WO2009122216A1

WO2009122216A1 - Assay system based on immunomagnetic separation for detecting microorganisms in a food sample

Info

Publication number: WO2009122216A1
Application number: PCT/GB2009/050327
Authority: WO
Inventors: Pradip Patel
Original assignee: Alaska Food Diagnostics Limited
Priority date: 2008-04-04
Filing date: 2009-04-02
Publication date: 2009-10-08
Also published as: GB0806147D0

Abstract

A method for detecting the presence of target micro-organisms in a food sample; said method comprising the following modules: 1. removing large particulates from a liquid comprising a finely divided food sample;2. forming a complex of magnetic solid beads of up to 1000nm (1µm) in size and microorganism in the liquid, wherein either 2i) the complex is formed by a process comprising incubating the resultant liquid product with solid beads and the resultant complex is passed through a column comprising a ferromagnetic fluid permeable matrix material of fibrous ferromagnetic material, said column being under the influence of a magnetic field so that complex is retained thereon; or (2ii) the magnetic solid beads are retained on a ferromagnetic fluid permeable matrix material of fibrous ferromagnetic material within a column under the influence of a magnetic field; and thereafter; 3. detecting target microorganisms retained on the column.

Description

ASSAY SYSTEM BASED ON IMMUNOMAGNETIC SEPARATION FOR DETECTING MICROORGANISMS IN A FOOD SAMPLE

The present invention relates to a method for detecting the presence of microorganisms such as bacteria and viruses in food products, as well as apparatus for use in the method and kits adapted to carry out the method. Background of the Invention

The detection of microorganisms such as bacteria or fungi is important in a wide variety of detection, diagnostic and health fields. For instance, the detection of microorganisms in consumer goods such as food, medicaments or cosmetic preparations is an important procedure to ensure quality control and public safety. Detection of microorganisms in samples such as clinical samples or samples collected for public health purposes may be important for diagnostic or health protection purposes. There is a need to detect even low levels of bacteria in these instances, in particular where the bacteria are pathogenic organisms, such as Salmonella, Listeria and E.coli such as toxigenic E. coli (and in particular the highly pathogenic strain E. coli 0157).

Classical culture techniques in which the presence of microorganisms is investigated by plating out the samples and allowing cultures to grow can take long periods of time. If potential colonies can be identified after a suitable period of time, confirmation of the identity of the colony for example using biochemical identification techniques and ultimately serology, must be carried out.

This process can take anything up to 5 days to complete. Delays of this type are unacceptable in situations where, for example, the substrate comprises a degradable foodstuff which has a limited shelf life.

Alternative commercial techniques (e.g. ELISAs, DNA probes and PCR) can detect the presence of pathogens at levels as low as approximately 10⁴- 10⁶ cfu per ml, which means for samples containing 1 - 10 colony forming unts (CFU)/ 25 g require at least 24 hours, but more often 48 hours, of cultural enrichment prior to rapid detection of the pathogen (Patel & Williams, (1994) "Evaluation of commercial kits and instruments for the detection of foodborne pathogens and bacterial toxins" in "Rapid Analysis Techniques in Food Microbiology". Ed. P. D. Patel. Blackie Academic, Glasgow). Use of micron- sized immunomagnetic particles for pathogen capture and concentration has increased vastly over the past 15 years since their use in food applications pioneered in early 1980's for staphylococcal enterotoxins, anti- staphylococcal enterotoxins and Salmonella. An alternative to micro-sized immunomagnetic particles is to use magnetic ferrofluidic materials (< 500nm) in batch systems as exemplified by a range of applications, including fungal purification (Patel et al. (1993). Rapid separation and detection of foodborne yeasts and moulds by means of lectins. In 'New techniques in food and beverage microbiology'. Kroll, R.G., Gilmour, A. & Sussman, M (Eds.), Blackwell Scientific Publicaitons, Oxford, pp. 31 - 42), enrichment of tumor cells from peripheral blood and bone marrow (Kemshead et al. (1994). Prog. Clin. Biol. Res., 389, 593 - 600), and selection of CD34+ cells (Fan et al. (1994). Prog. Clin. Biol. Res., 389, 309 - 315). Unlike the classical micro-sized immunomagnetic beads (> lμm), the advantages of ferrofluidic materials are that they do not require shaking, the reaction with the corresponding antigen is rapid and generally does not require removal of unbound excess reactants from the bound reagents. However, in these cases the applications are targeted to non-food applications, including mammalian cell purification and isolation of nucleic acids for clinical applications. Furthermore, when using such beads, it is generally accepted that they are better captured on matrices comprising closely stacked matrices of metallic spheres. It is reported (GB2425498) that the use of filamentous separation elements such as steel wool are problematic in these instances because they cause non-uniform pathways for the liquid through the element, and thus give a variable separation result. Furthermore they are said to give rise to the entrapment of substances other than target. Food samples present particular challenges however. Generally in these samples, the sample volumes being handled are very much higher than encountered during the analysis of clinical samples such as blood and tissue. For example, volumes handled in the clinical field may be up to 5ml in volume. However, in the analysis of food, the samples in excess of 5ml, for example from 10-250ml may need to be analysed This is mainly due to the fact that the concentration of target microorganisms in these samples may be very low as compared to the concentration of target cells found in other sample types. However, further complications exist because of the complex composition of the food samples. As compared with clinical samples, food samples are much more complex, containing as they do, a wide range of tissue types, varying amounts of biological components such as proteins, lipids, polysaccharides, carbohydrates, glycolipids, glycoproteins, and even solid materials as well as background microorganisms such as bacteria. Bacterial cells are much smaller than eukaryotic cells and have different types of cell wall. As a result, capture of these is particularly difficult when high volumes of liquids are being handled.

