Patent Application
20090136984
The present invention relates to polypeptide fragments of endolysin Ply511, which recognise and bind listeria irrespective of the serotype but which have no cell wall hydrolysing enzymatic activity. The invention further relates to methods for enrichment, removal, and detection of listeria.
Listeria are human and animal pathogenic bacteria, which are frequently present in food, in particular in fish, meat and milk products. The genus listeria comprises 6 different species with 16 different serotypes. In detail, these are L. monocytogenes having the serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 7; L. innocua having the serotypes 3, 6a, 6b, 4ab, U/S; L. ivanovii having the serotype 5; L. seeligeri having the serotypes 1/2a, 1/2b, 1/2c, 4b, 4c, 4d, 6b; L. welshimeri having the serotypes 1/2a, 4c, 6a, 6b, U/S, and L. grayi having the serotype Grayi. The two species L. monocytogenes and L. ivanovii are considered to be pathogenic. A third species, L. seeligeri, is regarded to be apathogenic; however, there is one case known, in which L. seeligeri caused meningitis in a human. The remaining species are considered to be apathogenic. About 90% of the listerioses are ascribed to L. monocytogenes serovar 1/2a, 1/2b, and 4b (Wing E J & Gregory S H, 2002: Listeria monocytogenes: Clinical and Experimental Update, J Infect Diseases 185 (Suppl 1): S18-S24).
Although only a small portion of the food related diseases is caused by listeria (about 1% in the USA), almost 30% of the annual fatal diseases, which are caused by food pathogens, are ascribed to this germ. Affected are above all immunosuppressed persons, e.g., elderly people, diabetics, and persons suffering from cancer and/or AIDS. Pregnant women and the unborn child represent about 25% of all cases of people diseased with invasive listerioses. Because of their ability to overcome the blood-brain barrier or the placental barrier listeria may cause meningitis, encephalitis, aborts, and stillbirths (Wing E J & Gregory S H, 2002, Listeria monocytogenes: Clinical and Experimental Update, J Infect Diseases 185 (Suppl 1): S18-S24; Doyle M E, 2001: Virulence Characteristics of Listeria monocytogenes, Food Research Institute, October 2001).
Listeria tolerate weak acids very well and are able to reproduce under relatively high salt concentrations and at temperatures from 1° C. to 45° C. The main source of infections are foods, in particular the ones which are not heat-treated prior to consumption, such as many milk products, kipper, meat products, and increasingly also ready-to-eat-products. While there is a zero tolerance for L. monocytogenes in ready-to-eat-foods, in many European countries, or also Canada, a contamination with listeria up to 100 cfu (colony forming units)/g food is allowable for certain foods. At any rate, the foods have to be tested for listeria contamination, however. Many of these foods, e.g., seafood, kipper, milk products or even raw vegetable ready-to-eat-products have only a limited shelf life. This frequently amounts to cost intensive product recalls, if these products are positively tested for a listeria contamination and a contamination beyond the allowed threshold value, respectively, after delivery. For said reason there is a great interest to significantly shorten the up to now relative long time period, which is necessary to detect a contamination.
For a sufficient control of the foods in respect to listeria, the detection of L. monocytogenes alone is not sufficient because cases of listerioses are known as well, which were caused by L. ivanovii and L. seeligeri. In addition, a testing of all listeria species may be used as a sanitation monitoring in connection with food production. A problem connected with the detection of listeria is the time, which is necessary for the detection. In particular, in food industry the detection time represents a major factor in regard to a short shelf life of some foods and a cost intensive storage, which is necessary until it has been made sure that the sample is not contaminated. Moreover, there can consistently be observed cost intensive product recalls, when contaminated goods are delivered ahead of schedule before the receipt of the control results. The standard detection times are more than 4-7 days according to ISO 11290-1:1996/FDAM 1:2004(E) and 4 to 7 days according to FDA and USDA/FSIS. For many other methods the enrichment takes up to 48 h in order to gain a sufficient amount of listeria for the detection without any interfering components of the food sample.
This problem was addressed by a multitude of detection methods for listeria, wherein many of them are also commercially available, e.g., PCR, ELISA, etc. (cf. US 2004/0197833 A1). Such a method is very promising in the case of pure cultures; however, it shows significant problems in the case of mixed cultures or complex matrices such as foods.
In this context, a method uses antibodies against the flagella proteins of L. monocytogenes (Skjerve, 1990; Skjerve E., Rorvik L M, Olsvik O.: Detection of Listeria monocytogenes in foods by immuno-magnetic separation. Appl Environ Microbiol. 1990 (11):3478-3481), which are immobilised on magnetic particles. Using a similar method in experiments with cheese (Uyttendaele et al., 2000; Uyttendaele M, Van Hoorde I, Debevere J.: The use of immuno-magnetic separation (IMS) as a tool in a sample preparation method for direct detection of L. monocytogenes in cheese. Int J Food Microbiol. 2000; 54(3):205-212) cell numbers of 0.5-1.5 cfu per gram cheese could be detected. However, it was found that the antibodies not only bind to the target cells. Such a method is indeed very promising in the case of pure cultures; however, it shows significant problems in the case of mixed cultures or complex matrices such as foods. Thusly, the portion of unbound non-listeria was very high. In addition, using said method sufficient listeria could only then be obtained, when the food homogenates were diluted, centrifuged, and enzymatically digested to enable binding of the cells to the particles. In a further experiment (Jung et al., 2003; Jung Y S, Frank J F, Brackett E R: Evalution of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes. J Food Prot. 2003; 66(7):1283-1287) Jung et al. showed that only 7%-23% of the listeria could be isolated from a buffer solution using the antibody coated magnetic beads.
Recovery rates of 5%-15% of the originally used listeria were also obtained in experiments by Fluit et al. (Fluit et al, 1993; Fluit A C, Torensma R, Visser M J, Aarsman C J, Poppelier M F, Keller B H, Klapwijk P, Verhoef J.: Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay. Appl Environ Microbiol. 1993; 59(5):1289-1293) with magnetic particles which were coated with monoclonal antibodies against whole listeria cells without flagella as well as with monoclonal antibodies against the listeria flagella protein. In addition, 10% of the listeria were not recognised by the antibodies.
A further method uses the cell binding domains (CBD) of two listeria phage endolysins (WO 00/11472; US2004/0197833 A1). Endolysins are phage encoded proteins, which are produced in the late phase of the phage maturation and which together with a membrane pore forming holin enables the lysis of the host cell for the release of the mature phage particles. Endolysins consist of two domains, a lytic domain and a cell binding domain (CBD). The lytic domain cleaves the peptidoglycane of the cell wall of gram positive bacteria. The CBD binds to the cell wall and determines the specificity of the endolysin.
