 fungal cells could be detected using fluorescence

PCR. 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. To resolve these inadequacies of existing tests, 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. The new protocol will make the need to culture organisms for detection obsolete, and could remedy shortcomings of traditional techniques by allowing rapid, sensitive, and specific identification of the pathogens of concern rather than indicator organisms. To establish the validity and efficacy of the approach, 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.

Statement of Results and Benefits:

Successful demonstration of the utility of the proposed real-time PCR test would bring biological testing of water quality up to the state-of-the art in modern microbiology, allowing inexpensive, rapid, direct, specific detection of harmful microorganisms via identification of specific genetic markers. These and additional tests based on this new technology will be expected to replace the archaic indirect-inference tests employing, laboratory culturing of indicator bacteria, and replace tests that require days with ones that can provide measurements within hours and therefore be used more routinely for real-time monitoring of water supplies. Turnaround time for detailed analyses to identify human pathogen bacteria, fungi and viruses is sometimes weeks. Microbiological analyses are conducted in-house often at major plants and main water sources. An enormous demand exits for faster, easier and more reliable tests using molecular probes specific for human pathogens in water supply sources. This area of application would contribute substantially to economic development, and improved security. In the short-term, the new test proposed here would most benefit researchers and manufacturers by allowing them to track the fate and source of pathogenic organisms in water to be determined and traced to identifiable sources. Example 60 Application to Testing of Surfaces

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. To resolve these inadequacies of existing tests, 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. 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. To establish the validity and efficacy of the approach, 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.

Statement of Results and Benefits:

Successful demonstration of the utility of the proposed real-time PCR test would bring biological testing of surfaces up to the state-of-the art in modern microbiology, allowing inexpensive, rapid, direct, specific detection of harmful micro-organisms via identification of specific genetic markers. These and additional tests based on this new technology would be expected to replace the archaic indirect-inference tests employing, laboratory culturing of indicator bacteria, and replace tests that require days with ones that can provide measurements within hours and therefore be used more routinely for real-time monitoring of surfaces. Turnaround time for detailed analyses to identify human pathogen bacteria, fungi and viruses is sometimes weeks. Microbiological analyses are conducted in- house often at major plants and main production lines. An enormous demand exits for faster, easier and more reliable tests using molecular probes specific for human pathogens on surfaces. This area of application would contribute substantially to economic development, and improved security. In the short-term, the new test proposed here would most benefit researchers and manufacturers by allowing them to track the fate and source of pathogenic organisms surfaces in pharmaceutical production environment to be determined and traced to identifiable sources.

Example 61 Application to Testing of Pharmaceutical Packaging Material

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. To resolve these inadequacies of existing tests, 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. 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. To establish the validity and efficacy of the approach, 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.

Statement of Results and Benefits:

Successful demonstration of the utility of the proposed real-time PCR test would bring biological testing of pharmaceutical packaging material quality up to the state-of-the art in modern microbiology, allowing inexpensive, rapid, direct, specific detection of harmful micro-organisms via identification of specific genetic markers. These and additional tests based on this new technology would be expected to replace the archaic indirect-inference tests employing, laboratory culturing of indicator bacteria, and replace tests that require days with ones that can provide measurements within hours and therefore be used more routinely for real-time monitoring of pharmaceutical packaging material. Turnaround time for detailed analyses to identify human pathogen bacteria, fungi and viruses is sometimes weeks. Microbiological analyses are conducted in-house often at pharmaceutical packaging material. An enormous demand exits for faster, easier and more reliable tests using molecular probes specific for human pathogens in pharmaceutical packaging material. This area of application would contribute substantially to economic development, and improved security. In the short-term, the new test proposed here would most benefit researchers and manufacturers by allowing them to track the fate and source of pathogenic organisms in pharmaceutical packaging material to be determined and traced to identifiable sources.

Example 62 Application to Testing of Pharmaceutical Products

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. To resolve these inadequacies of existing tests, 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. 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. To establish the validity and efficacy of the approach, 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.

Statement of Results and Benefits:

Successful demonstration of the utility of the proposed real-time PCR test would bring biological testing of pharmaceutical products quality up to the state-of-the art in modern microbiology, allowing inexpensive, rapid, direct, specific detection of harmful micro-organisms via identification of specific genetic markers. These and additional tests based on this new technology would be expected to replace the archaic indirect-inference tests employing, laboratory culturing of indicator bacteria, and replace tests that require days with ones that can provide measurements within hours and therefore be used more routinely for real-time monitoring of pharmaceutical products. Turnaround time for detailed analyses to identify human pathogen bacteria, fungi and viruses is sometimes weeks. Microbiological analyses are conducted in-house often at pharmaceutical products. An enormous demand exits for faster, easier and more reliable tests using molecular probes specific for human pathogens in pharmaceutical products. This area of application would contribute substantially to economic development, and improved security. In a short-term, the new test proposed here would most benefit researchers and manufacturers by allowing them to track the fate and source of pathogenic organisms in pharmaceutical products to be determined and traced to identifiable sources.

