DE202012005258U1 - Means for rapid quantitative and qualitative determination of fungi and yeasts - Google Patents

Abstract

A lysis buffer for the lysis of mold and / or yeast, the lysis buffer being suitable for lysing the zeolite and yeast.

Description

Field of engineering

The present invention is in the field of bioanalytics. More particularly, the invention relates to the detection and quantification of fungi by PCR and DNA hybridization.

background

The taxonomic detection of molds and yeasts is fundamentally associated with difficulties. Classically, the taxonomy was done by staining and microscopy based on colony morphology and color, since lower fungi show a different habit on different media. However, the form of this taxonomy is insufficient.

Modern DNA-based methods make the assignment unique. With this methodology, considerable time can be saved in the field of analytics, since the fungi on nutrient media no longer have to grow to the visual colony size. This can be of considerable importance in diagnostics in the medical field and in the selection of the subsequent therapy.

The cell wall digestion of the fungi concerned, however, is a difficulty as it varies from fungus to fungus. Originally, cell wall lysis of fungi was adapted to the species of interest. Examination of samples with unknown fungi or their spores has created a desire for a universal combination of enzymes for the lysis of cell walls. There were first approaches in the field of cooking lysis, which does not always lead to the lysis of the fungal cell wall. Therefore, there is still a need for lysis buffers and methods that make it possible reliably to lyse taxonomically removed molds and / or yeasts in one step and to prepare them for taxonomic investigation.

Brief description of the invention

Therefore, in a first embodiment, the invention comprises a lysis buffer for the lysis of molds and / or yeasts, wherein the lysis buffer is suitable for lysing the cell walls of taxonomically distant molds and yeasts. The lysis buffer preferably comprises chitinase, the chitinase optionally being present in a concentration of from 0.1 μg / ml to 3.0 μg / ml in the lysis buffer, and the chitinase preferably being present in a concentration of approximately 0.44 μg / ml in the lysis buffer , The lysis buffer may further comprise 5mM to 200mM Tris / HCl, 10mM to 250mM NaCl, and 2mM to 50mM MgCl ₂ , which lysis buffer may have a pH of 4.5 to 7.5.

In a further embodiment, the invention comprises a kit for detecting molds and / or yeasts, the kit comprising a first lysis buffer as described above; a second lysis buffer, wherein the second lysis buffer comprises lyticase, wherein the lyticase is optionally present in a concentration of 300 to 2000 U / ml in the second lysis buffer and wherein the lyticase is preferably present in a concentration of about 700 U / ml in the second lysis buffer; and at least one reaction vessel, wherein at least one type of nucleic acid probe is bound to the inside of the at least one reaction vessel, each type of nucleic acid probe being specific for a mold or yeast and comprising a binding sequence complementary to a genomic or complementary sequence of a genomic sequence of the mold or yeast residing in a DNA region comprising the ITS1, the 5.8S rDNA and the ITS2.

In a preferred embodiment of the kit according to the invention, the second lysis buffer further comprises 10 mM to 250 mM Tris / HCl, 2 mM to 50 mM EDTA and 5 mM to 100 mM β-mercaptoethanol, the second lysis buffer having a pH of 6.0 to 9, 0 has.

In another embodiment, the molds and / or yeasts may be selected from the group consisting of the Zygomycetes, the Ascomycetes and the Deuteromycetes.

In a further embodiment of the kit according to the invention, a single type of nucleic acid probe is bound to each of the at least one reaction vessel. In another embodiment, at least one reaction vessel, several types of nucleic acid probes are bound.

In a further preferred embodiment, the kit according to the invention further comprises a reaction mixture comprising at least one DNA polymerase, dNTPs, a first primer and a second primer, wherein the first primer at the 3 'end contains a sequence which is specific to a target sequence of a mold or a yeast that resides in the 18S rDNA, and wherein the second primer at the 3 'end contains a sequence that specifically binds to a targeting sequence of the mold or yeast that resides in the 28S rDNA. The DNA polymerase may be selected from the group consisting of Taq polymerase, Pfu polymerase and Tbr polymerase.

In another preferred embodiment, a label group is attached to at least one of the two primers, the kit further comprising: an enzyme to which is attached a binding group capable of specifically binding the labeling group of the first and / or second primers, the enzyme being suitable is to catalyze a reaction whose sequence is optically detectable; and a substrate that can be reacted by the enzyme in the reaction. The labeling group may be biotin and the binding group streptavidin. The labeling group can also be an antigen and the binding group can be an antibody specific for the antigen. The enzyme may be selected from the group consisting of alkaline phosphatase, horseradish peroxidase and firefly luciferase. In a preferred embodiment, the enzyme is alkaline phosphatase and the substrate is p-nitrophenyl phosphate (pNPP) or a mixture of 5-bromo-4-chloro-3-indoxyl phosphate with nitroblue tetrazolium chloride (BCIP / NBT).

In a further preferred embodiment, the kit further comprises: at least one centrifugation column for the solid phase extraction of nucleic acids; a first wash buffer capable of removing molecules other than nucleic acids from the spin column; and a second wash buffer capable of removing DNA not bound to the nucleic acid probes from a reaction vessel. The first wash buffer may comprise 70-90% ethanol and 2mM to 10mM potassium phosphate buffer at a pH of 7.5 to 8.5, preferably about 80% ethanol and about 5mM potassium phosphate buffer at a pH of 8.0. The at least one centrifugation column may comprise a solid phase of silica. The second wash buffer may comprise PBS with 0.1-2.0 vol% Tween 20.

Description of the pictures

shows the location of the intergenetic regions ITS1 and ITS2 within the ribosomal DNA of fungi.

FIG. 3 illustrates a flow chart of a detection method in which lysis buffers or kits of the invention can be used.

Schematic representation of a preferred detection reaction in NucleoLink ^™ microtiter strips. 1: coupling specific probes to the surface of NucleoLink ^™ microtiter strips; 2: hybridization of the specific probe with the biotinylated amplificate; 3: attachment of streptavidin-alkaline phosphatase; 4. Cleavage of pNPP by the alkaline phosphatase, resulting color development

: Agarose gel electrophoresis of the PCR after boiling lysis compared Candida albicans (C1, C2), Penicillium chrysogenum (P1, P2), Aspergillus fumigatus (A1, A2).

: Agarose gel electrophoresis of the PCR after enzymatic digestion with lyticase Candida albicans (C1, C2), Penicillium chrysogenum (P1, P2), Aspergillus fumigatus (A1, A2).

: Agarose gel electrophoresis of PCR after enzymatic digestion with chitinase and lyticase from Candida albicans (C1-C5).

: Agarose gel electrophoresis of PCR after enzymatic digestion with chitinase and lyticase from Saccharomyces cerevisiae (S1-S5).

: Agarose gel electrophoresis of PCR after enzymatic digestion with chitinase and Rhizopus stolonifer lyticase (R1-R5).

: Agarose gel electrophoresis of PCR after enzymatic digestion with chitinase and lyticase of Absidia glauca (A1-A5).

: Agarose gel electrophoresis of PCR after enzymatic digestion with chitinase and lyticase of Aspergillus fumigatus (Af1-Af5).

: Agarose gel electrophoresis of PCR after enzymatic digestion with chitinase and lyticase of Penicillium chrysogenum (P1-P5).

: The length standard used in the lysis experiments. The figure shows the position of the 500bp fragment of phage λ compared to the length standard consisting of a mix of HindIII-cut λ DNA and HaeIII-cut ΦX174 DNA.

: Optical density of the dilution series of specific amplificates of A. fumigatus, C albicans and P. chrysogenum in an individual experiment.

: Measurement of the Optical Density of Cross Experiments in C. albicans Probe Coated Strips After 20 h Color Reaction.

: Measurement of the Optical Density of the Cross Experiments in P. chrysogenum Probe Coated Strips After 20 h Color Reaction.

: Measurement of the Optical Density of Cross Experiments in A. fumigatus Probe Coated Strips After 20 h Color Reaction.

Detailed description of the invention

DEFINITIONS

For the purposes of the application, "lysis buffer" refers to solutions which are capable of lysing prokaryotic and / or eukaryotic cells. In this case, lysing means the breaking of the cell walls and / or cell membranes, so that the cytoplasm is released. For example, a lysis buffer may contain surfactants such as sodium dodecyl sulfate (SDS) or Triton X. A lysis buffer may have an alkaline pH.

"Mushrooms" in this application, unless expressly stated otherwise, refers to molds and yeasts. Under "molds" in the sense of the application filamentous mushrooms are understood. "Yeasts" are unicellular fungi that can reproduce asexually. In particular, molds and yeasts within the meaning of this application include Ascomycetes, Zygomycetes and Deuteromycetes.

A "nucleic acid" in the sense of the invention may be any nucleic acid of any length. Examples of nucleic acids are deoxyribonucleic acids (DNA), ribonucleic acids (RNA) or peptide nucleic acids (PNA). Nucleic acids may be single-stranded, double-stranded, or partially single-stranded and partially double-stranded.

For the purposes of the invention, "DNA polymerase" means any enzyme which is capable of synthesizing a DNA strand that is complementary to a single-stranded DNA strand. Examples of DNA polymerases which can be used in embodiments of the invention are Taq polymerase, Pfu polymerase, Tth polymerase, Tma polymerase, Tne polymerase, Tfl polymerase, Pwo polymerase, Bst polymerase, Bca polymerase Polymerase, Sac polymerase, Tac polymerase, Mth polymerase, Tru polymerase, Tbr polymerase, Stoffel fragment, VENT ^™ DEEPVENT ^™ or DYNAZYME ^™ , or functional variants or fragments of such polymerases.