In this method (WO 00/11472; US2004/0197833 A1) fusions of a green fluorescence protein (GFP) with the CBD (CBD118 and CBD500) of the endolysins Ply118 and Ply500 from the listeria phages A118 and A500 were used. These proteins were immobilised on magnetic particles and tested for their ability to enrich listeria. The CBD118/CBD500-system was tested with different food samples in comparison to the IDF standard method (IDF, 143A:1995). It was shown that by using the coated particles the pre-enrichment period could be significantly shortened in order to detect listeria in foods.
In regard to the binding properties of the CBD it was shown, however, that neither CBD118 nor CBD500 alone recognise all serotypes. Thus, it is necessary to use always a mixture with both CBD linked to particles for the enrichment, because it is not known beforehand, which serotype and which serotypes, respectively, the listeria represent in the samples. Thereby the amount of particles, which is to be used per food test, is duplicated resulting in an increase in cost of the system and an increase of the risk of non-listeria adhering unspecifically to the particles.
Thus, the problem underlying the present invention is to provide a method and the means to perform said method with which listeria of all serotypes may be detected easily and fast and be removed.
The problem is solved by the subject-matter defined in the patent claims.
The following figures illustrate the invention.
a show the comparison of the nucleic acid sequence of endolysin Ply511 with the polypeptide fragments CBD511_f1, CBD511_f2, and CBD_f3.
The term “listeria” as used herein means all bacteria, which are assigned to the genus listeria. In particular, the term “listeria” encompasses the species L. monocytogenes having the serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 7; L. innocua having the seroptypes 3, 6a, 6b, 4ab, U/S; L. ivanovii having the serotype 5; L. seeligeri having the serotypes 1/2a, 1/2b, 1/2c, 4b, 4c, 4d, 6b; L. welshimeri having the serotypes 1/2a, 4c, 6a, 6b, U/S and L. grayi having the serotype Grayi.
The term “depletion of listeria” or “listeria removal”, as used herein, means the total or partial removal of listeria from sample material.
The term “pre-enrichment” or “enrichment”, as used herein, means the growing of listeria e.g., in a food sample, which was spiked with respective nutrient medium, having the aim to increase the concentration/amount of listeria in said sample, so that a respective detection step enables an unambiguous positive or unambiguous negative statement.
The term “sample material” or “sample”, as used herein, comprises all solutions in which listeria are to be detected or from which listeria are to be removed. Exemplary for samples is the following listing: aqueous solution and mixtures of water and organic solvents, food, media, blood, blood products, plasma, serum, urine, protein solutions, water-ethanol-mixtures. Furthermore, comprised are also solutions in which non-aqueous, solid substances were solved, which are to be examined or to be isolated, for example protein, DNA, RNA, sugars, salts, food, food-media-homogenates, medicaments, vaccines, organic and inorganic chemicals (e.g., NaCl, MgCl2, purines, pyrimidines, etc.).
The term “endolysin”, as used herein, relates to a naturally phage encoded enzyme which serves for the release of new phages at the end of a respective phage reproduction cycle. These endolysins consist of an enzymatic active domain and a domain binding to the cell wall of the respective host cell. In addition, said term is understood to comprise also the similarly composed autolysins. These are bacteria encoded and consist also of an enzymatic active cell wall hydrolysing domain and a domain binding to the cell wall of the target bacterium.
The term “CBD”, as used herein, relates to polypeptide fragments, wherein the respective amino acid sequence corresponds to a portion in endolysins. Said portion is responsible for the binding of the endolysins to the listeria cell wall. Said polypeptide fragments are not enzymatically active. The CBD may also be present as a gene fusion with a spacer molecule (GFP, MBP, biotinylation domains) with and without an affinity tag (His-Tag, Strep-Tag, Avi-Tag, biotinylation domains) or also as a gene fusion only with affinity tag (His-Tag, Strep-Tag, Avi-Tag, biotinylation domains).
The term “unspecific immobilisation” or “undirected immobilisation”, as used herein, means that the coupling of a CBD to a matrix is achieved via amino acid residues (e.g., primary amines), which are dispersed over the entire polypeptide surface. The selection of the residue, which is used for the coupling of the single polypeptide molecule, is performed randomly. In this case the CBD may either be coupled directly to activated groups on a matrix (e.g., binding to Dynabeads M-270 Epoxy, Dynale), or a group may be chemically introduced into the CBD (e.g., introduction of biotin with EZ-link-sulfo-NHS-LC-LC-Biotin, Pierce), and via said group the CBD may be bound to a matrix, which is coated with a ligand for the introduced group (e.g., streptavidin).
The term “directed immobilisation”, as used herein, means that the coupling of a CBD is performed via amino acid residues or other residues (e.g., glycosylations of the protein) whose position in the protein (e.g., N- or C-terminal) is known. The selection of these groups for coupling is performed by the selection of suitable reaction partners/linkers, which react preferably with said residues (e.g., coupling of sulfhydryl residues to iodoacetate residues; iodoacetate reacts a thousand times faster with sulfhydryl residues than with amino residues). The term means moreover that the nucleotide sequence of the CBD is fused with the nucleotide sequence of an affinity tag (e.g., Strep-Tag or His-Tag), which binds to a specific matrix (e.g., streptavidin coated magnetic particles or nickel-chelate-ligands). Said term means moreover that the nucleotide sequence of the CBD is fused to the nucleotide sequence of a polypeptide, which is recognised by other proteins, in which said other proteins introduce a molecule at a defined position (e.g., Avi-Tag or the biotinylation domain of oxalacetate decarboxylase of Klebsiella).
The term “surface” or “carrier”, as used herein, comprises all materials to which a coupling or adhesion of a CBD-molecule is possible, such a glass surfaces, chromatography materials, e.g., agarose or sepharose, plastic surfaces, e.g., polystyrene or polypropylene, filter materials, e.g., cellulose.
The term “1-step-method”, as used herein, relates to a method in which the CBD were either directedly or undirectedly immobilised to a suitable carrier or surface already prior to the addition of a sample. After incubation of the immobilised CBD with the sample the listeria CBD-carrier-complex is removed from the sample and subsequently optionally washed.
The term “2-step-method”, as used herein, relates to a method in which suitable non-immobilised CBD are brought into contact and incubated with the sample. The formed listeria-CBD-complexes are subsequently brought into contact with a suitable carrier or a surface so that the listeria-CBD-complexes are bound to the carriers or surfaces via CBD. Subsequently, the listeria-CBD-carrier-complexes are removed from the sample and optionally washed. Suitable CBD are modified with a polypeptide or a chemical group in such a way that they specifically bind to a carrier or a surface, which are coated with the respective binding partner of the polypeptide or the chemical group.
The present invention relates to polypeptide fragments, which are derived from the endolysin Ply511 of the phage A511 and are responsible for the binding of the phage to the cell wall of listeria. The amino acid sequence of the full-length endolysin Ply511 is depicted in SEQ ID NO:2 and the nucleic acid sequence coding therefore is depicted in SEQ ID NO:1. Surprisingly, the isolated polypeptide fragments of the endolysin Ply511 according to the invention exhibit a binding spectrum, which deviates from the lysis spectrum of the full-length endolysin Ply511. Whereas the endolysin Ply511 hydrolyses also at least a portion of the genus bacillus besides all serotypes of listeria, exclusively listeria are recognised and bound by the isolated polypeptide fragments according to the invention.