The commercial product available from Pathatrix, uses a flow-through mode with micron- sized immunomagnetic beads for food analysis. However, there are several limitations with this technology. For instance, the immunomagnetic beads added to large volume of the food enrichment broths are captured and concentrated as a pellet within a few minutes of flowing the broths through the flow cell harbouring a small circular magnet. Thus, the beads are not in direct contact with the whole volume of the enrichment broth (and consequently the target pathogens) during the capture process. In fact, the rest of the process continuously flows the sample through the flow cell containing the concentrated beads. This may result in low capture efficiency for target pathogens. In addition, the technology is not particularly suited to high- throughput formats, as at any given time only small numbers of samples, for example 5 samples can be processed. Furthermore, the technology is generally applied after an overnight cultural incubation step (e.g. 18 - 24h for Salmonella), thus reliable results are not obtained within a working day (<10h).

The applicants have developed a process for the concentration of target microorganisms in food which is fast and is amenable to high-throughput operation.

Summary of the Invention

According to the present invention there is provided a method for detecting the presence of target micro-organisms in a food sample; said method comprising the following modules:

1. removing large particulates from a liquid comprising a finely divided food sample;

2. forming a complex of magnetic solid beads of up to lOOOnm (lμm) in size and microorganism in the liquid, wherein either

2i) the complex is formed by a process comprising incubating the resultant liquid product with solid beads and the resultant complex is passed through a column comprising a ferromagnetic fluid permeable matrix material of fibrous ferromagnetic material, said column being under the influence of a magnetic field so that complex is retained thereon; or

(2ii) the magnetic solid beads are retained on a ferromagnetic fluid permeable matrix material of fibrous ferromagnetic material within a column under the influence of a magnetic field; and thereafter;

3. detecting target microorganisms retained on the column. The method provides an integrated modular system for the real-time capture and concentration of low levels of food borne microorganism, in particular bacteria or viruses, and especially pathogenic microorganisms. The modular design of the procedure readily adaptable for high-throughput procedures ideally suited to the large volumes of samples such as food enrichment broths, which are required for the analysis of food samples.

Furthermore, the applicants have found that the use of small beads in step (ii) combined with the use of fibrous matrix material in step (iii) provides an unexpectedly good results with specifically food samples.

There may also be provided a method for detecting the presence of target micro-organisms in a food sample; said method comprising: a. removing large particulates from a liquid comprising a finely divided food sample; b. forming a complex of solid beads and microorganism by a process comprising incubating the resultant liquid product with solid beads; c. passing the complex formed in step (b) through a column comprising a ferromagnetic fluid permeable matrix material, said column being under the influence of a magnetic field so that complex is retained thereon; and thereafter; d. removing the column from the magnetic field and eluting complex therefrom; and e. detecting target microorganisms in the eluate from step (d). Suitably food samples which are intended to be analysed are first mixed with a liquid such as a broth and homogenised for example in a homogeniser or Stomacher. They may also be subject to a preliminary incubation step, with or without shaking, to enrich the amount of microorganisms present. This process may take place under conventional conditions, for example over a period of from 5 to 24 hours at 37⁰C. However, due to the efficiency of the present method, extensive pre-incubation may not be necessary and thus the speed of results may be significantly quicker.