By "primer" in the sense of the application is meant a single-stranded DNA or RNA which can be used in the amplification of nucleic acids. Here, the sequence of the primer, or at least one partial sequence comprising the 3 'end, is complementary to a target sequence at one of the ends of the nucleic acid segment which is to be amplified. The complementary partial sequence at the 3 'end of the primer must be of sufficient length to be specific for the target sequence of the nucleic acid segment to be amplified. The complementary partial sequence is preferably at least 10 bp in length. For example, the complementary partial sequence may comprise between 10 and 50 bp, between 12 and 40 bp, between 15 and 30 bp or between 20 and 25 bp. For the purposes of this application, a "partial sequence" that does not have bases that are complementary over their entire length at all positions to the target sequence but that nevertheless has sufficient homology to the specific binding is also considered "complementary" to a target sequence to ensure the target sequence. For example, a partial sequence of the primer may have ≥ 80%, ≥ 85%, ≥ 90%, ≥ 92%, ≥ 95%, ≥ 97%, ≥ 98%, or ≥ 99% identity to the complementary strand of the target sequence.

A "marker group" in the sense of the application is a molecule which can be attached to a nucleic acid and specifically bound by a "binding group". Preferably, the labeling group is covalently bound to the nucleic acid. In principle, the labeling group and the bonding group can be any pair of molecules, as long as a specific binding reaction between the two molecules is possible. For example, the binding group may be a receptor and the labeling group may be its specific ligand. The binding group may be streptavidin and the labeling group biotin. The labeling group may be an antigen and the binding group a specific antibody. However, the binding group can also be a DNA-binding protein, where the labeling group is then a nucleic acid comprising the specific binding sequence of the DNA-binding protein. Labeling and binding group can also be two complementary nucleic acid strands.

"ITS1" and "ITS2" refer to the two internal transcribed spacers in the ribosomal DNA of eukaryotes, which separate the 18S rDNA and the 5.8S rDNA and the 5.8S rDNA and the 28S rDNA, respectively. A description of the ITS regions in mushrooms can be found z. B. at McCullough et al. (Journal of Clinical Microbiology 1998, 36 (4): 1035-1038) and White et al. (Pp. 315-322 in "PCR protocols: a guide to methods and applications" by Innis, Gelfand, Sninsky and White; San Diego 1996, Academic Press). ,

"Optically detectable" is a reaction in the sense of this invention if its course can be followed by means of light-measuring analysis methods, for example spectroscopically. These include color reactions in which the absorption coefficients of starting material (s) and product (s) differ at least one wavelength. When the starting materials and products are soluble, color reactions can be monitored, inter alia, by measuring the optical density of a solution at a suitable wavelength. However, it is also possible to detect optically reactions in which colored precipitates form or chemiluminescence occurs.

By "room temperature" in this application temperatures between 18 ° C and 30 ° C understood. Preferably, the room temperature is between 20 ° C and 25 ° C.

lysis buffer

In one aspect, the invention provides lysis buffers suitable for lysing the cell walls of taxonomically distant molds and yeasts. This contributes to a significantly faster detection of fungal cells and spores, as cells of all or almost all fungal species occurring in the sample can be lysed and analyzed together in one step. Less starting material is also needed because a single reaction batch is sufficient to test for the presence of all fungi of interest.

The time required for lysis may vary and may be less than 24 hours. Preferably, the required time is less than 12 hours, more preferably less than 6 hours, even more preferably less than 4 hours, and most preferably less than 3 hours. In some embodiments, the lysis is carried out at room temperature. In other embodiments, lysis may also be carried out at lower temperatures, e.g. B. between 2 ° C and 8 ° C, or at higher temperatures, for. B. between 35 ° C and 40 ° C, are performed.

For example, suitable buffers of the invention may lyse cells from such distant organisms as Aspergillus fumigatus, Absidia glauca, Candida albicans, Penicillium chrysogenum, Rhizopus stolonifer or Saccharomyces cerevisiae.

In a particularly preferred embodiment, two different lysis buffers are used in succession, the first lysis buffer containing chitinase and the second lysis buffer containing lyticase.

The first lysis buffer contains chitinase in a concentration of 0.05-10 μg / ml, preferably 0.1-3.0 μg / ml; more preferably 0.2-1.0 μg / ml, and most preferably about 0.44 μg / ml. The first lysis buffer should have an acidic, neutral or slightly alkaline pH. Preferably, pH's are between 4.5 and 7.5, more preferably between 5.0 and 7.0, more preferably between 5.5 and 6.5, most preferably pH's of about 6.0. As the buffer system, Tris / HCl can be used, preferably at a concentration of 5-200 mM, more preferably 10-100 mM, more preferably 20-50 mM, and most preferably of about 30 mM. The first lysis buffer may further contain salts, for example NaCl (preferably 10-250 mM, more preferably 20-100 mM, more preferably 35-70 mM and most preferably about 50 mM) and / or MgCl ₂ (preferably 2-50 mM, more preferably 4-20 mM, more preferably 7-15 mM and most preferably about 10 mM).

The second lysis buffer contains lyticase in a concentration of 100-3000 U / ml, preferably 300-2000 U / ml, more preferably 500-1000 U / ml, and most preferably about 700 U / ml. The second lysis buffer should have a slightly acidic, neutral or alkaline pH. Preferably, pH values are between 6.0 and 9.0, more preferably between 6.5 and 8.5, more preferably between 7.0 and 8.0, most preferably pH values of about 7.5. As the buffer system, Tris / HCl can be used, preferably at a concentration of 10-250 mM, more preferably 20-100 mM, more preferably 35-70 mM, and most preferably about 50 mM. The second lysis buffer may further contain EDTA (ethylenediamine tetraacetate, preferably 2-50 mM, more preferably 4-20 mM, more preferably 7-15 mM and most preferably about 10 mM) and / or β-mercaptoethanol (preferably 5-200 mM, more preferably 10-100 mM, more preferably 20-50 mM, and most preferably about 28 mM).

METHOD

The lysis buffers of the invention enable faster and improved methods of detecting fungal cells and spores. Therefore, there is also disclosed a method of detecting molds and / or yeasts, comprising taking a sample of interest; Cultivating molds and / or yeasts contained therein; Lysis of the increased molds and / or yeasts using lysis buffers according to the invention; Extraction of nucleic acids from the lysate; Amplification of a DNA region comprising the ITS1, the 5.8S rDNA and the ITS2 of the extracted nucleic acids by PCR and at least one primer; Contacting the amplicon in at least one reaction vessel with at least one type of nucleic acid probe, each type of nucleic acid probe being bound to the inside of a reaction vessel and comprising a binding sequence identical or complementary to a genomic sequence of a mold and / or yeast present in a DNA Comprising the ITS1, the 5.8S rDNA and the ITS2 of the mold and / or the yeast; and detecting specific hybridization between a nucleic acid probe and a nucleic acid from the amplificate in a reaction vessel.

The detection of the specific hybridization can be carried out by contacting the at least one reaction vessel with an enzyme to which is attached a linking group capable of specifically binding the labeling group, which enzyme is capable of catalyzing a reaction the course of which is optically detectable; and contacting the at least one reaction vessel with a substrate that can be reacted by the enzyme in the reaction.

Figure 4 shows a general flow diagram of one embodiment of such methods. First, a sample of interest is taken and the fungi contained therein are cultured. This is typically done on solid culture medium, such as on agar plates, with fungal colonies forming during culture. However, it is also possible to cultivate the fungal germs from the sample in liquid medium. The cultivation conditions may differ depending on the type of fungi to be detected. In particular, the temperature should correspond to the optimum growth conditions of the fungi to be detected. The culture time is also different depending on the type of fungi to be detected. It is typically 2-3 days, but a longer or shorter cultivation period is possible.

Once the fungal germs have been propagated from the sample to the desired scale, nucleic acid, preferably DNA, is extracted from the fungi. For this purpose, at least one lysis buffer according to the invention is used, which makes it possible to lyse the cell walls of taxonomically far distant molds and yeasts and thereby make the nucleic acids of many different fungi accessible in one step. In this step, fungal cells are incubated with a suitable amount of lysis buffer.

In a preferred embodiment, a first incubation with a chitinase-containing first lysis buffer takes place first. The first incubation period can be less than 12 hours. Preferably, the incubation time is less than 6 hours, more preferably less than 3 hours, even more preferably less than 2 hours and most preferably about 1 hour. The first incubation is preferably carried out at room temperature (about 25 ° C). This is followed by a second incubation with a lyticase-containing second lysis buffer. The second incubation time may be less than 6 hours. Preferably, the incubation time is less than 3 hours, more preferably less than 1.5 hours, even more preferably less than 1 hour and most preferably about 30 minutes. The second incubation is preferably carried out at about 37 ° C.