The present invention relates to polypeptide fragments of the endolysin Ply511 having the property to bind to the cell wall of listeria, wherein the polypeptide fragments do not exhibit any enzymatically active cell wall hydrolysing regions anymore. Furthermore, the invention relates to the nucleic acid sequences coding for the polypeptide fragments according to the invention. The polypeptide fragments according to the invention are referred to in the following also as “cell wall binding domains” (CBD). The CBD recognise and bind all listeria irrespective of their serotype, however, in addition, they do not recognise and bind any other species.
Preferably, the polypeptide fragments according to the invention exhibit an amino acid sequence (referring to the full-length sequence according to SEQ ID NO:2) at least from position 116 to 341 and at the most from 180 to 341. The polypeptide fragments according to the invention hence correspond at the N-terminal end to any position in the range from position 116 to position 180 and at the C-terminal end to the position 341. The range from position 116 to 180 is not exactly fixed on this region but may be shifted by a few amino acid positions in the direction to the N- or C-terminus as long as the polypeptide fragments do not exhibit any cell wall hydrolysing activity and the cell wall binding activity is sustained. Preferably, the invention relates furthermore to nucleic acid molecules encoding the described preferred polypeptide fragments.
The present invention relates furthermore to modified polypeptide fragments and the nucleic acid sequences coding for the modified polypeptide fragments according to the invention.
Especially the CBD may be coupled to low molecular substances, e.g., biotin. It may be chemically introduced into the CBD or by fusion of the CBD with a polypeptide, in which biotin is introduced in vivo or in vitro using another protein. Such polypeptides are, e.g., biotinylation domains, i.e., regions in naturally occurring polypeptides, which are biotinylated. Such biotinylation domains are exhibited, e.g., by the oxalacetate decarboxylase of Klebsiella (U.S. Pat. No. 5,252,466 and EP 0511747), the Salmonella typhimurium oxalacetate decarboxylase, the Propionibacterium shermanii transcarboxylase subunit, the biotin carboxyl carrier protein of the Escherichia coli acetyl-CoA carboxylase, the Saccharomyces cerevisiae pyruvate carboxylase or the Saccharomyces cerevisiae acetyl-CoA carboxylase. Such a polypeptide may, however, also be the Avi-Tag (avidity-patents U.S. Pat. No. 5,932,433, U.S. Pat. No. 5,874,239, and U.S. Pat. No. 5,723,584). Furthermore, a biotin may be chemically specifically coupled to a group by fusion with a polypeptide which carries said group, which is not or seldom—but hardly accessible—present in the protein (e.g., cysteine). Furthermore, instead of biotin, the so-called Strep-Tag (Skerra, A. & Schmidt, T. G. M. Biomolecular Engineering 16 (1999), 79-86, U.S. Pat. No. 5,506,121) may be used, which is a short amino acid sequence and binds to streptavidin. Furthermore, the His-Tag may be used. It is also possible to combine different tags and in such a way to use the different binding affinities of the different tags, e.g., Strep-Tag and His-Tag, or biotinylation domain and His-Tag. The biotinylation domains as well as the Avi-Tag, the Strep-Tag as well as the His-Tag are preferably coupled to the CBD using DNA-recombination technology. Preferably, the fusion protein consists of the biotinylation domain of the oxalacetate decarboxylase from Klebsiella or the Avi-Tag, the Strep-Tag or the His-Tag, which are bound to the N-terminal end of the CBD at their C-terminal end. Such a fusion, however, may also be one of the above-mentioned tags, with whose C-terminal end the N-terminus of another protein, which is used as a kind of “spacer molecule”, is coupled, e.g., GFP or maltose binding protein. In this case, the CBD may be coupled via its N-terminal end to the C-terminal end of said other protein.
However, the biotinylation domains may also act as “spacer molecule” such as GFP or MBP, because they are bigger than the other mentioned tags such as His-Tag, Strep-Tag, or Avi-Tag. They may kind of fulfil a double function. Since CBD represent fragments from larger proteins, they are usually relatively small (about 100 to 300 amino acids). Thus, it may be sensible or necessary to introduce a kind of spacer between the CBD domain and the group, which is responsible for the immobilisation on the carrier. This may prevent, on the one hand, that the CBD is denatured by the immobilisation, which is associated with the loss of its binding activity to the bacterial surface; on the other hand, the accessibility of the bacteria to the CBD may be improved as well as unspecific binding to the surfaces reduced, if the CBD are in a distance from the surfaces. Furthermore, the spacers may contribute to the fact that the groups, which are responsible for the immobilisation to the surfaces, are more accessible if they are not directly fused to the CBD.
The above-mentioned coupling may be performed directedly, e.g., at the N- or C-terminus, or undirectedly. The directed coupling is performed using a suitable, reactive amino acid, which in CBD does not frequently occur as a surface exposed amino acid such as cysteine, which was specifically introduced at a suitable position. Preferably, the coupling may be also performed in a direct way by using other amino acids, or, as it is also the case with cysteine, by using a “spacer” or “cross linker” (monofunctional or bifunctional) in an indirect way.
In case of the cysteine coupling all bifunctional cross linkers having NH- and SH-reactive groups are possible, with or without intermediate spacers, e.g., 11-maleimidoundecanoic acid sulfo-NHS or succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy-[6-amido]caproate. In the case that no spacers are present 8-12 C-atom-spacers having a terminal NH-group may be introduced. Preferably, the cysteine coupling is performed using a specific biotinylation of the cysteine using, e.g., EZ-link-PEO-maleimide activated biotin (Pierce).
The polypeptide fragments according to the invention may be used for the methods for enrichment, removal, and detection of listeria as described hereinafter.
The present invention relates to a method for the enrichment of listeria from a sample (so-called 1-step-method), the method comprising the following steps:
a) incubating or contacting a sample with a CBD, which is unspecifically or directedly immobilised to a solid carrier
b) separating the carrier-CBD-listeria-complex from the sample, and
c) optionally washing away of sample components unspecifically adhering to the carrier-CBD-listeria-complex.
For the enrichment method according to the invention the CBD, which is the polypeptide according to the invention, are coupled to solid carriers. The solid carriers may be magnetic or non-magnetic particles as well as filling materials for chromatography columns (e.g., sepharose materials), cellulose, filtration media, glass particles, centrifugation or sedimentation materials (e.g., agarose particles).