Samples obtained in this way are suitably greater than 5ml in volume, for example greater than 10 ml in volume. In particular they may be from 5-250ml in volume, for example about 100ml in volume.

The samples are then treated to remove any large particulates that would clog or otherwise interfere with the passage of liquid through the fluid permeable matrix of the column in step 2 or (c) above. This step should be carried out so that the levels of the microbial flora present are generally maintained. The size of particles which therefore need to be removed in this step depends to some extent on the nature of the specific matrix being used, as discussed further below. However, in general, particulates of greater than lOOμm, such as greater than 5μm, in particular of greater than 3μm, or for example, 2μm or more in size are removed in the filtration step 1 or (a).

Removal of particulates may be done by centrifugation as is conventional in the art. The samples are centrifuged so that the larger particles drop to the bottom of the centrifuge tube and the supernatant is then removed for further processing. However, in a particular embodiment, the particulates are removed by filtration. A particularly suitable filter arrangement comprises a filter column and in particular a graded filter syringe column which, in a particular embodiment, is disposable. In a particular embodiment, the filtration step of step 1 or (a) is carried out using a disposable 'food filter' comprising a graded filter syringe column for the gross clarification of food matrices. The graded filter syringe column suitably comprises a glass fibre filter of the desired porosity, for example of less than 3μm, and in particular of about 2μm, which is arranged between two porous pads, for example of a compressed foam material. The pads will ensure that very large particulates do not reach and clog the filter. This arrangement of pad and glass fibre filter is suitably provided in a suitable tube which may comprise the body of a suitably sized syringe. Liquid from the homogeniser or stomacher is added to the tube or body of the syringe and allowed to pass or preferably forced through the filter using for example the plunger of the syringe. Thus material emerging from the syringe has been clarified and may be used in the next stage of the process.

Such a graded filter syringe column, as illustrated herein after, and its use form a further aspect of the invention. The beads used in step 2 or (b) are suitably ferromagnetic nanobeads of from

5nm to lOOOnm size such as diameter, suitably having an average size of 500nm or less, for example from 5nm to 500nm. Such beads are available from commercial sources such as Miltenyi, Biotec and Immunicon. These small beads are particularly efficient for the capture of small microorganisms such as bacteria. However, they have generally not been thought to be useful for the analysis of food samples in the past due to the difficulties of handling these in very large sample volumes. Beads of larger sizes (>500 um) within a range 500 nm - 10 μm may also be used with the arrangement in step c.

The beads may be coated with a binding agent, and in particular a specific binding agent for a particular target microorganism, such as a Salmonella, Listeria or E.coli bacteria. Suitable specific binding agents include immunoglobulins such as antibodies or binding fragments thereof. These may be immobilised on the beads using conventional methods.

These include (a) direct non-specific adsorption; (b) covalent coupling via a spacer chemical linkage such as a hydrocarbon chain and (c) by first binding an antibody binding protein such as Protein A or Protein G to the support before application of the binding antibody. In a preferred embodiment, a protein comprising an antibody binding domain is applied to the surface of the beads, and the binding antibody applied subsequently. Where the beads are coated with a specific binding agent, they may simply be incubated in the liquid product of step 1 or (a) for a sufficient period of time to allow the complex to form. In a particular embodiment, the complex in step (2i) or (b) is formed by first incubating the liquid product from step 1 or (a) respectively with a first specific binding agent such as an antibody, which is suitably specific for a target microorganism, for a period sufficient to form a microorganism/antibody complex, for example for 5 minutes or more. Subsequently the microorganism/antibody complex is incubated with beads for a period sufficient to form a complex therewith, which is generally 5 minutes or more. The beads suitably carry a secondary binding agent such as a secondary antibody, which binds the first specific binding agent for a period sufficient to allow a complex of for instance bead/secondary antibody/target specific antibody/microorganism to form. This allows standard secondary antibody coated beads to be used for the detection of a range of different microorganisms.