The subsequent extraction of the nucleic acids from the lysate can be carried out by standard methods. One possibility is solid phase extraction, in which the lysate is contacted with a solid substrate and the nucleic acid is adsorbed to the substrate. Subsequently, the substrate is washed with a wash buffer which removes proteins and other unwanted molecules from the substrate. In a further elution step, the nucleic acid is then desorbed from the substrate and released in an eluate. For example, silicon dioxide can serve as the solid phase. Highly ethanol-containing potassium phosphate buffers are suitable as washing buffer, for example a washing buffer with 70-90% ethanol and 2 mM to 10 mM potassium phosphate buffer at a pH of 7.5 to 8.5. Preferred is a wash buffer with about 80% ethanol and about 5 mM potassium phosphate buffer at a pH of 8.0. For elution, distilled water can be used. Another possibility of nucleic acid extraction is alcoholic precipitation (for example with ice-cold ethanol or isopropanol), the nucleic acid being obtained as a solid precipitate. The person skilled in the well-known methods for nucleic acid extraction.

Subsequently, a region from the ribosomal DNA of the existing fungi is amplified by means of a PCR. The structure of ribosomal DNA is highly conserved in fungi and is expressed in shown. In this case, the sections of the 18S rDNA, the 5.8S rDNA and the 28S rDNA are separated by two internal transcribed spacers (ITS), namely ITS1 and ITS2. While ITS1, ITS2, and 5.8S rDNA show sequence differences between different fungal species, both 18S rDNA and 28S rDNA contain conserved regions that are identical in all fungi. Therefore, using primers that specifically bind in these conserved sections, an approximately 650 bp sequence comprising each of the ITS1, ITS2, and 5.8S rDNA can be amplified from each fungus to be detected. Suitable primers for such an amplification, for example in the fungal species Aspergillus fumigatus, Absidia glauca, Candida albicans, Penicillum chrysogenum, Rhizopus stolonifer or Saccharomyces cerevisiae, are the primers "ITS1"(5'-TCCG TAGGTGAACCTGCGG-3 ') and "ITS4" ( 5'-TCCTCCGCTTATTGATATGC-3 ') as in McCullough et al. (Journal of Clinical Microbiology 1998, 36 (4): 1035-1038) described.

Preferably, at least one primer is provided with a labeling group, so that at least one strand in the resulting amplified DNA contains the labeling group. The labeling group can be, for example, biotin. The labeling group can also be an antigen.

After amplification, the double-stranded amplificates are denatured by heating, and aliquots of the amplificates are subsequently pipetted into at least one reaction vessel, to which side of each reaction vessel specific single-stranded nucleic acid probes are bound. As a reaction vessel is any vessel to which nucleic acid probes can be stably bound. Preferably, the reaction vessel is made of plastic. Particularly suitable are reaction vessels with at least one transparent, flat boundary, which may be one side or the bottom. Examples of suitable reaction vessels are ELISA strips, microtiter plates (preferably flat bottom) and cuvettes.

The nucleic acid probes are preferably covalently bound to the inside of the at least one reaction vessel. Particularly preferred is a bond by means of carbodiimide condensation.

Each nucleic acid probe comprises a binding sequence linked to an amplified target sequence from the region. ITS1, ITS2 and / or 5.8S, rDNA can hybridize to a yeast or a mold of interest. In this case, the binding sequence should be complementary to the strand of the target sequence containing the labeling group, so that when present from the corresponding fungus-derived sequence in the amplified DNA strands are bound to the probe. The binding sequence must be of sufficient length to be specific for the target sequence. The binding sequence is preferably at least 8 bp in length. For example, the binding sequence may be between 8 and 50 bp, between 12 and 30 bp, between 15 and 25 bp, or between 18 and 22 bp. For the purposes of this application, a binding sequence which does not have bases that are complementary over its entire length at all positions to the target sequence, but nevertheless has sufficient homology to ensure specific binding to the target sequence, is considered to be "complementary" to a target sequence. For example, a binding sequence may have ≥50%, ≥65%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, or ≥98% identity to the complementary strand of the target sequence.

The specificity of the binding sequence can also be adapted to be somewhat degenerate, ie, to hybridize to different but related target sequences from different fungi. The nucleic acid probe may be specific for a family, genus, species, strain or any other group of fungi, depending on the fungi to be studied. If a probe is also intended to be specific to several strains or species at the same time, the complex domain may also be so be chosen to bind to a relatively conserved region found in all fungi of the particular strain or species, but not others. Alternatively, several probes of different specificity can be mounted in the same vessel on the strip, so that several different fungi can be detected.

For example, a probe having the sequence 5'-ACCACGTATATCTTCAAACTTT-3 'specific for Candida albicans, a probe having the sequence 5'-CACCCAACTTTATTTTTACTA-3' specific for Aspergillus fumigatus and a probe having the sequence 5'-CTTGCCCATCAACCCAAAT-3 'is specific for Penicillium chrysogenum.

The hybridization time for the amplificates with the probes may vary depending on the conditions chosen, and may be less than 6 hours, for example. In preferred embodiments, the hybridization time is less than 3 hours, more preferably between 30 minutes and 90 minutes. Particularly preferred is a hybridization time of about 1 h. The incubation temperature depends on the melting temperature (T _m ) of the probes used. Methods for determining the melting temperature of oligonucleotides are known in the art. After hybridization, the vessels of the strips are washed and then treated with an enzyme to which a specific binding group for the labeling group is bound. If the labeling group is biotin, the binding group can be streptavidin. If the label group is an antigen, the binding group may be an antigen specific to the antigen.

The enzyme is any enzyme that can catalyze an optically detectable reaction in solution. In preferred embodiments of the invention, the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase and firefly luciferase. The incubation time of the enzyme with the bound amplicons may vary depending on the conditions and the labeling or binding group, and may be less than 4 hours. In preferred embodiments, the hybridization time is 1-2 h. The hybridization temperature is also dependent on the chosen binding partners (labeling and binding group) and the enzyme used. When streptavidin and biotin are chosen as the binding partner and alkaline phosphatase as the enzyme, hybridization can occur at about 37 ° C.

After incubation, the tubes of the strips are washed and treated with a solution containing a suitable substrate of the enzyme which can be reacted by the enzyme in an optically detectable reaction. The substrate may also be a mixture of different compounds. The solution should also contain all other reagents and cofactors required for the enzymatic reaction (eg, ATP, Mg ²⁺ , NADH / H ⁺ , etc., depending on the reaction). If. For example, when the enzyme is alkaline phosphatase, the substrate may be, for example, p-nitrophenyl phosphate (pNPP) or a mixture of 5-bromo-4-chloro-3-indoxyl phosphate with nitroblue tetrazolium chloride (BCIP / NBT).

When the desired fungi were present in the sample, the enzyme in the vessels containing the corresponding nucleic acid probes binds to the corresponding amplicons and catalyzes the optically detectable reaction. This can be followed with the naked eye or with a suitable detection instrument and provide quantitative and / or qualitative information about the fungi in the sample. For qualitative statements about the presence of the fungi of interest, even the observation of the reaction, such as a color change in the corresponding vessels, may be sufficient with the naked eye. For more reliable and / or quantitative statements, the reaction can be followed, for example, in a spectrometer. The quantification of the fungi in one batch then takes place in relative percentage terms via the measured optical density in the vessel. This makes it possible, for example, for air samples by comparison of outside air to indoor air a mold fungus load after VDI 6022 to determine.

Such methods can be used, for example, for detecting foreign yeasts in beer production (eg Saccharomyces bayanus, Saccharomyces exiguus, Saccharomyces partorianus), dermatophytes in clinical analysis or airborne molds in the context of hygiene checks of ventilation systems.

KITS

In another aspect, the invention also includes kits for facilitating the above-described methods.

In one embodiment, a kit for the rapid detection of molds and / or yeasts comprises at least one lysis buffer according to the invention and at least one reaction vessel.

In preferred embodiments, the kit comprises a chitinase-containing first lysis buffer and a lyticase-containing second lysis buffer. The first lysis buffer contains chitinase in a concentration of 0.05-10 μg / ml, preferably 0.1-3.0 μg / ml, particularly preferably 0.2-1.0 μg / ml, and most preferably about 0 , 44 μg / ml. The first lysis buffer should have an acidic, neutral or slightly alkaline pH. Preferably, pH's are between 4.5 and 7.5, more preferably between 5.0 and 7.0, more preferably between 5.5 and 6.5, most preferably pH's of about 6.0. As the buffer system, Tris / HCl can be used, preferably at a concentration of 5-200 mM, more preferably 10-100 mM, more preferably 20-50 mM, and most preferably about 30 mM. The first lysis buffer may further contain salts, for example NaCl (preferably 10-250 mM, more preferably 20-100 mM, more preferably 35-70 mM and most preferably about 50 mM) and / or MgCl ₂ (preferably 2-50 mM, more preferably 4-20 mM, more preferably 7-15 mM and most preferably about 10 mM).

The second lysis buffer contains lyticase in a concentration of 100-3000 U / ml, preferably 300-2000 U / ml, more preferably 500-1000 U / ml, and most preferably about 700 U / ml. The second lysis buffer should have a slightly acidic, neutral or alkaline pH. Preferably, pH values are between 6.0 and 9.0, more preferably between 6.5 and 8.5, more preferably between 7.0 and 8.0, most preferably pH values of about 7.5. As the buffer system, Tris / HCl can be used, preferably at a concentration of 10-250 mM, more preferably 20-100 mM, more preferably 35-70 mM, and most preferably about 50 mM. The second lysis buffer may further contain EDTA (ethylenediamine tetraacetate, preferably 2-50 mM, more preferably 4-20 mM, more preferably 7-15 mM and most preferably about 10 mM) and / or β-mercaptoethanol (preferably 5-200 mM, more preferably 10-100 mM, more preferably 20-50 mM, and most preferably about 28 mM).