The coupling of the polypeptide according to the invention, in the following referred to also as CBD, may be performed unspecifically, or it can be preferentially performed in a directed manner, via, e.g., a selective biotinylation, or coupled using a spacer or a linker. The CBD may be unspecifically bound to chemically activated solid phases, e.g., solid phases with epoxy groups, tosyl- or NHS-groups using a reaction with amino acid side chains of the CBD. The accessibility of the CBD for listeria may be increased for example by the fact that the CBD is present in a protein fusion with a polypeptide chain which exhibits a higher affinity to specific activated surfaces than the CBD (e.g., GFP on activated polystyrene). In particular, the polypeptide according to the invention may exhibit the above described properties and modifications.
The enrichment of the listeria may be performed using a magnetism based method or using chromatographic methods or using the so-called “batch” method.
The duration of the incubation of the sample with the respective carrier material coupled to CBD has to be adapted to the respective sample and may vary between 1 min and 24 h, in particular, for about 5-60 min or about 30-180 min or even over night if needed.
If the CBD coated carrier material consists of magnetic particles the sample is incubated with the carrier material. Subsequently, the carrier-CBD-listeria-complex is magnetically separated form the sample by applying a magnetic field. In case of the batch-method the listeria contaminated sample is mixed with the carrier material, to which the CBD according to the invention is covalently coupled, and incubated together. Subsequently, the carrier-CBD-listeria-complex may be centrifuged or sedimented away from the sample or be filled onto a column and eluted or filtrated away. The enrichment using the batch-method, i.e., with pre-incubation of sample and with the respective CBD-coupled carrier materials, may be sensible especially in the case of very low listeria concentrations.
The enrichment of listeria using chromatography columns may be performed also in a pure flow through method, however. For this purpose firstly the CBD loaded carrier material is put onto a chromatography column. The listeria-contaminated sample is applied to said column and flows through it, whereby the listeria bind to the CBD and remain on the column. The sample itself does ideally not show any interaction with chromatography material and may be found in the flow through. The flow rate is dependent on the volume and geometry of the column. Furthermore, the flow rate is dependent on the volume and the amount of listeria in the sample, in order to achieve an efficient depletion even in the case of low listeria concentrations by a long as possible contact time between column and listeria. In this context the contact time is the time, which is needed by the sample from applying it onto the column until its outflow. The listeria bound to the column may be removed from the column again by washing with suitable buffers, so that the columns may be repeatedly reused.
The present invention relates furthermore to a method for the enrichment of listeria from a sample (so-called 2-step-method), the method comprising the following steps:
a) incubating or contacting a sample with CBD, which is fused to a polypeptide or modified with a chemical group, so that it binds specifically to a carrier, which is coated with the respective binding partner of the polypeptide or the chemical group
b) contacting and incubating the listeria-CBD-complex with a carrier which is coated with the respective binding partner of the polypeptide or the chemical group
c) separating the carrier-CBD-listeria-complex from the sample, and
d) optionally washing away of sample components unspecifically adhering to the carrier-CBD-listeria-complex.
In particular, the polypeptide according to the invention, hereinafter also referred to as CBD, may exhibit the above described properties and modifications.
The coupling of the CBD to another protein, by which the biotin is introduced, may be performed directedly, e.g., at the N- or C-terminus, or may be performed undirectedly. The directed coupling is performed using a suitable, reactive amino acid, which in CBD does not frequently occur as a surface exposed amino acid such as cysteine, which was specifically introduced at a suitable position. Preferably, the coupling may be also performed in a direct way using other amino acids, or, as it is the case with cysteine, using a “spacer” or “cross linker” (monofunctional or bifunctional) in an indirect way.
In case of the cysteine coupling all bifunctional cross linkers having NH- and SH-reactive groups are possible, with or without intermediate spacers, e.g., 11-maleimidoundecanoic acid sulfo-NHS or succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy-[6-amido]caproate. In the case that no spacers are present 8-12 C-atom-spacers having a terminal NH-group may be introduced. Preferably, the cysteine coupling is performed using a specific biotinylation of the cysteine using, e.g., EZ-link-PEO-maleimide activated biotin (Pierce). Furthermore, the coupling may be performed using known coupling reactions to protein residues such as for example carboxyl, amino, hydroxyl, or sulfhydryl residues.
The duration of the incubation and the separation of the carrier-CBD-listeria-complex are performed as for the above described 1-step-method.
The methods according to the invention are not only suitable for the enrichment but also for the removal of listeria from a sample.
Furthermore, the present invention relates to a method for the detection of listeria in a sample. The listeria detection comprises further steps following the above described method steps for enrichment, wherein the following detections may be performed both with the 1-step-method and with the 2-step-method.
For example the following detections may be used: detection of the listeria in the carrier-CBD-listeria-complex or after stripping them off the carrier material, using selective growth conditions, e.g., plating and incubation on selective media plates; detection of the target cell DNA, e.g., using PCR or DNA hybridisation methods; detection of the target cell wall or components thereof, e.g., using cell binding domains of endolysins or antibodies; detection of target cell components, e.g., proteins, using ELISA or enzymes via their activity, or of, e.g., ATP; detection using a target cell specific bacteriophage, e.g., A511-luxA. Preferably, the detection of the target cell DNA is performed, e.g., using PCR or the detection of target cell components, e.g., of proteins, is performed using ELISA after cracking the listeria open using a listeria-specific endolysin, e.g., Ply511.
Furthermore, the detection of the listeria in the carrier-CBD-listeria-complex or after stripping them off the carrier material may be performed using a further CBD, which is coupled to an enzymatically active polypeptide, so that the reaction product may be detected using the enzymatic reaction. Further, the additional CBD may be fused to a fluorescent protein and with a tag (e.g., StrepTag-HisTag-GFP-CBD), which is subsequently bound by a conjugate of an enzyme and a protein, which binds specifically to the tag (e.g., streptavidin-alkaline phosphatase conjugate).
The methods according to the invention are characterised by the following advantages:
Furthermore, the present invention relates to a kit, comprising a carrier immobilised with the polypeptide fragments according to the invention as well as the buffer solutions, e.g., washing buffer, stripping buffer, and/or cell cracking buffer, required for the enrichment and the detection of the listeria. Furthermore, the present invention relates to a kit, comprising a modified polypeptide fragment according to the invention, a carrier immobilised with the respective binding partner of the modification of the polypeptide fragment as well as the buffer solutions, e.g., washing buffer, stripping buffer, and/or cell cracking buffer, required for the enrichment and the detection of the listeria.
The following examples illustrate the invention and are not to be considered to be limiting. Unless otherwise indicated, molecular biological standard methods were used, such as described for example in Sambrook et al., (1989): Molecular cloning: A Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Experiment 1: Detection of the Binding of CBD511 Fusions having Different Lengths to listeria.