Alternatively, in step 2i or (b) the liquid is first incubated with a specific binding agent that is bound to the beads.

Another alternative is where the specific binding agent bound to beads is held in place in the ferromagnetic column used in step 2ii by means of a magnetic field (so that the column acts as an affinity column) and the liquid from step 1 is simply passed through this affinity column. In this instance, the complex comprising the microorganism will form in situ on the column as the liquid flows through the column. Suitable fluid permeable ferromagnetic matrix material used to fill the column used in step 2 or (c) is a fibrous material such as steel wool or wire matrix.

Ferromagnetic beads such as those described in WO90/07380 are alternative matrices, but these are less suitable for use in the method described herein. In a particularly preferred embodiment, the matrix material is steel wool. Although it has previously been reported that such matrices give rise to non-specific binding or entrapment, and variable separation due to non-uniform pore sizes, the applicants have found that in the context of the present method, these act more efficiently than matrices made up of ferromagnetic particles or beads.

Suitably, the steel wool is a coarse grade material, with a grading of at least 3#, for example from 3-6#. Such steel wool comprises fibres having a width of at least 0.0889 mm, for example from 0.0889mm to 0.1143mm (3#), or from 0.1143-

0.1778mm(4#). Such material allows the relatively large volumes of samples used when analysing food materials to pass through under gravity. Furthermore, the risk of clogging as a result of the presence of residual food particulates is minimised.

The matrix material is suitably placed or packed into a column (which may be of various shapes so long as liquid can flow through it), but will generally be a cylindrical column which is suitably a disposable column, for example of plastics material or glass.

Where cylindrical column is used, it may be of relatively small diameter, for example of from 3 to 20mm diameter, for example about 5mm diameter. This may be packed with matrix material to a height of from 3 to 25mm for example from about 5- 10 mm.

In order for the application of a magnetic field to the matrix, the column must be surrounded by a series of high strength magnets which ensure that the complex of beads and microorganism is captured on the surface of the fibrous material, providing for rapid capture and concentration of the microorganisms. Generally, when very small beads and in particular nanobeads are used in the process, very high strength electromagnets are required to ensure that they are captured in many instances. These may be large, cumbersome and expensive. However the applicants have found that by using a matrix material that induces a high magnetic field gradient locally at the surface of the matrix such as steel wool, conventional high strength permanent magnets may be employed.

One or more columns may be provided and arranged for high throughput of samples, for example by being placed within the same housing. In a particular embodiment, each housing carries at least 18 columns, and is provided with a corresponding number of holes to allow the introduction of sample into each column simultaneously. The holes are suitable arranged in lines, with permanent bar magnets interposed between the rows within the housing. Each row may contain for example 8, 12 or even 24 holes, so as to accommodate conventional multiwell plate arrangements.

The holes are arranged so that columns located in them have the fibrous magnetic matrix directly adjacent one or more bar magnets within the housing. This may be achieved by retaining means such as annular flanges or lips that are provided on either the column itself or on the housing, so that the column and the housing interlock when the column is in the correct position within the hole in the housing. Samples may be passed repeatedly down a column in step 2 or (c) to ensure maximal capture of the microorganisms. However, a single pass may be sufficient. It is important to ensure that sufficient volume of sample is applied to ensure that sufficient target microorganisms are captured. For a food broth, this will suitably mean that a sample size in excess of 10ml, and preferably in excess of 25ml is used. Sample sizes in the range of from l-250ml, for example from 25-150ml such as about 100ml are used. The use of coarse grade fibrous material in particular, allows the passage of such volumes of food broths to pass through under gravity in a reasonable timescale, without clogging.

Once completed, the columns are suitably washed prior to step 3 or (d), for example using clean broth sample such as BPW. This washing step removes potential background interference such as food particulates, components or non-target microorganism.

Thereafter, step 3 can be carried out by detecting target microorganism directly on the column, or by first removing the complex from the column. The removal step or step (d) above can be effected quite simply by inactivating or removing the magnetic field around the column or housing. For instance, where the column has been lodged in a housing comprising a permanent magnet, it may simply be removed from the housing to isolate it from the magnetic field.

The resultant complex can then be eluted readily from the column using for example a clean sample of the incubation buffer or broth.