As a reaction vessel is any vessel to which nucleic acid probes can be stably bound. Preferably, the reaction vessel is made of plastic. Particularly suitable are reaction vessels with at least one transparent, flat boundary, which may be one side or the bottom. Examples of suitable reaction vessels are ELISA strips, microtiter plates (preferably flat bottom) and cuvettes.

At least one type of nucleic acid probe is bound to the inside of the at least one reaction vessel as described above. The nucleic acid probes are preferably covalently bound to the inside of the at least one reaction vessel. Particularly preferred is a bond by means of carbodiimide condensation.

Each type of nucleic acid probe is specific for a mold or yeast and includes a binding sequence that is complementary to a genomic sequence or the complementary sequence of a genomic sequence of the mold or yeast present in a DNA region comprising the ITS1, the 5.8S rDNA and the ITS2 is located. The binding sequence must be of sufficient length to be specific for its target sequence. The binding sequence is preferably at least 8 bp in length. For example, the binding sequence may be between 8 and 50 bp, between 12 and 30 bp, between 15 and 25 bp, or between 18 and 22 bp. For the purposes of this application, a binding sequence which does not have bases that are complementary over its entire length at all positions to the target sequence, but nevertheless has sufficient homology to ensure specific binding to the target sequence, is considered to be "complementary" to a target sequence. For example, a binding sequence may have ≥50%, ≥65%, ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, or ≥98% identity to the complementary strand of the target sequence.

Each of the at least one reaction vessel may contain nucleic acid probes each having the same specificity. Alternatively, at least one vessel may contain several types of nucleic acid probes with different specificity.

For example, a probe having the sequence 5'-ACCACGTATATCTTCAAACTTT-3 'specific for Candida albicans, a probe having the sequence 5'-CACCCAACTTTATTTTTACTA-3' specific for Aspergillus fumigatus and a probe having the sequence 5'-CTTGCCCATCAACCCAAAT-3 'is specific for Penicillium chrysogenum.

In a preferred embodiment, the kit additionally comprises a reaction mixture containing at least one DNA polymerase, dNTPs, and a first primer and a second primer. Contains this the first primer at the 3 'end binds a sequence specific to a targeting sequence of the mold or yeast residing in the 18S rDNA and the second primer at the 3' end a sequence specific to a targeting fungus of the yeast or yeast binds, which lies in the 28S rDNA. The primers bind to strongly conserved regions in the 18S rDNA or the 28S rDNA, which are identical in all fungi. With the help of such primers, an approximately 650 bp long DNA region can be amplified, which includes the ITS1, the 5.8S rDNA and the ITS2. Suitable primers for such an amplification, for example in the fungal species Aspergillus fumigatus, Absidia glauca, Candida albicans, Penicillum chrysogenum, Rhizopus stolonifer or Saccbaromyces cerevisiae, are the primers "ITS1"(5'-TCCGTAGGTGAACCGCGG-3') and "ITS4" (5 '-TCCTCCGCTTATTG ATATGC-3') as in McCullough et al. (Journal of Clinical Microbiology 1998, 36 (4): 1035-1038) described.

In a further preferred embodiment, a labeling group is bound to at least one of the two primers. The labeling group can be, for example, biotin or an antigen. In such an embodiment, the kit may also include an enzyme covalently linked to a linking group that can specifically bind the label group of, and a substrate for the enzyme. The binding group may be, for example, streptavidin or an antibody. The enzyme is capable of catalyzing an optically detectable reaction of the substrate. Examples of suitable enzymes are alkaline phosphatase, horseradish peroxidase and firefly luciferase.

The enzyme can be in any form from which a solution of the functional enzyme can be made. For example, the enzyme may already be in the functional form in a suitable buffer in solution. The buffer may contain reagents which stabilize the enzyme in the functional form, for example protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF). The enzyme can also be present in a highly concentrated glycerol solution. Alternatively, the enzyme may also be provided as a dry protein, for example freeze-dried or lyophilized.

Likewise, the substrate may also be provided in any suitable form, provided it is physically separated from the enzyme. The substrate can also be dissolved. The substrate may also be in dry form, for example as a powder or in tablet form. The substrate may also be a mixture of different compounds. Examples of suitable substrates are p-nitrophenyl phosphate (pNPP) and a mixture of 5-bromo-4-chloro-3-indoxyl phosphate with nitroblue tetrazolium chloride (BCIP / NBT).

A kit according to the invention may also comprise further constituents. Thus, at least one centrifugation column for solid phase extraction of nucleic acids from the cell lysate can also be contained in the kit. The centrifugation column (s) may comprise a solid phase of silica.

Further, the kit may also comprise a first wash buffer suitable for removing molecules other than nucleic acids from the centrifugation column. It is preferably an alcoholic wash buffer, for example comprising 70-90% ethanol and 2 mM to 10 mM potassium phosphate buffer at a pH of 7.5 to 8.5. Most preferred are about 80% ethanol and about 5 mM potassium phosphate buffer at a pH of 8.0.

Also, the kit may contain a second wash buffer suitable for removing DNA not bound to the nucleic acid probes from the ELISA strips. Preferably, the second wash buffer comprises PBS with 0.1-2.0 vol% Tween 20.

Examples

A. Material and Methods

A1 material

A1.1 fungal strains

All fungal cultures, except for Penicillium chrysogenum, were obtained as active cultures from the German Collection of Microorganisms and Cell Cultures (DSMZ). description DSM no. Aspergillus fumigatus 819 Candida albicans 1665 Absidia glauca 63295 Rhitiopus stolonifer 63011 Saccharomyces cerevisiae 1334

The Penicillium comes from an air sample of the company biotec GmbH, Elbrachtsweg 76, 33332 Gütersloh. A determination of 285 rDNA ribotyping at the Heart and Diabetes Center North Rhine-Westphalia revealed 99% of Penicillium chrysogenum.

A1.2 primers and probes

Sonden:

The primers and sands used in the experiments were purchased from Invitrogen Life Technologies. primer:

probes:

A1.3 polymerase

DyNazyme ^TM II DNA polymerase (polymerase gene from Thermus borckianus (F55) expressed in E. coli) from Finnzymes.

A1.4 Buffers, culture media and solutions

A1.4.1 Buffer TAE buffer 50 × 242.0 g Tris base 57.1 ml acetic acid 100 ml 0.5 M EDTA, with H ₂ O to 1000 ml Adjust pH 8.0 Phosphate buffer 7.0 g K ₂ HPO ₄ 3.0 g KH ₂ PO ₄ 4.0 g NaCl with H ₂ O to 1000 ml Adjust pH 7.0 Autoclave for 15 min at 121 ° C hybridization buffer 7.00 g Sarkosyl 7.1 g Na ₂ HPO ₄ with H ₂ O bidist. to 100 ml Adjust pH 7.2 Autoclave for 15 min at 121 ° C wash buffer 100 ml 1 × PBS 50 μl Tween 20 Autoclave for 15 min at 121 ° C PBS 8.0 g NaCl 0.2 g KCl 0.2 g KH ₂ PO ₄ 1.26 g Na ₂ HPO ₄ × 2 H ₂ O with H ₂ O to 1000 ml Adjust pH 7.0 Autoclave for 15 min at 121 ° C antibody conjugate streptavidin Alcaline Phosphatase (Sigma Aldrich) 1: 2000 with Dilute DIAPROPS buffer DIAPROPS buffer 80 mM Tris-HCl 20 mM Tris base 150 mM NaCl 0.1% Tween 20 Adjust pH 7.5 Autoclave for 15 min at 121 ° C Color detection buffer 10 mg / ml pNPP (p-Nirophenyl phosphate) 1 m diethanolamine 1 mm MgCl ₂ pH 9.8

A1.4.2 Culture media

The nutrient medium used to culture the fungal strains is a malt-yeast-tract agar suitable for culturing and maintaining molds and yeasts. malt medium 25.0 g malt extract firmly 2.0 g yeast extract 20.0 g Agar Agar with H ₂ O to 1000 ml (pH 5.6 ± 0.2 at 25 ° C) Autoclave for 15 min at 121 ° C Allow to cool to 48 ° C Add 100 μg / ml ampicillin and stir Hand warm A1.4.3 Solutions blue marker 50.00% (v / v) glycerin 0.05% (w / v) bromophenol 50.00 mM EDTA pH 8.0 Lyticase Solution 50.00 mM Tris base, pH 7.5 10.00 mM EDTA 28.00 mM β-mercaptoethanol 700 U / ml lyticase Chitinase Solution 30.00 mM Tris HCl 50.00 mM NaCl 10.00 mM MgCl ₂ 0.44 μg / ml chitinase Adjust pH 6.0 Methylimidazole solution 82 μl 1-methylimidazole 100 μl H ₂ O bidist. EDAC solution (40 mM) 0.38 g 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide 50.0 ml 10 mM methylimidazole wash solution 40.0 ml 1N NaOH 60.0 ml H ₂ O bidist. 250 μl Tween 20

A2 methods

A2.1 Forms of cell lysis in Aspergillus fumigatus, Absidia glauca, Candida albicans, Penicillium chrysogenum, Rhizopus stolonifer and Saccharomyces cerevisiae

The work-up of the cell walls of said mushrooms is carried out by three methods, which are shown in A2.1.1-A2.1.3. The starting material for all three cell lyses was obtained by cultivating the fungal strains for 72 hours at 28 ° C.