The different CBD511 fusion proteins StrepTag-HisTag-GFP-CBD511_f1, StrepTag-HisTag-GFP-CBD511_f2 and StrepTag-HisTag-GFP-CBD511_f3—heterologously expressed in E. coli HMS174 (DE3)—were purified using Ni-affinity chromatography (Qiagen) following the instructions of the manufacturer of the column chromatography material. 50 μl of an overnight culture of Listeria monocytogenes ScottA (serovar 4b) or Listeria monocytogenes ProCC679 (serovar 1/2a) were mixed with about 2 μg of the purified protein and incubated for 2 min at RT. After addition of 1 ml of PBST (10 mM sodium phosphate pH 8, 150 mM NaCl, 0.05% Tween 20) the cells were centrifuged, 2× washed in 0.5 ml and resuspended in 50 ml of PBST. The binding was controlled under the fluorescence microscope. It was shown that the HisTag-GFP-CBD-variants bind both listeria serotypes 4b and 1/2a.
The listeria were grown overnight in 2 ml of listeria enrichment broth acc. FDA (Profos AG) at 30 or 37° C., Bacillus, Chryseobacterium, Citrobacter, Escherichia, Enterobacter, Klebsiella, Cocuria, Pseudomonas, Salmonella, Stenotrophomonas, and Yersinia were grown in Lauria-Bertani bouillon (Profos AG) at 30 or 37° C. Campylobacter was grown in Campylobacter enrichment bouillon (Profos AG) under semi-aerobic atmosphere (CampyGen-Oxoid) at 42° C., Clostridium was grown in TYG medium at 42° C. under anaerobic atmosphere (Genbox anaer, Biomerieux). Lactobacillus was cultivated at 37° C. in MRS-bouillon (Profos AG), Staphylococcus was cultivated in BHI bouillon (Profos AG). The cells were centrifuged, 1× washed in 1 ml of TBST (10 mM Tris HCl, pH 8, 150 mM NaCl, 0.05% Tween 20), resuspended in 200 μl of PBST and heat inactivated for 15 min at 80° C.
Examination of the binding of the fusion proteins to immobilised listeria cells:
About 20-30 μl of these suspensions were spotted on a nitrocellulose membrane (Sartorius AG) and baked for 30 min at 80° C. After wetting with TBST the membrane was blocked for 30 min in BSA solution (1% w/v in TBST) and incubated with StrepTag-HisTag-GFP-CBD511_f2, StrepTag-HisTag-GFP-CBD500 or StrepTag-HisTag-GFP-CBD118 (10 μg/ml each) at RT under gentle agitation for 30 min. Subsequently, the membrane was washed 2×15 min in TBST and incubated for 30 min with streptaktin-AP-conjugate (IBA) in TBST. After washing (4×15 min TBST) the membrane was incubated in staining solution (100 mM Tris HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2, 0.18 mg/ml NBT (Applichem) and 0.22 mg/ml BCIP (Applichem)) for about 15-30 min. The reaction solution was removed, the membrane washed in water and dried.
Table 1 shows the results of binding of the cell binding domain CBD511 to non-listeria.
Bacillus cereus
Bacillus mycoides
Bacillus sp.
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus vallismortis
Bacillus subtilis
Campylobacter lari
Campylobacter jejuni
Clostridium perfringens
Clostridium perfringens
Clostridium perfringens
Clostridium sordelli
Chryseobacterium meningosepticum
Chryseobacterium sp.
Citrobacter amalonaticus
Citrobacter freundii
Citrobacter freundii
Enterobacter aerogenes
Enterobacter amnigenus
Enterobacter cloacae
Enterobacter asburiae
Enterococcus durans
Escherichia coli
Escherichia coli
Klebsiella granulomatis
Klebsiella oxytoca
Klebsiella pneumoniae
Kocuria rhizophila
Lactobacillus casei (393)
Lactobacillus casei (862)
Lactobacillus casei (864)
Lactobacillus plant (2410)
Micrococcus luteus
Pseudomonas fluoreszens
Ralstonia picketti
Salmonella enteritits SZ 15
Salmonella dublin
Salmonella berta
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Examination of the binding of listeria to magnetic particles via the fusion proteins:
0.5 ml of a fresh ON culture were added to 2 ml of listeria enrichment broth acc. FDA (Profos AG) and cultivated to an OD600 of about 1 at 37° C. (corresponds to 0.5 to 1×109 cfu/ml). The cultures were diluted in PBST to about 1×104 cfu/ml. 1 μg of StrepTag-HisTag-GFP-CBD511_f3 fusion protein was added to 1 ml of the respective cell dilutions and briefly mixed. After addition of 50 μg MagPrep-Streptavidin (Merck) the mixtures were incubated for 20 min in an overhead rotator at RT. Subsequently, the complexes of magnetic particles, fusion protein and listeria were collected in a magnetic field at the vessel wall and the supernatant (SN1) was transferred to a sterile vessel. The particle-protein-listeria-complex was washed in 1 ml of PBST for 10 min in the overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was added to SN1. The complex was resuspended in 1 ml of PBST. Subsequently, serial dilutions in PBST of the combined supernatants and of the resuspended complexes were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the portion of the listeria adhering to the magnetic particles was calculated in percent of the in toto recovered listeria.
Table 2 shows the result of the serotype dependency of the binding properties of different cell binding domains from listeria endolysins to strains of the genus listeria. CBD118 stands for the cell binding domain of the endolysin Ply118; CBD500 stands for the cell binding domain of the endolysin Ply500; CBD511_f2 and CBD511_f3 stand for the variants of the cell binding domain of the endolysin Ply511; NC test means: the binding of the respective StrepTag-HisTag-GFP-CBD-fusions were determined via the binding to cells immobilised on nitrocellulose membrane (experiment 2a) and detection over streptaktin-alkaline-phosphatase-conjugate; LB-test means: the binding was determined via the removal of listeria from solutions using streptavidin coated magnetic particles and the mono-biotinylated cell binding domain Bio-GFP-CBD511f3 (experiment 2b).
L. monocytogenes
L. innocua
L. ivanovii
L. seeligeri
L. welshimeri
L. grayi (ssp. g
indicates data missing or illegible when filed
Experiment 3: Comparison of the Cell Binding Property of the StrepTag-HisTag-CBD511_f3 with StrepTag-HisTag-CBD500
0.5 ml of a fresh ON culture (L. monocytogenes ScottA, serovar 4b; L. monocytogenes EGDe, serovar 1/2a) were added to 2 ml of listeria enrichment broth acc. FDA (Profos AG) and cultivated to an OD600 of about 1 at 37° C. (corresponds to 0.5 to 1×109 cfu/ml). The cultures were diluted in PBST to about 1×104 cfu/ml. 1 μg, 5 μg and 10 μg of StrepTag-HisTag-GFP-CBD511_f3 fusion protein and StrepTag-HisTag-CBD500 fusion protein, respectively, were added to 1 ml of the respective cell dilutions and briefly mixed. After addition of 50 μg MagPrep-Streptavidin (Merck) the mixtures were incubated for 20 min in an overhead rotator at RT. Subsequently, the complexes of magnetic particles, fusion protein and listeria were collected in a magnetic field at the vessel wall and the supernatant (SN1) was transferred to a sterile vessel. The particle-protein-listeria-complex was washed in 1 ml of PBST for 10 min in the overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was added to SN1. The complex was resuspended in 1 ml of PBST. Subsequently, serial dilutions in PBST of the combined supernatants and of the resuspended complexes were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the portion of the listeria adhering to the magnetic particles was calculated in percent of the in toto applied cells.