The eluate contains the concentrated complex, which, if the binding agent is a specific binding agent, will comprise a particular target microorganism. If it is required that multiple micoorganisms are detected, then beads carrying specific binding agent for each said microorganism may be used in the process. Preferably however, the liquid product from step 1 or (a) sample is contacted with multiple specific binding agents such as antibodies, each of which binds to a common secondary antibody, and the complex formed by contacting the resultant antibody/microorganism mixture with beads carrying the secondary antibody. For example, beads carrying anti-mouse, or anti-goat or anti-rabbit antibodies will bind a range of specific antibodies if they are all derived from the appropriate mouse, goat or rabbit species. Depending upon whether step 2i or 2ii is used, the liquid product may be incubated with beads in solution or they may be retained on the column when the contact occurs. The material retained on the column as well as any eluate or product from step

(d) if used, will be concentrated in respect of the target microorganisms. However, due to possible cross-reactivity of binding agents used in the process, other microorganisms may be carried over and form a "background" level of microorganisms. If the specific nature of the target microorganism is important to the assay being carried out, it is preferable then that the detection carried out in step 3 or

(e) is specific for that target organism. This is acceptable in the analysis of food samples. The procedure of steps 1-2 or (a)-(d) will lead to significant concentration of the target organism as compared to the starting samples which will facilitate detection in step 3 or (e) respectively. Detection in step 3 or (e) may be carried out in any conventional manner. For example, the beads may be plated onto suitable nutrient medium and any resultant cultures detected and if necessary identified. However, other methods conventional in the art, such as those based upon detection of specific proteins such as ELISA, or those based upon the detection of nucleic acids, in particular methods that utilise nucleic acid amplification reactions such as polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), rolling circle DNA amplification, multiplex ligation-dependent probe amplification (MLPA) and multiple displacement amplification. Such methods are well known in the art, and a skilled person would be in a position to adapt any of these to provide detection in the context of the method described herein.

If required, these may be carried out in a quantitative manner, as is well known in the art, so as to allow the concentration of the specific microorganism in the sample to be measured. Particular quantitative assays include the TAQMAN™ system which utilises PCR, but others may be available.

Where a single specific binding agent is used in step 2 or (b), concentration of the particular target microorganism is effectively carried out. This may mean that any microorganism detected in a subsequent step 3 or (e) would be of the target type. However, where multiple target microorganisms are being detected and/or to avoid the possibility that some non-specific binding has occurred, it may be preferable to utilise a method in step 3 or (e) in which a specific target microorganism, such as a bacteria is detected.

One method which allows for specific identification is described in WO9406931, the entire content of which is incorporated herein by reference. In this method, in essence, a sample is incubated in the presence of a bacteriophage which specifically infects a particular target bacteria, so as to cause lysis of the bacteria. At this point, cellular components are released from the bacteria, and detection of any of these is indicative of the presence of the specific bacteria in the initial sample. The enhanced purification opportunities afforded by the use of the method described above, such as a proteolytic step, is extremely beneficial here, in that it will ensure that no false positives are generated as a result of contaminants which may be retained upon the support.

Assays for a wide variety of cellular components including hormones, enzymes etc. are reported in the art. One cellular component which may be conveniently detected however is the nucleotide adenosine triphosphate or ATP. ATP is conveniently detected using a bio luminescent assay, such as the well known bio luminescent assay based upon the reaction of luciferase and luciferin.

However, in a particular embodiment, the cellular component released on cell lysis which is detected is adenylate kinase. This enzyme catalyses the following equilibrium reaction in cells:

Mg.ATP + AMP <-> 2ADP

WO9417202 and WO9602667 describes how the detection of this particular enzyme produces a greatly amplified signal, and the entire content of these documents is incorporated herein by reference. In brief, adenylate kinase is detected by adding an excess of pure ADP to the sample, so the equilibrium is driven towards the right and ATP is created. This can readily be detected using a variety of assays, but in particular a bio luminescent assay, such as that based upon the reaction of a luciferase enzyme on its substrate luciferin. In the presence of ATP, this interaction occurs and a light signal is generated. The fact that this type of assay is so sensitive means that low levels of microorganisms can be detected, and therefore the need for extended culture periods can be reduced. This allows the determination of microorganisms and particularly specific target microorganisms to be effected rapidly and accurately. Thus the method described above is highly advantageous in the field of food testing where speedy results are helpful in determining whether particular food products meet acceptable standards or not. However, it is also clear that any contaminating adenylate kinase in the sample may cause false positives to occur. Thus in this particular case, the use of a proteolytic purification step, for example as described in WO 2007/113583 which remove this contaminant may allow the assay to be used more reliably.