A2.1.1 cooking lysis

For the cooking lysis, first colony material is removed from the agar plate with a sterile toothpick and redistilled in 1 ml H ₂ O. suspended. The suspension is vortexed for 30 sec. And the titer is determined in a counting chamber. After 10 min. Incubation at 95 ° C in a water bath, the suspension is vortexed again for 30 sec. And 1 min. centrifuged at 13,000 rpm and room temperature (RT) in the eppifuge. Following the purification of the DNA extract by NucleoSpin ^® Food Kit takes place (see A2.2).

A2.1.2 Enzymatic digestion with lyticase

Colonial material is removed from the agar plate with a sterile toothpick and redistilled in 1 ml H ₂ O. suspended, vortexed for 30 sec and the titer determined by means of a counting chamber. The suspension is at 13,000 rpm and RT for 1 min. centrifuged in the eppifuge. The supernatant is removed and the pellet resuspended in 500 μl lyticase solution. The suspension incubated for 30 min. at 37 ° C. Afterwards it will be again 1 min. centrifuged at 13,000 rpm and RT in the eppifuge. The supernatant is discarded and the pellet is redistilled in 500 μl H ₂ O. washed, again 1 min. centrifuged at 13,000 rpm at RT in the Eppifuge. Done the purification of the DNA extract by the NucleoSpin ^® Food Kit (see A2.2).

A2.1.3 Enzymatic digestion with chitinase and lyticase

First, remove colony material from the agar plate using a sterile toothpick and place in 1 ml H ₂ O bidist. suspended. As with the other two methods, vortexing is carried out for 30 sec. And the titer is determined by counting. The suspension is left for 1 min. centrifuged at 13,000 rpm at RT in the Eppifuge and the supernatant removed. The pellet is resuspended in 500 μl chitinase solution. The enzymatic digestion by the chitinase is carried out for 1 hour at 25 ° C in a water bath.

Following the suspension is again 1 min. Centrifuge at RT in the eppi tube at 13,000 rpm, discard the supernatant, and distil with 500 μl H ₂ O bidist. washed and centrifuged again under the same conditions. In the following steps, the enzymatic digestion of the pellet by the lyticase takes place and corresponds to the method description from A2.1.2. The purification of the DNA extract is also carried out by means of the NucleoSpin ^® Food Kit (see A2.2).

A2.2 purification of the DNA extract by means NucleoSpin ^® Food Kit

The purification of the DNA extract from the cell lysis is carried out by means of the NucleoSpin ^® Food Kit from Macharey-Nagel. It is a column purification through a membrane, according to the protocol to the kit containing reagents (CF, buffer to bind the DNA to the membrane, C4, wash buffer, CQW, wash buffer, C5, buffer for dissolving the DNA from the membrane and CE, to bind the DNA in the buffer). For this purpose, the pellets produced by the respective lysis technique are dissolved in 500 .mu.l of CF and incubated for 30 min at 65.degree. C. in a waterbath. This is followed by a ten-minute centrifugation at 13,000 rpm at RT in the eppi-tube. It is a mixture of 300 ul supernatant, 300 ul C4 and 200 ul ethanol (reinst) created. 750 μl of this mixture are added to the column and incubated at 10,000 rpm and RT for 1 min. centrifuged. The supernatant is discarded. This is followed by washing steps with 400 μl washing buffer CQW and 700 μl washing buffer C5. Between the washing steps in each case 1 min. centrifuged at 10,000 rpm and RT in the effervescent tube and the supernatant discarded. Again, it is washed with 200 μl washing buffer C5. The centrifugation is carried out for 2 min. In the eppi tube at 13,000 rpm and RT. There are 100 ul elution buffer CE, previously 5 min. was heated to 70 ° C, added to the column and 5 min. incubated. Subsequently, the column 1 min. centrifuged at 13,000 rpm and RT in the eppifuge. The flow contains the purified DNA extract that can be used for the PCR.

A2.3 Polymerase Chain Reaction (PCR)

For the polymerase chain reactions carried out in these experiments, the master mixes are prepared and used according to the following instructions. A2.3.1 PCR approach D1-D2 region 36.25 μl H ₂ O Millipore 5.00 μl 10x PCR buffer 2.00 μl dNTPs 0.50 μl Primer 1 0.50 μl Primer 2 (biotin) 0.25 μl Taq polymerase 5.00 μl DNA extract A2.3.2 PCR approach for ITS1-ITS2 region 36.25 μl H ₂ O Millipore 5.00 μl 10x PCR buffer 2.00 μl dNTPs 0.50 μl Primer 1 0.50 μl Primer 2 (biotin) 0.25 μl Taq polymerase 5.00 μl DNA extract

To detect the species, a biotin-labeled PCR is first performed. The reverse primer is labeled with biotin and the PCR is set up as described in A2.3.2 and the program is used as described in A2.3.3.

The PCR programs used in these experiments are from a recommendation of the Heart and Diabetes Center, Bad Oeynhausen, Dr. med. Three. It comprises the steps described in A2.3.3 for the PCR of the D1-D2 region. For the ITS1-ITS2 region they are listed in A2.3.4. A2.3.3 Program for the PCR for the amplification of the D1-D2 region of the 28S rDNA 1. 94 ° C 300 s Second 94 ° C 15 s Third 54 ° C 30 s 4th 72 ° C 30 s 5th 72 ° C 300 s

Step 2.-4. with 35 repetitions

A2.3.4 PCR program to amplify the ITS1-ITS2 region of the 28S rDNA 1. 94 ° C 180 s Second 94 ° C 60 s Third 60 ° C 60 s 4th 72 ° C 150 s 5th 94 ° C 180 s

Step 2.-4. with 30 repetitions

A2.4 agarose gel electrophoresis

For the separation of a 500 bp fragment, an agarose gel with an agarose concentration of 1.5% (w / v) is suitable. An amount of agarose required for this ratio is dissolved in 1 × TAE electrophoresis buffer by heating. After cooling the solution to about 50 ° C, 18 ml are pipetted onto a gel carrier of dimensions 7 × 8 cm. After the gel has cured, the gel chamber is filled with 1X TAE electrophoresis buffer until the gel is covered with buffer for 1 to 2 mm, and the gel comb is pulled. 5 μl-10 μl of the PCR product are mixed with 3 μl bromophenol blue and pipetted into the gel pockets. 3 μl of the length standard containing λ-DNA HindIII cut and ΦX174-DNA HaeIII cut is pipetted into the outer gel pockets. The voltage at the gel chamber is 5 V / cm electrode gap. After electrophoresis, the gel is stained in a singly concentrated SYBR Gold solution for 30-40 minutes, placed on the transluminator and photographed with the Polaroid GelCamSystem.

A2.5 Quantitative nucleic acid determination by UV measurement

P:

Proteinmenge in mg/ml

d:

Schichtdicke in cm

N:

Nukleinsäuremenge in mg/ml

Tabelle 1: UV-Bestimmung von Nukleinsäuren neben Protein E₂₈₀/E₂₆₀ T F E₂₈₀/E₂₆₀ T F 1,75 0 1,118 0,86 0,052 0,671 1,60 0,0030 1,078 0,84 0,056 0,650 1,50 0,0056 1,047 0,82 0,061 0,628 1,40 0,0087 1,011 0,80 0,066 0,605 1,30 0,0126 0,969 0,78 0,071 0,581 1,25 0,0146 0,946 0,76 0,076 0,555 1,20 0,0175 0,921 0,74 0,085 0,528 1,15 0,0205 0,893 0,72 0,093 0,500 1,10 0,024 0,836 0,70 0,103 0,470 1,05 0,028 0,831 0,68 0,114 0,438 1,00 0,033 0,794 0,66 0,128 0,404 0,96 0,037 0,763 0,64 0,145 0,368 0,92 0,043 0,728 0,62 0,166 0,330 0,90 0,046 0,710 0,60 0,192 0,289 0,88 0,049 0,691 According to Warburg and Christian, total quantities of nucleic acids can be determined spectrometrically in addition to protein quantities. For this, the absorbance of the sample has to be measured at 260 and 280 nm. The protein and nucleic acid levels can then be calculated using the following formulas. The factors required for this finding are T, starting from the difference between the extinction at 280 nm (E280) to the absorbance at 260 nm (E260), from Table 1. P = E ₂₈₀ * F / d N = T * P / (1-T)

P:

Protein amount in mg / ml

d:

Layer thickness in cm

N:

Nucleic acid amount in mg / ml

Table 1: UV determination of nucleic acids in addition to protein E ₂₈₀ / E ₂₆₀ T F E ₂₈₀ / E ₂₆₀ T F 1.75 0 1,118 0.86 0,052 0.671 1.60 0.0030 1,078 0.84 0.056 0,650 1.50 0.0056 1,047 0.82 0,061 0.628 1.40 0.0087 1,011 0.80 0.066 0.605 1.30 0.0126 0.969 0.78 0,071 0,581 1.25 0.0146 0.946 0.76 0,076 0,555 1.20 0.0175 0.921 0.74 0.085 0.528 1.15 0.0205 0,893 0.72 0.093 0,500 1.10 0.024 0,836 0.70 0.103 0,470 1.05 0.028 0.831 0.68 0.114 0.438 1.00 0.033 0,794 0.66 0,128 0.404 0.96 0.037 0.763 0.64 0.145 0.368 0.92 0.043 0.728 0.62 0.166 0,330 0.90 0.046 0,710 0.60 0.192 0,289 0.88 0,049 0.691

Table 1 lists the factors F and T needed to calculate total nucleic acids besides protein.