Bibliographic references cited in the patent specification

Abee T, Wouters JA., 1999: Microbial stress response in minimal processing. Int. J. Food. Microbiol. 15: 65-91

Chun J, Bae KS., 2000: Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. : Antonie Van Leeuwenhoek;78: 123-127

Hancock RE, Siehnel R, Martin N., 1990: Outer membrane proteins of Pseudomonas. Mol. Microbiol.; 4:1069-1075

De Vos D, Lim A Jr, Pirnay JP, Struelens M, Vandenvelde C, Duinslaeger L,

Vanderkelen A, Cornells P., 1997: Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprl and oprL. J. Clin. Microbiol.; 35:1295-1299

Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, Kobayashi I, Cui L, Oguchi A, Aoki K, Nagai Y, Lian J, Ito T, Kanamori M, Matsumaru H, Maruyama A, Murakami H, Hosoyama A, Mizutani-Ui Y, Takahashi NK, Sawano T, Inoue R, Kaito C, Sekimizu K, Hirakawa H, Kuhara S, Goto S, Yabuzaki J, Kanehisa M, Yamashita A, Oshima K, Furuya K, Yoshino C, Shiba T, Hattori M, Ogasawara N, Hayashi H, Hiramatsu K, 2001: Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet; 357:1225-1240

McDaniels AE, Rice EW, Reyes AL, Johnson CH, Haugland RA, Stelma GN Jr., 1996: Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase. Appl. Environ. Microbiol.; 62: 3350-3354

Melese T, Xue Z., 1995: The nucleolus: an organelle formed by the act of building a ribosome. Curr. Opin. Cell. Biol.; 7: 319-324

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

Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galan JE, Ginocchio C, Curtiss

R 3rd, Gyles CL. , 1992: Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol. Cell. Probes; 6:271-279

Reiss E, Tanaka K, Bruker G, Chazalet V, Coleman D, Debeaupuis JP, Hanazawa

R, Latge JP, Lortholary J, Makimura K, Morrison CJ, Murayama SY, Naoe S, Paris S, Sarfati J, Shibuya K, Sullivan D, Uchida K, Yamaguchi H., 1998: Molecular diagnosis and epidemiology of fungal infections. Med. Mycol.; 36 Suppl 1:249-257

Relman DA., 1993: The identification of uncultured microbial pathogens. J. Infect.

Dis.; 168:1-8

Smith DK, Kassam T, Singh B, Elliott JF., 1992: Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci. J. Bacteriol.;174: 5820-5826

Claims

1. 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; reuO-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: (i) for Escherichia coli:

SEQ ID NO: 10 as forward primer

SEQ ID NO:l 1 as probe and

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; (iii) for Bacillus subtilis:

SEQ ID NO:26 as forward primer SEQ ID NO:27 as probe and

SEQ ID NO:28 as reverse primer (iv) for Salmonella enterica:

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 and

SEQ JJD NO:48 as reverse primer (vi) for Aspergillus niger: SEQ ID NO:50 as forward primer

SEQ ID NO:51 as probe and

SEQ J-D NO:52 as reverse primer (vii) for Candida albicans: SEQ ID NO: 54 as forward primer

SEQ J-D NO:55 as probe and

SEQ ID NO: 56 as reverse primer (viii) for Bacteria:

SEQ ID NO:58 as forward primer SEQ J-D NO:59 as probe and

SEQ ID NO:60 as reverse primer (ix) for Fungi:

SEQ ED NO: 62 as forward primer

SEQ ID NO:63 as probe and 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; and quantifying the emitted fluorescence signal as a measure of the number of the living microbial contaminants present in the sample.

2. 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 ID NO: 11 as probe and

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; (iii) for Bacillus subtilis:

SEQ ID NO:26 as forward primer SEQ J_D NO:27 as probe and

SEQ ID NO:28 as reverse primer (iv) for Salmonella enterica:

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 and SEQ J-D NO:48 as reverse primer (vi) for Aspergillus niger:

SEQ ID NO:50 as forward primer SEQ J-D 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 and SEQ ID NO:56 as reverse primer (viii) for Bacteria:

SEQ ID NO:58 as forward primer SEQ J-D NO:59 as probe and

SEQ ID NO: 60 as reverse primer (ix) for Fungi:

SEQ ID NO:62 as forward primer SEQ ID NO:63 as probe and SEQ ED NO:64 as reverse primer.