According to a further aspect, the invention provides a kit for carrying out the method as described above, said kit comprising magnetic beads as described above in particular beads of up to lOOOnm in size, such as nanobeads coated with either (i) a specific binding agent for a microorganism which is a pathogen and which may be found in food, or (ii) with a secondary binding agent, able to bind to a first binding agent, and wherein in the case of (ii), the kit further comprises a first binding agent such as an antibody, which is specific for said microorganism.

Particular binding elements etc. are as described above. In the case of (i), the specific binding agent for a microorganism may be directly bound to the beads, or may be bound by way of a secondary binding agent, such as a non-specific binding antibody. Other elements useful in the method described above may also be included in the kit. For instance, kits may also comprise means for detecting the target microorganisms in step 3 or (e). In a particular embodiment, the kit may further comprise a bacteriophage which specifically infects and lyses a target bacteria, which is used in step 3 or (e) for the specific determination of the target bacteria as described above.

Furthermore, it may further comprise ADP, suitably in pure form, in order to act as a basis for the AK assay described above. Bioluminescent reagents activated by ATP, such as luciferin and a luciferase may also be included in order to allow the specific sensitive AK assay described above to be incorporated into the kit. Kits may also include food filters, in particular as described above. Apparatus for carrying out the method described herein in a high-throughput manner is also novel and forms a further aspect of the invention. Thus it comprises a housing holding a plurality of columns packed with fibrous ferromagnetic material, the housing having a plurality of openings therein which allow large volumes of sample to be delivered simultaneously to the columns, and means for creating a magnetic field able to fix beads and in particular nanobeads to said fibrous ferromagnetic material.

In particular the apparatus comprising a housing comprising rows of holes, which are each adapted to accommodate a single column. Each hole passes through the housing so that a column retained in the hole in an orientation such that the magnetic field is applied to the fibrous ferromagnetic material packed therein. For example, the column may be appropriately shaped to seat in the base of the hole in the housing. Permanent bar magnets are interposed between the rows within the housing. Such housings for use in the apparatus form yet a further aspect of the invention.

Detailed Description of the Invention The invention will now be particularly described by way of example, with reference to the accompanying diagrammatic drawing in which;

Figure 1 is a schematic diagram showing an arrangement for sampling which may be used in an embodiment of the invention:

Figure 2 is a schematic diagram showing an arrangement of a food filter used in the method described herein, which forms an aspect of the invention,

Figure 3 is a plan view of a magnetic housing for use in the method of the invention; and

Figure 4 is a section on line A-A in Figure 3, with pipettes in position with the holes. Example 1: Separation and concentration of Salmonella enteritidis from a mixed culture containing Hafnia alveii using immunomagnetic nanobead technology Method Overnight 10 ml NB cultures of Salmonella enteriditis SA029 and Hafnia alveii FI002 were incubated at 37⁰C.

Following incubation, these cultures were appropriately diluted and inoculated into 70 ml BPW/T to given final concentrations (estimated) of 10⁴ cfu/ml Salmonella and 10⁵ cfu/ml Hafnia. Viable counts from each pre-enrichment bag were carried out by plating appropriate dilutions on NA and XLD. Plates were incubated overnight at 37⁰C.

Ten ml triplicate samples from the Salmonella/Hafnia culture were used. The first three (1-3) were designated negative controls whereas samples 4-6 were test samples. To samples 4-6, 2.5 μl of a 4 mg/ml biotin-conjugated anti-Salmonella group antigen antibody (Biogenesis, Cat. No. 8209-4011) was added, corresponding to a final antibody concentration of 1 μg/ml. Bacteria and antibody were incubated together on the MSR (Magnetic Sample Rotator) at 37⁰C for 15 min at a speed setting of 5. No antibody was added to samples 1-3, but from hereon all samples were treated identically. Following incubation, 20 μl of Miltenyi anti-biotin microbeads (Cat. No. 130-

090-485) were added to the antibody/bacteria mix, and incubation continued with rotation on the MSR as previously for 15 min.