A2.6 Total protein test with biuret method

The total protein test was carried out by means of the biuret method (kit from Merck). The test follows the information sheet included in the kit.

A2.7 Determination of the minimum detection limit

The aim of the experiment is to determine the minimum number of cells, which after agglutination of the fungi and subsequent PCR allows representation in agarose gel electrophoresis. For this purpose, cultures of the species Candida albicans and Aspergillus fumigatus, grown fresh on malt agar, were skimmed off with 2 ml of phosphate buffer. After the sample stood on the shaker for 10 minutes, 1 ml was taken and the conidite titer contained therein was determined. Dilution series of 10 ⁰ to 10 ^-5 were prepared from the samples with phosphate buffer. These dilutions were processed using the cell lysis method described in A2.1.3 and subjected to PCR (A2.3.1 / A2.3.3). The number of cells determined from the dilution, at which just one visible signal can be seen in the electrophoresis, is assumed to be the minimum detection limit.

A2.8 Determination of minimum growth period

For practical investigations, it is interesting to determine after which growing period a fungus can be detected with the described methodology. For this purpose, samples of a defined conidia number are prepared. For this purpose, 5 ml of phosphate buffer were added to agar with one mushroom species grown fresh on malt agar.

After the plate for 10 min. standing on the shaker, 1 ml of suspension was removed and the titer determined. The suspension was diluted until it contained 1000 conidia / ml. The exact conidia number is determined by plating out in each case 100 .mu.l of the respective suspension on five malt agar plates (control plates) by averaging. In addition, 100 .mu.l of the respective suspension are plated on another seven malt agar plates and also incubated at 28.degree. After 8, 16, 24, 32, 40, 48 and 72 hours, 2 ml of phosphate buffer were added to a plate. After the plate stood on the shaker for 10 minutes, 1 ml suspension was taken and the titer was determined. Next, the suspension of the in A2.1.3 subjected to the method described for cell lysis. The result according to the PCR described in A2.3.1 / A2.3.3 is shown in B3.

A2.9 Detection reactions

The methodology for the detection of fungal species is based on the DIAPROPS technology (Nunc). This is done in three steps. First, a specific probe is coupled to the surface of the NucleoLink ^™ microtiter strips (A2.9.1). Then, the hybridization with the biotin-labeled amplificate from the PCR (A2.9.2) and finally the detection reaction, in this case using SAP and pNPP (A2.9.3). The three steps are described in more detail in points A2.17.2-C2.17.4 and are in shown schematically.

A2.9.1 Coupling of Specific Probes to the Surface of NucleoLink ^™ Microtiter Strips

For detection of the species it is necessary to bind specific probes to the surface of the NucleoLink ^™ wells for the respective types of fungi to be detected. This is done as follows: First, the specific oligonucleotides are redistilled with H ₂ O. Diluted to a final concentration of 7.5 μg / ml. 1/10 of the volume is added in the form of cold methylimidazole solution. Each well of the NucleoLink ^™ strip is pipetted with 75 μl of this solution. There are also 25 μl of EDAC solution per well. The strip is sealed with an adhesive film (Tape 8, Nunc) and incubated overnight in a humid chamber or in the incubator at 50 ° C. The strips are then washed five times with prewarmed to 50 ° C washing solution. They are easily knocked out between the washing steps. The strips are now slightly dried and sealed with adhesive film (Tape 8, Nunc). They can be stored at 4 ° C for up to two months.

A2.9.2 Hybridization of the specific probes with the biotinylated amplicon

For the detection of the respective species it is necessary to hybridize the biotinylated amplificate from the PCR to the respective probes. For this, 5 μl PCR product (A2.3.2 / A2.3.4b) with 95 μl hybridization buffer are pipetted into the probe-coated NucleoLink ^™ wells and sealed with adhesive tape (Tape 8, Nunc). As a control reaction, 100 μl of hybridization buffer without PCR product are used. The wells are incubated for one hour at 49 ° C. Subsequently, unbound amplificates are removed by washing three times with washing buffer. The wells are watered for 15 minutes at 50 ° C with washing buffer and then washed again three times and gently tapped on a paper.

A2.9.3 Detection reaction using SAP and pNPP

After the hybridization of specific probes with the PCR product, the detection is finally carried out by optical detection. This is achieved by the cleavage reaction of pNPP by the alkaline phosphatase. For this purpose, SAP 1: 2000 is diluted in DIAPROPS buffer and 100 μl of the solution are pipetted into each well. The strips are incubated at 37 ° C for one to two hours. Subsequently, the wells are washed three times with washing buffer, watered for five minutes and washed again three times. Each well is pipetted with 100 μl of color detection buffer. Color development occurs in the dark at room temperature. The optical density is measured spectrometrically at 405 nm.

A2.10 Tests for detection reaction

The detection system developed in these experiments (see A2.9) consists of four individual reactions. In order to exclude sources of error such as non-specific binding of the individual components, the reactions must be individually tested in advance. Thus, detection of the mixed culture is first preceded by detection of the individual species in the wells provided for this purpose. The hybridization of the respective probe with the homologous biotinylated amplicon from the PCR (A2.3.2, A2.3.4) is carried out. A detailed description of the experiment is given in A2.10.1. Following the single-fungus experiments, the specificity of the probes prepared for the respective species of fungus can be tested in a cross-experiment. For this purpose, all mushrooms are raised in the form of isolated individual plants. The amplificates derived from the cell lysis as described in A2.1.3 following PCR are subjected to the detection system in the designated probes coated wells. It is important that the amplicons of each fungus are tested on each probe.

A2.10.1 Detection of specific amplicons of A. fumigatus, P. chrysogenum and C. albicans in an individual experiment

The detection system described in A2.9 must first be tested on fungi grown in isolated single colony. Following cell lysis (A2.1.3) and PCR with biotinylated primer (A2.3.2, A2.3.4), the specific probes in the wells are hybridized only with the homologous amplicons. For this, the described detection method is first carried out individually for each fungus. In each half of NucleoLink ^™ strips (four wells), which are coated with the specific probe of each one fungus, 100 ul hybridization buffer are pipetted. In addition, 15 μl of the PCR product consisting of the amplicon specific for the probe are added in three wells. For example, in a well with a C. albicans probe it hybridizes only with amplicons from the PCR of C. albicans. The control is one well per strip, in which no PCR product is pipetted into the hybridization buffer. After hybridization, the detection reaction is carried out using SAP and pNPP as described in A2.9.3. The reaction is carried out for A. fumigatus, P. chrysogenum and C. albicans and repeated three times. The result is shown in B4. At this point also raises the question of how large the amount of DNA used must be to cause a clear color reaction. For this, a test with NucleoLink ^™ strips from all three probes was performed. In this case, 15 .mu.l, 10 .mu.l, 7.5 .mu.l, 5 .mu.l, 2.5 .mu.l and 1 .mu.l of the amplificate from the PCR are pipetted in the hybridization mixture, in each case in a well, with the respective gene section homologous to the probe. The two remaining wells per strip serve as negative control and are set up without amplicons. After hybridization for one hour at 48 ° C, the detection reaction is carried out as described in A2.9.3. The results are listed in B4.2.

A2.10.2 Detection of specific amplicons of A. fumigatus, P. chrysogenum and C. albicansim cross test

After a detection reaction of the specific fungal DNA in the wells, which were coated with the probes, was carried out (A2.10.1), the specificity of the probes is now tested in a cross experiment. In doing so, the question will be investigated whether foreign DNA also hybridizes to the respective probes. For this purpose, in each case one well of 15 μl of the PCR product of one of the three fungi to be detected is added to 100 μl of hybridization buffer in the wells per half a NucleoLink ^™ strip. That is, for example, in a half strip (four wells) with A. fumigatus probe, the well hybridization approach is carried out with 15 .mu.l C. albicans amplificate, one with 15 .mu.l P. chrysogenum amplicon, and one with 15 .mu.l of A. fumigatus -Amplifikat set. As a control reaction, only hybridization buffer without PCR product is added to the remaining well and the detection reaction (A2.9) is carried out with the strip. The cross test is carried out with the probes of A. fumigatus, P. chrysogenum and C albicans and their amplicons from the PCR, of the respective fungi in single colony, on the basis of four test strips. The results are shown in B5.

A2.11 Identification of A. fumigatus, C. albicans and P. chrysogenum from mixed culture

Furthermore, a test of the detection reaction with an amplicon mixture from the species used in these experiments, A. fumigatus (A), C. albicans (C) and P. chrysogenum (P), is required.

Here are. in strips (eight wells) of each probe hybridization approaches of 100 ul hybridization buffer and 15 ul of a mixture of the amplificates created. Three wells with one amplicon mixture of all three fungi (APC) and three others each with a mixture of only two fungal amplicons (PC, AC and AP) are created. The two remaining wells per strip serve as negative control without PCR product. After hybridization, one hour at 48 ° C, the detection system is applied as described in A2.9.3. The result is shown in B6.

B. Results

B1 workup methods

The comparison of the work-up methods described in A2.1.1, A2.1.2 and A2.1.3 was performed twice each with Candida albicans (C1 and C2), Penicillium chrysogenum (P1 and P2) and Aspergillus fumigatus (A1 and A2) and five times with the one in A2 .1.3 enzymatic digestion with chitinase and lyticase.