In contrast to CBD500, listeria of the serotype 1/2a could be enriched in a protein concentration dependent manner using CBD511. The serotype 4b is enriched both with the CBD511 and the CBD500 in a protein concentration dependent manner.
Experiment 4: Dependency of the Binding of listeria to Magnetic Particles in the 2-Step-Method on the Concentration of Biotinylated CBD511 Constructs—Bio-OD-CBD511_f3 and Bio-Av-GFP-CBD511_f3.
Either the biotinylation domain of the α subunit of the Klebsiella pneumoniae oxalacetate decarboxylase (GenBank Accession No. J03885, U.S. Pat. No. 5,252,466, EP 0511747, Schwarz et al., Journal of Biological Chemistry 263 (1988), 9640-9645) as biotinylation domain or the Avi-Tag (U.S. Pat. No. 5,723,584; U.S. Pat. No. 5,874,239) were N-terminally cloned to the CBD511_f3 using molecular biological standard techniques. Because of the small size of the Avi-Tag GFP was additionally introduced as “linker domain”, as depicted in
The listeria-particle-complex was resuspended in 1 ml of PBST. Subsequently, serial dilutions of the combined supernatants and of the resuspended complexes in PBST were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the portion of listeria adhering to the magnetic particles was calculated in percent of the in toto applied cells.
It was shown that using the Bio-OD-CBD_f3 (A) the maximal yield of listeria is achieved in the 2-step-method starting from a concentration of 0.3 μg/ml and remains constant over a wide concentration range up to 40 μg/ml, in the case of Bio-Av-GFP-CBD_f3 (B) the maximum yield of bound listeria is achieved starting from about 2 μg/ml protein.
0.5 ml of a fresh ON culture (L. monocytogenes ScottA, serovar 4b; L. monocytogenes EGDe, serovar 1/2a) were added to 2 ml of listeria enrichment broth acc. FDA (Profos AG) and grown to an OD600 of about 1 at 37° C. (corresponds to 0.5 to 1×109 cfu/ml). The cultures were diluted in buffer X (10 mM sodium citrate, 10 mM sodium phosphate), adjusted to the respective pH-value, to about 1×104 cfu/ml. 5 μg of the Bio-Av-GFP-CBD_f3 fusion proteins were added to 1 ml each of the cell dilutions and briefly mixed. After addition of 50 μg MagPrep-Streptavidin (Merck) the mixtures were incubated for 20 min in an overhead rolator at RT. Subsequently, the complexes of magnetic particles, fusion protein and listeria were collected in a magnetic field at the vessel wall and the supernatant (SN1) was transferred to a sterile vessel along with a 1/10 volume of 10×PBST. The particle-protein-listeria-complex was washed in 1 ml of buffer X for 10 min in an overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was added to SN1. The complex was resuspended in 1 ml of PBST. Subsequently, serial dilutions of the combined supernatants and of the resuspended complexes in PBST were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the portion of the listeria adhering to the magnetic particles was calculated in percent of the in toto applied cells.
It was shown that the 2-step-method for enrichment of listeria of serotype 4b and serotype 1/2a results in consistent yields over the pH range from 5 to 10.
0.5 ml of a fresh ON culture (L. monocytogenes ScottA, serovar 4b; L. monocytogenes EGDe, serovar 1/2a) were added to 2 ml of listeria enrichment broth acc. FDA (Profos AG) and grown to an OD600 of about 1 at 37° C. (corresponds to 0.5 to 1×109 cfu/ml). The cultures were diluted in 10 mM sodium phosphate, pH 8, 0.05% Tween 20 and a salt content of 0, 100, 200, 400, 600, 800, and 1000 mM NaCl to about 1×104 cfu/ml. 5 μl of the StrepTag-HisTag-GFP-CBD511_f3 fusion protein were added to 1 ml each of the cell dilutions and briefly mixed. After addition of 50 μg MagPrep-Streptavidin (Merck) the mixtures were incubated for 20 min in an overhead rotator at RT. Subsequently, the complexes of magnetic particles, fusion protein and listeria were collected in a magnetic field at the vessel wall and the supernatant (SN1) was transferred to a sterile vessel. The particle-protein-listeria-complex was washed in 1 ml of PBST for 10 min in an overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was added to SN1. The complex was resuspended in 1 ml of PBST. Subsequently, serial dilutions in PBST of the combined supernatants and of the resuspended complexes were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the portion of the listeria adhering to the magnetic particles was calculated in percent of the in toto applied cells.
Whereas the enrichment efficiency in the case of serotype 1/2 a remains constant almost over the entire range from 0 to 1 M NaCl, it declines in the case of serotype 4b with increasing salt concentration.
Covalent coupling of Bio-Av-GFP-CBD_f3 to the magnetic particles Dynabeads M-270 Epoxy (Dynal): the dynabeads were resuspended in diglym according to the manufacturer's instructions and washed according to the instructions prior to coupling, equilibrated and taken up in 250 μl of sterile sodium phosphate (0.1 M, pH 7.5). Subsequently, 1 ml of 3 M ammonia sulphate solution in 0.1 M sodium phosphate pH 7.4 and 1 ml protein solution (StrepTag-HisTag-CBD511_f2 in 0.1 M sodium phosphate pH 7.4) were added, mixed and incubated overnight at 4° C. and subsequently for 8 h at RT in a rolator. The magnetic particles were collected in a magnetic field, the supernatant was removed and the magnetic particles were washed 2× in 10 mM sodium phosphate pH 7.5, 150 mM sodium chloride, 0.1% bovine serum albumin and 0.02% sodium azide for 20 min at RT and subsequently stored in this buffer at 4° C. 0.5 ml of a fresh ON culture (L. monocytogenes ScottA, serovar 4b; L. monocytogenes EGDe, serovar 1/2a) were added to 2 ml of listeria enrichment broth acc. FDA (Profos AG) and grown to an OD600 of about 1 at 37° C. (corresponds to 0.5 to 1×109 cfu/ml). The cultures were diluted in PBST to about 1×104 cfu/ml.
1-step-method: 300 μg/ml of the Bio-Av-GFP-CBD_f1 coated magnetic particles (Dynabeads Epoxy) were added to 1 ml each of the cell dilutions, and the mixtures were incubated for 5, 10, 20, 40, and 60 min in an overhead rolator at RT.
2-step-method: 5 μg of the Bio-Av-GFP-CBD_f3 fusion protein were added to 1 ml each of the cell dilutions and briefly mixed. Subsequently, MagPrep-Streptavidin-particles (Merck) were added to 50 μg/ml, and the mixtures were incubated for 5, 10, 20, 40, and 60 min in an overhead rolator at RT.
Subsequently, the particle-listeria-complexes were collected in a magnetic field at the vessel wall and the supernatant (SN1) was transferred to a sterile vessel. The particle-listeria-complex was washed in 1 ml of PBST for 10 min in an overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was added to SN1. The complex was resuspended in 1 ml of PBST. Subsequently, serial dilutions in PBST of the combined supernatants and the resuspended complexes were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the portion of the listeria adhering to the magnetic particles was calculated in percent of the in toto applied cells.
In case of the 2-step-method the maximal enrichment efficiency is obtained already after an incubation time of 5 min. In case of the 1-step-method the same enrichment efficiency as found with the 2-step-method is obtained after 60 min only.
Experiment 8: Detection of listeria in Camembert Using the 1-Step- and the 2-Step-Method
300 g of Camembert from a supermarket were sterilely divided into 25 g portion units and stored in Stomacher bags at −80° C. One portion unit was examined for presence of listeria according to the norm ISO: 11290-1:1996 FDAM 1. In the case of absence of a listeria contamination 5 portion units were thawed at RT and infected with different amounts of L. monocytogenes ScottA. For this purpose an ON culture was diluted 1/5 and incubated to an OD600 of about 1 at 37° C. Subsequently, serial dilutions were performed in sterile PBST. Under the assumption of 0.5 to 1×109 cfu/ml at OD600 of 1 the portion units were contaminated with 0, 1-10, 11-50, 50-100, and 100-500 cfu/25 g Camembert and stored ON at 4° C. For the exact determination of the cell numbers duplicates of the dilutions were plated on Oxford agar (Profos AG), the plates were incubated for 24 h at 37° C. and counted. 225 ml Fraser ½ medium (Profos AG) were sterilely added to the portion units, homogenised for 1 min in a Stomacher and incubated at 30° C. After an incubation time of 4 h, 6 h, and 24 h 1 ml was removed per mixture.
1-step-method: 300 μg/ml of the StrepTag-HisTag-CBD511_f2 coated magnetic particles (Dynabeads Epoxy) were added to 1 ml homogenate, and the mixture was incubated for 20 min in an overhead rotator at RT.
2-step-method: 5 μg of the StrepTag-HisTag-GFP-CBD511_f3 fusion protein were added to 1 ml homogenate and briefly mixed. Subsequently, MagPrep-Streptavidin-particles (Merck) were added to 50 μg/ml, and the mixtures were incubated for 20 min in an overhead rotator at RT.
Subsequently, the particle-listeria-complexes were collected in a magnetic field at the vessel wall and the supernatant was removed. The particle-listeria-complex was washed 3× with 1 ml of PBST for 10 min in an overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was discarded each time. The particle-listeria-complexes were resuspended in 100 μl of PBST and plated on Oxford agar (Profos AG). After 24 h and 48 h at 37° C. the plates were counted and the portion of listeria adhering to the magnetic particles was calculated in percent of the in toto applied cells. In parallel, the contaminated mixtures were examined according to norm ISO: 11290-1:1996 FDAM 1 for listeria. For this purpose, 100 μl were added to 10 ml Fraser medium (Profos AG) at the indicated time points, incubated for 24 h at 37° C. in a roler and subsequently plated on Oxford agar (Profos AG). All mixtures were performed in quadruplets.
It was shown that both using the 1-step-method and the 2-step-method the necessary enrichment times are significantly shorter than using the method according to ISO: 11290-1: 1996, in order to detect minor listeria contaminations in Camembert. Concerning the reduction of the enrichment time the results for the 2-step-method are better than for the 1-step-method.
Experiment 9: Detection of listeria in Raw Ham and in Shrimps According to the 1 ml- and the 10 ml-2-Step-Method
300 g raw ham and 300 g shrimps from a supermarket were divided sterilely in 25 g portion units and stored in Stomacher bags at −80° C. One portion unit each was examined for the presence of listeria according to the norm ISO: 11290-1:1996 FDAM 1. In case of absence of a listeria contamination 5 portion units were thawed at RT and infected with different amounts of L. monocytogenes ScottA. For this purpose, an ON culture was diluted 1/5 and incubated to an OD600 of about 1 at 37° C. Subsequently, serial dilutions were performed in sterile PBST. Under the assumption of 0.5 to 1×109 cfu/ml at an OD600 of 1 the portion units were contaminated with 0, 1-10, 11-50, 50-100, and 100-500 cfu/25 g food and stored ON at 4° C. For the exact determination of the cell numbers duplicates of the dilutions were plated on Oxford agar (Profos AG), the plates were incubated for 24 h at 37° C. and counted.
225 ml Fraser ½ medium (Profos AG) were added sterilely to the portion units, homogenised for 1 min in a Stomacher and incubated at 30° C. After an incubation time of 4 h and 6 h 2×1 ml and 2×10 ml were taken per portion unit. 2.5 μg/ml of StrepTag-HisTag-GFP-CBD511_f3 fusion protein were added to the homogenate and briefly vortexted. Subsequently, MagPrep-Steptavidin-particles (Merck) were added to 100 μg/ml in a 1 ml mixture and to 50 μg/ml in a 10 ml mixture, and the mixtures were incubated for 20 min in an overhead rotator at RT. The particle-listeria-complexes were collected in a magnetic field at the vessel wall and the supernatant was removed. The particle-listeria-complex was washed 2× with 1 ml of PBST by pipetting it up and down several times and resuspended in 100 μl of PBST and plated on Oxford agar (Profos AG). After 24 h and 48 h at 37° C. the plates were counted and the portion of listeria adhering to the magnetic particles calculated in percent of the in toto applied cells. In parallel, the contaminated mixtures were examined for listeria according to the norm ISO: 11290-1:1996 FDAM 1. For this purpose, 100 μl were added to 10 ml Fraser medium (Profos AG) at the indicated time points, incubated for 24 h at 37° C. in a roler and subsequently plated on Oxford agar (Profos AG). All mixtures were performed in duplicates. It became apparent that listeria in ham and shrimps can be detected faster by enrichment using the 2-step-method than using the method according to ISO: 11290-1:1996. A further reduction of the enrichment time may be accomplished using the 10 ml-2-step-method.
Experiment 10: Detaching the listeria from the Magnet Particles
0.5 ml of a fresh ON culture of L. monocytogenes ScottA were added to 2 ml of listeria enrichment broth acc. FDA (Profos AG) and grown to an OD600 of about 1 at 37° C. (corresponds to about 0.5 to 1×109 cfu/ml). The cultures were diluted in PBST to about 1×104 cfu/ml.