In the agarose gels, 3 μl of bromophenol blue (BPB) were always mixed with 5 μl of PCR product and pipetted into a bag when comparing the work-up methods. In the outer pockets were always 3 μl length standard (M) pipetted (see A2.4). As control (K), 5 μl of a PCR batch without DNA was used, which was applied like a sample with 3 μl BPB.

B1.1 cooking lysis

In the gel in For example, the PCR products of the six batches (two per species) are plotted after boiling lysis (see A2.1.1). Table 2: Conidient titre of the batches before cooking lysis sample C1 C2 P1 P2 A1 A2 Konidientiter 3.5 · 10 ⁸ 3,4 · 10 ⁸ 2.8 · 10 ⁸ 2.9 · 10 ⁸ 1,2 · 10 ⁸ 1.7 · 10 ⁸

The polaroid uptake of the agarose gel of the three fungal species after boiling lysis and PCR, as described in A2.1.1, shows that this work-up method was only successful with C. albicans (C1 and C2) at approximately comparable titers. Also, higher titers of P. chrysogenum (P1, P2) and A. fumigatus (A1, A2) did not result in PCR amplicons (results not shown). The amplificate corresponds to an expected size and banded at the height of the 603 bp sands of the marker (cf. )

B1.2 Enzymatic digestion with lyticase

On the gel photo in the PCR products of the six batches of the species to be compared are to be seen after the enzymatic digestion by lyticase (see A2.1.2). Table 3: Conidient titres of the batches before enzymatic digestion by lyticase sample C1 C2 P1 P2 A1 A2 Konidientiter 3.6 · 10 ⁸ 3.1 · 10 ⁸ 4.5 · 10 ⁸ 3.8 · 10 ⁸ 2.2 · 10 ⁸ 2.8 · 10 ⁸

The polaroid uptake of the agarose gel of the three fungal species after enzymatic digestion by means of lyticase, as described in A2.1.2, shows that the work-up method at approximately comparable titers was also successful only in C albicans (C1 and C2). Even higher titers of P. chrysogenum (P1, P2) and A. fumigatus (A1, A2) did not result in PCR amplicons (results not shown).

B1.3 digestion using chitinase and lyticase

The work-up method with enzymatic digestion by chitinase and lyticase described in A2.1.3 was tested five times on each fungus species. In the agarose gels in - the PCR products of the species A. fumigatus, A. glauca, C. albicans, P. chrysogenum, R. stolonifer and S. cerevisiae are applied. Each gel contains the five PCR products of one species and one negative control each. The length standard is in described. The conidient titer of the mixtures of the species before enzymatic digestion with chitinase and lyticase is shown in Table 4. Table 4: Conidient titers of the batches before enzymatic digestion with chitinase and lyticase sample 1 2 3 4 5 Konidientiter: C. albicans 1.4 · 10 ⁸ 2.8 · 10 ⁸ 1.0 · 10 ⁸ 1.0 · 10 ⁸ 0.9 · 10 ⁸ S. cerevisiae 1,2 · 10 ⁸ 1.8 · 10 ⁸ 1.5 · 10 ⁸ 2.3 · 10 ⁸ 1.0 · 10 ⁸ R. stolonifer 3.2 · 10 ⁸ 4.6 · 10 ⁸ 1.7 · 10 ⁸ 0.9 · 10 ⁸ 2.3 · 10 ⁸ A. glauca 2.6 · 10 ⁸ 0.8 · 10 ⁸ 4.4 · 10 ⁸ 3.2 · 10 ⁸ 2,7 · 10 ⁸ A. fumigatus 4.3 · 10 ⁸ 5.3 · 10 ⁸ 0.9 · 10 ⁸ 5.2 · 10 ⁸ 0.8 · 10 ⁸ P. chrysogenum 1.1 · 10 ⁸ 1.3 · 10 ⁸ 1.9 · 10 ⁸ 1.0 · 10 ⁸ 1.4 · 10 ⁸

Polaroid photographs of agarose gels after PCR show that this method of lysis and purification by NucleoSpin ^® kit at the compared fungal species A. fumigatus, A. glauca, C. albicans, P. chrysogenum, R. stolonifer and S. cerevisiae successfully was. The area to be amplified is expected to be about 600 bp in size. Variations in size are possible depending on the species. Since this work-up procedure (C) has a positive result in all the species tested, the further experiments were carried out at the D1-D2 region for the lysis of fungi exclusively with this lysis and work-up procedure and with the PCR program used (A2.3.3). For the experiments for detection at the ITS1-ITS2 region, the above-described lysis and work-up procedure (enzymatic digestion with chitinase and lyticase, A2.1.3) was also used, but the PCR protocol and program from A2.3.2 or A2.3.4 ,

B2 Determination of the minimum detection limit

The method for determining the minimum detection limit is described in A2.7. In the agarose gels for this test series, in each case 3 μl of BPB were mixed with 5 μl of PCR product and applied. 3 μl of marker with 3 μl BPB were applied to the outer pockets.

In addition, a negative control (K) was included.

B2.1 Candida albicans

The conidia suspension of C. albicans was diluted in increments of 10 and the dilution steps were subjected to Lysis Procedure C. The lysis products were subjected to purification by NucleoSpin ^® Food Kit subjected (cf.. A2.2). The dilution series of Candida albicans conidia suspension showed up to a Konidientiter of 1.9 · 10 ⁴ visible in the agarose gel result. Candida albicans can therefore be detected by enzymatic digestion by chitinase and lyticase to a titer of about 19,000 cells / ml by PCR.

B2.2 Aspergillus fumigatus

The conidia suspension of A. fumigatus was diluted in increments of 10 and the dilution levels were subjected to Lysis Procedure C. The lysis products were subjected to purification by NucleoSpin ^® Food Kit subjected (cf.. C2.13. The dilution series of Aspergillus fumigatus conidia suspension pointed to a Konidientiter of 4.2 × 10 ³ in a visible result on agarose gel. Aspergillus fumigatus can consequently with a enzymatic digestion by chitinase and lyticase to a titer of about 4,200 cells / ml are detected by PCR.

B2.3 Penicillium chrysogenum

The conidia suspension of P. chrysogenum was diluted in increments of 10 and the dilution steps were subjected to Lysis Procedure C. The lysis products were subjected to purification by NucleoSpin ^® Food Kit subjected (cf.. A2.2). The dilution series of the Penicillium chrysogenum conidia suspension showed up to a Konidientiter of 1.6 · 10 ⁴ visible in the agarose gel result. Aapergillus fumigatus can thus be detected by enzymatic digestion by chitinase and lyticase to a titer of about 16,000 cells / ml by PCR.

B3 Definition of the minimum growth period necessary for a proof

B3.1 Control of the output titer

Conidial suspensions were diluted in phosphate buffer so far that after plating 0.1 ml aliquots, approximately 10 colonies per agar plate were expected. 11 plates were inoculated per batch, of which 5 plates were used for the titering. The remaining 6 plates were incubated and each plate was washed away at 5, 8, 15, 20, 24 and 29 hours. The suspensions were subjected to the developed procedure for cell lysis and purification (see A2.8).

B3.2 Candida albicans

For Candida albicans, it was found that with an average colony count of 7 CFU / plate and 20 hours of growth time, total spore and total cell counts in the washed-out suspension was measurable and detectable in the agarose gel. The experiments were repeated several times with the same result {results not shown).

B3.3 Aspergillus fumigatus

Aspergillus fumigatus was found to be detectable in the agarose gel with an average colony count of 12 cfu / plate and incubation for 40 hours. Microscopic conidia can also be visualized at this time. The experiments were repeated several times with the same result {results not shown).

B4 identification via NucleoLink ^™ Wells

B4.1 Detection of specific amplicons of A. fumigatus, C. albicans and P. chrysogenum in an individual experiment

The method described in A2.10.1 for the detection of the amplicons of A. fumigatus, C albicans and P. chrysogenum is carried out in individual tests of the respective species. The aim of the experiment is to ensure the binding of the specific probes and the corresponding amplicons. Table 5 plots the optical densities measured after 20 hours at 405 nm. The experiment was repeated three times. Table 5: Optical density averages at 405 nm after 20 h of single-mode amplified A. fumigatus, C albicans and P. chrysogenum amplitudes in the NucleoLink ^™ wells Coating the probe with: OD after hybridization with specific amplificates n = 9 standard deviation Significance in the t-test with an error probability of α A. fumigatus 1,092 0.057 0.0001 P. chrysogenum 1,960 0.169 0.0001 C. albicans 2,153 0.331 0.0001 Negative control without amplificate 0.078 0.033

The optical densities after hybridization with the amplificates differ significantly from the negative control (significance in the t-test with an error probability of α = 0.0001, at FG = 8). This allows a clear conclusion on a positive hybridization of the amplicons of A. fumigatus, C albicans and P. chrysogenum with the respective specific probe.

B4.2 Verification of the sensitivity of the detection reaction

In the following experiment, the sensitivity of the detection reaction was tested. For this purpose, decreasing volumes (15 μl, 10 μl, 7.5 μl, 5 μl, 2.5 μl and 1 μl) of the amplificates were used for the hybridization. The detection reaction is carried out according to the procedure described in A2.9.3. The optical density was measured at 405 nm after a reaction period of 20 h at RT. The data collected in Table 6 is stored in shown. Table 6: Optical density of the dilution series of specific amplificates of A. fumigatus, C. albicans and P. chrysogenum in an individual experiment 15 μl 10 μl 7.5 μl 5 μl 2.5 μl 1 μl 0 μl 0 μl Aspergillus 1,814 1,550 1,026 0.709 0,330 0,250 0,120 0.057 Penicillium 3,224 3,598 3,198 1,238 0,770 0.201 0,099 0.086 Candida 3,554 3,253 2,877 1,092 0,975 0,181 0.097 0,049

The in Table 5 and The data shown in the data show that the rDNA levels achieved after amplification provide a sufficient sensitivity for species detection at a test volume of 10 μl-15 μl. Only at volumes of about 5 μl-2.5 μl, the test specificity decreases significantly. The volumes used do not correspond to the band strength of the amplificates in the agarose gel (Gelfoto DNA levels of about 6.6 ng / μl A. fumigatus PCR product, about 7.7 ng / μl PCR product for P. chrysogenum and about 8.3 ng / μl PCR product for C albicans.

B5 Verification of the specificity of the detection system

Homologous amplificates show a significant response compared to the control (B4.1). The specificity of the hybridization probes was tested below. For this purpose, amplificates of the species A. fumigatus, C. albicans and P. chrysogenum were subjected to hybridization with each probe used in the experiments and further tested with the detection system. For example, in the homologous system of the Candida probe it was hybridized with Candida amplicons. In the heterogeneous system of the Candida probe, it was hybridized with Aspergillus or Penicillium amplificates. The control used was a reaction mixture which was prepared with 15 μl of water instead of the amplificate. One control reaction was carried out per probe. The exact.

Experiment description is detailed in C2.18.2. The results from the measurements of the optical density (OD) at 405 nm of the cross experiments are for the species A. fumigatus, C. albicans and P. chrysogenum in the - and were performed in the spectrometer after 20 h color reaction. The dark highlighted columns in the diagrams represent the mean value of the measurements (light columns) of the various approaches. The mean value is additionally indicated in numbers in the diagrams. The cross experiment was performed with C. albicans probe, P. chrysogenum probe and A. fumigatus probe and the respective amplicons of the 28S rDNA.

Candida albicans probe:

From the results, presented in , it can be seen that the amplified products homologous to the probes (with Candida amplificate) show the strongest reaction in all cases. In comparison, the heterogeneous amplified products show a significantly weaker color reaction. In the batches without any amplificate, the negative controls, the color reaction goes to zero.

Penicillium chrysogenum probe

In the homologous system (Penicillium amplificate with Penicillium probe), a stronger reaction can also be observed in the cross experiment with the Penicillium probe than in the heterogeneous system ( ). Although the reaction strength in the homogeneous system is about 60% higher than in the heterogeneous system, the Candida amplificates show in individual cases relatively high optical densities (1.036 / 0.955).

Aspergillus fumigatus probe

clarifies the result for A. fumigatus. It is also shown for the Aspergillus probe that the color reaction in the wells is much stronger with the Aspergillus amplicons homologous to the probes than in the wells with the heterogeneous amplicons. On average, the OD of the samples in the wells two and three (heterogeneous system) is less than half or only one third of the OD of the homogeneous system. The color reaction in the non-amplified batches, which serve as the negative control, approaches zero.

From the experiments to verify the specificity in the detection system, presented in - , it can be seen that in all cases the amplicon homologous to the probes show the strongest response. Meanwhile, the heterogeneous amplicons produce significantly weaker detection reactions, but their OD ranked above that of the controls without amplificate. A comparison of the three species shows that there is clear evidence for the probes of C. albicans and P. chrysogenum. The negative control shows in all cases only a very low optical density.

B6 Identification of A. fumigatus, C. albicans, and P. chrysogenum from mixed culture

The test for the detection reaction from the mixed culture is carried out with an amplificate mixture of A. fumigatus (A), C. albicans (C), and P. chrysogenum amplicons (P) and is described in detail in A2.11. In this case, amplicon mixtures of all amplified products (APC) or only two (AP, PC, AC) are used in the hybridization and subjected to the detection system. As a result, the measurements of the optical density at 405 nm after 20 hours of incubation with the color buffer in Table 7 are tabulated. Table 7: Optical densities of the detection reaction from the mixed culture APC APC APC PC AC AP Without amplificate Without amplificate Asp 0.458 0.509 0.556 0.118 0.437 0,381 0.056 0,052 Pen 0.637 0.837 0.573 0.562 0.098 0.499 0.103 0.098 Can 1,055 0.643 0,599 0.498 0.584 0.162 0.097 0,101

The measurement of the optical densities revealed a stronger color reaction in the wells containing the amplicon homologous to the probe (values highlighted) than in those containing heterogeneous amplicons. The reaction strength of the heterogeneous amplified products is approximately in the range of the controls. Overall, it can be shown that detection is also successful from mixed culture.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

• McCullough et al. (Journal of Clinical Microbiology 1998, 36 (4): 1035-1038) [0035]
• White et al. (Pages 315-322 in "PCR protocols: a guide to methods and applications" by Innis, Gelfand, Sninsky and White, San Diego 1996, Academic Press). [0035]
• McCullough et al. (Journal of Clinical Microbiology 1998, 36 (4): 1035-1038) [0050]
• VDI 6022 [0060]
• McCullough et al. (Journal of Clinical Microbiology 1998, 36 (4): 1035-1038) [0071]

Claims (17)

A lysis buffer for the lysis of molds and / or yeasts, wherein the lysis buffer is suitable for lysing the cell walls of taxonomically far-reaching molds and yeasts. The lysis buffer of claim 1, wherein the lysis buffer comprises chitinase, optionally wherein the chitinase is present at a concentration of from 0.1 μg / ml to 3.0 μg / ml in the lysis buffer, preferably wherein the chitinase is present in a concentration of about 0.44 μg / ml is present in the lysis buffer. The lysis buffer of claim 2 further comprising 5 mM to 200 mM Tris / HCl, 10 mM to 250 mM NaCl and 2 mM to 50 mM MgCl ₂ , the lysis buffer having a pH of 4.5 to 7.5. A kit for the detection of molds and / or yeasts, comprising: i) a first lysis buffer according to any one of the preceding claims; ii) a second lysis buffer, wherein the second lysis buffer comprises lyticase, optionally wherein the lyticase is present in a concentration of 300 to 2000 U / ml in the second lysis buffer, preferably wherein the lyticase is present in a concentration of about 700 U / ml in the second lysis buffer; and iii) at least one reaction vessel, wherein at least one kind of nucleic acid probe is bound to the inside of the at least one reaction vessel, each kind of nucleic acid probe being specific for a mold or a yeast and comprising a binding sequence that is complementary to a genomic sequence or the complementary sequence of a genomic Sequence of the mold or yeast residing in a DNA region comprising the ITS1, the 5.8S rDNA and the ITS2. The kit of claim 4, wherein the second lysis buffer further comprises 10 mM to 250 mM Tris / HCl, 2 mM to 50 mM EDTA, and 5 mM to 100 mM β-mercaptoethanol, the second lysis buffer having a pH of 6.0 to 9, 0 has. The kit of any one of claims 4 or 5, wherein the molds and / or yeasts are selected from the group consisting of the Zygomycetes, the Ascomycetes and the Deuteromycetes. The kit of any one of claims 4-6, wherein each of said at least one reaction vessel is bound to a single type of nucleic acid probe, or wherein at least one reaction vessel is attached to a plurality of types of nucleic acid probes. The kit of any of claims 4-7, further comprising: iv) a reaction mixture comprising at least one DNA polymerase, dNTPs, a first primer and a second primer, wherein the first primer at the 3 'end contains a sequence which specifically binds to a target fungus or yeast sequence described in 18S rDNA, and wherein the second primer at the 3 'end contains a sequence that specifically binds to a targeting sequence of the mold or yeast residing in the 28S rDNA. The kit of claim 8, wherein the DNA polymerase is selected from the group consisting of Taq polymerase, Pfu polymerase and Tbr polymerase. The kit of any of claims 4-9, wherein at least one of the two primers is linked to a labeling group, the kit further comprising: v) an enzyme to which is attached a linking group capable of specifically binding the labeling group of the first and / or second primer, which enzyme is capable of catalyzing a reaction the course of which is optically detectable; and vi) a substrate that can be reacted by the enzyme in the reaction. The kit of claim 10, wherein the labeling group is biotin and the binding group is streptavidin or wherein the labeling group is an antigen and the binding group is an antibody specific for the antigen. The kit of any one of claims 10 or 11, wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase and firefly luciferase. The kit of claim 12, wherein the enzyme is alkaline phosphatase and the substrate is p-nitrophenyl phosphate (pNPP) or a mixture of 5-bromo-4-chloro-3-indoxyl phosphate with nitroblue tetrazolium chloride (BCIP / NBT). The kit of any of claims 4-13, further comprising: vii) at least one centrifugation column for the solid phase extraction of nucleic acids; viii) a first wash buffer suitable for removing molecules other than nucleic acids from the centrifugation column; and ix) a second washing buffer which is suitable for removing DNA not bound to the nucleic acid probes from a reaction vessel. The kit of claim 14, wherein the first wash buffer comprises 70-90% ethanol and 2mM to 10mM potassium phosphate buffer at a pH of 7.5 to 8.5, preferably wherein the first wash buffer contains about 80% ethanol and about 5mM potassium phosphate buffer a pH of 8.0. The kit of any one of claims 14 or 15, wherein the at least one centrifugation column comprises a solid phase of silica. The kit of any one of claims 4-16, wherein the second wash buffer comprises PBS with 0.1-2.0% Tween 20 by volume.

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