1 μg/ml StepTag-HisTag-GFP-CBD511_f3 fusion protein was added to 0.5 ml each of the cell dilutions and briefly mixed. After addition of 50 μg/ml MagPrep-Streptavidin (Merck) the mixtures were incubated for 20 min in an overhead rotator at RT. Subsequently, the complexes of magnetic particles, fusion protein and listeria were collected in a magnetic field at the vessel wall and the supernatant (SN1) was transferred to a sterile vessel. The particle-protein-listeria-complex was washed in 1 ml of PBST for 10 min in an overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was added to SN1.
Half of the mixtures were resuspended in 100 μl of PBST, the other half was resuspended in 100 μl of 50 mM sodium phosphate pH11. After 5 min at RT the magnetic particles were removed and the supernatants were added to 400 μl of PBST. The magnetic particles were resuspended in 0.5 ml.
Serial dilutions with PBST of the supernatants and the resuspended magnetic particles were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the portion of the listeria adhering to the magnetic particles was calculated in percent of the in toto recovered listeria.
90% of the listeria which were bound to the magnetic particles via the cell binding domain were detached with the pH 11 buffer.
Experiment 11: Enrichment of listeria and Detection via PCR
0.5 ml of a fresh ON culture of L. monocytogenes ScottA were added to 2 ml of listeria enrichment broth acc. FDA (Profos AG) and grown to an OD600 of about 1 at 37° C. (corresponds to about 0.5 to 1×109 cfu/ml). The culture was diluted in listeria enrichment broth acc. FDA (Profos AG) to about 1×106 and 1×105 cfu/ml.
For each cell dilution in listeria enrichment broth acc. FDA (Profos AG) 14 mixtures à 1 ml were prepared. 4 mixtures of them were centrifuged (for 5 min at 13,000 rpm in a table top centrifuge), the supernatants were discarded, the cells washed 1× in PBST and pelleted. The cell pellets were stored on ice until further processing.
The cells were isolated from 10 mixtures using the 2-step-method: 20 μg of the StrepTag-HisTag-GFP-CBD511_f2 fusion protein were added to 1 ml each of the cell dilutions and briefly mixed. Subsequently, streptavidin-magnetic-particles (Roche) were added to 100 μg/ml, and the mixtures were incubated for 20 min in an overhead rotator at RT. Subsequently, the particle-listeria-complexes were collected in a magnetic field at the vessel wall, the supernatant was removed and discarded. The particle-listeria-complex was washed in 1 ml of PBST for 10 min in an overhead rolator, collected in a magnetic field at the vessel wall; the supernatant was discarded.
The particle-listeria-complex of 2 mixtures was resuspended in 1 ml of PBST. Serial dilutions thereof were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. In addition, serial dilutions of the starting cell dilutions having estimated 106 and 105 cfu/ml were plated on Oxford agar (Profos AG) and incubated for 24 h at 37° C. The plates were counted and the actual cell number (1.4×105 and 1.4×106 cfu/ml) and the portion of the listeria adhering to the magnetic particles were calculated in percent of the in toto applied cells (85-95%).
The particle-listeria-complex of 4 mixtures was resuspended in 20 μl of 150 mM sodium phosphate pH 11, incubated for 15 min at RT; subsequently the magnetic particles were collected in a magnetic field at the vessel wall and the supernatant was added quantitatively to 10 μl of 150 mM sodium phosphate pH 6 (mixtures A). The particle-listeria-complex of the 4 remaining mixtures was resuspended in 20 μl of PBST (mixtures B). Each of the 4 mixtures of the centrifuged cells was also resuspended in 20 μl of PBST (mixtures C).
10 μl of PBST were added to 2 mixtures of A, B, and C each; to each of the remaining 2 mixtures 10 μl of PBST with Ply511 (2 μg/ml) were added and incubated at 40° C. for 5-10 min. Subsequently, 5 μl of proteinase K (1 mg/ml PBST) were added to all 4 mixtures, incubated for 5 min at 56° C. and then for 5 min at 94° C. The magnetic particles of the mixtures B were removed in a magnetic field and 5 μl of all mixtures A, B, and C were used in a PCR reaction according to Aznar & Alarcon (Aznar R & Alarcon B (2002): On the specificity of PCR detection of Listeria monocytogenes in food, System. Appl. Microbiol. 25, 109-119), and subsequently the reaction products were separated using a 1% agarose gel.
It could be shown that in all 3 cases (centrifugation, 2-step-method with cracking at the magnetic particles and 2-step-method with detaching from the magnetic particles) the addition of endolysin Ply511 to the cracked cells significantly improves the sensitivity of the detection. Whereas with the lysis of the listeria at the magnetic particles the same sensitivity is obtained as in the case when the cells are centrifuged, the signal strength in the PCR is weaker if the cells are detached from the magnetic particles prior to cracking using a pH 11 buffer.
Experiment 12: Detection of listeria in Frankfurter Sausages and Mozzarella
225 ml FDA medium were each added to 25 g Frankfurter and mozzarella, respectively and the portions were sterilely homogenised in Stomacher bags. The samples were incubated overnight at 30° C. Prior to the listeria detection the samples were buffered each with 1/10 volume of PBST.
1-step-method: 300 μg/ml of the magnetic particles (Dynabeads M270 Epoxy) coated with Bio-Av-GFP-CBD511_f3 were added to 1 ml homogenate, and the mixture was incubated for 20 min in an overhead rotator at RT.
2-step-method: 0.5, 2, 5, or 10 μg of the Bio-Av-GFP-CBD511_f3 fusion protein were each added to 1 ml homogenate and briefly mixed. Subsequently, MagPrep-Streptavidin-particles (Merck) were added to 50 μg/ml, and the mixtures were incubated for 20 min in an overhead rotator at RT.
Subsequently, the particle-listeria-complexes were collected in a magnetic field at the vessel wall and the supernatant was removed. The particle-listeria-complex was washed 1× with 1 ml of PBST for 10 min in an overhead rolator, collected in a magnetic field at the vessel wall and the supernatant was discarded in each case. The particle-listeria-complexes were resuspended in 100 μl of PBST and plated on Oxford agar (Profos AG). After 24 h at 37° C. the plates were counted and the portion of listeria adhering to the magnetic particles were calculated in percent of the in toto applied cells. All mixtures were performed twofold.
It was shown that both from Frankfurter sausages and also from mozzarella listeria could be isolated with the help of the Bio-Av-GFP-CBD511_f3 fusion protein. In the case of mozzarella this was achieved with the strain EGDe significantly better than with ScottA. Slightly higher concentrations of protein are used with food in order to achieve a high binding efficiency. Whereas the 1-step-method in the case of Frankfurter sausages and the strain EGDe exhibits a high binding efficiency, under all other conditions the 2-step-method is suited better.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2005 040 347.6 | Aug 2005 | DE | national |
This application is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/DE2006/001480 filed Aug. 23, 2006, which claims priority to German Patent Application No. DE 10 2005 040 347.6 filed Aug. 25, 2005. The entire text of each of the above-referenced disclosures is specifically incorporated herein by reference without disclaimer.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DE2006/001480 | 8/23/2006 | WO | 00 | 8/29/2008 |