Six Miltenyi MACS separation columns (Cat. No. 130-091-506) were placed the Miltenyi Octomax magnet with the outlet nozzles leading into red-capped tubes. Each sample was applied to one column and broths allowed to drain by gravity flow. Samples were re-circulated three times.

Columns were washed once with 10 ml BPW/T.

Once all broth had passed through the columns, in turn the columns were removed from the magnet and eluted using 1 ml BPW/T as an elution buffer with the plunger provided with the columns. Eluates were enumerated by plating serial dilutions on XLD to assess Salmonella growth, and on NA to assess total numbers of bacteria present.

Results All samples passed easily through the filters. No agglutination was seen, and recirculation was successful. Table 1 (below) shows the level of Salmonella and Hafnia in the starting culture, and cell numbers following elution from the columns. Flow through rate was approximately lml/min.

Table 1. Start levels of bacteria and elution levels

Discussion

Low levels of Salmonella (~10 •\4 //ml) are captured and concentrated following immunomagnetic capture on the column. The levels of Hafnia only appear to be modestly reduced in those samples with added antibody. The levels of Salmonella and Hafnia recovered in the absence of antibody are broadly similar per ml compared to what was applied, but in real terms this shows a 10-fold reduction owing to volume differences. The same effect is seen in the Salmonella samples with antibody, where, although the number of bacteria per ml appear to be 10-fold increased upon elution, there is ten times less volume in the elution fraction. Nevertheless, the overall effect seen is that Salmonella numbers have increased by 1 log and Hafnia numbers have decreased by half a log upon elution, compared to initial input, showing that the method is effective at concentrating target bacteria, in this case, Salmonella.

Example 2: Preparation of Food samples

Food broths comprising food samples which may contain bacterial species are prepared in a conventional manner.

For example, a test food sample (25g) is weighed into a sterile plastic filter bag, to which is added 225 ml of broth medium (TSB+AGS) and the mixture homogenised in a Stomacher for 30 seconds. The sample is then incubated in a shaker-incubator at 41.5⁰C and 120rpm for 10 to 12 hours.

In an alternative ISO method, 225 ml Buffered Peptone Water (BPW) is added to a 25g test food sample, which is then stomached for 30s and incubated for 18-24h at 37°C.

A sample of the homogenate (2) suitably of 10 ml or more may then be drawn into a sterile pipette tube (1) (Figure 1) which is suitably of a plastic material. The pipette is graduated to allow accurate sample measurement.

The pipette is provided with a suction device (3) able to draw liquid in and also expel it.

Once filled, the pipette tip (4) is pushed into a bung (5) of a filter syringe (6). The body of the syringe (6) is packed with a glass fibre filter medium (7) of the desired porosity, (which may for example be 2μm, interposed between two pads of compressed fluid permeable foam material (8,9). The sample is then expelled from the pipette tube (1) so that it passes through the syringe body (6) and out through an opening (10) into an appropriately positioned tube (11), which is suitably capped immediately. The clarified sample may then be analysed for target microorganisms in the method described herein.

Example 3 : Recovery of Salmonella from 10-100 ml enrichment broths (pure culture and turkey mince) using steel wool columns and anti-Salmonella beads

Coarse grade wool (13) (Oakey, UK), was used to prepare mini columns in glass Pasteur pipettes (12) (Figure 4). The columns were packed to a depth of approximately 5-8 mm with a 5 mm internal diameter. The columns were used in combination with anti-Salmonella beads and a permanent magnet to separate and concentrate Salmonella from 10-100 ml of enrichment broths. Methods

BcMag carboxyl derivatised beads from Bioclone are supplied as an aqueous suspension of magnetic iron oxide beads coated with carboxyl groups for covalent coupling of proteins using EDC, a zero length cross linker that is amine- and carboxyl-reactive.

BcMag characteristics: bead size: 1 μm; concentration ~20mg/ml

Coupling of anti-salmonella antibody to BcMag bead

1. 0.5 ml of BcMag carboxyl derivatised beads (Bioclone; product no. FBlOl, lot 80011) were washed three times with an equal volume of phosphate buffered saline (PBS; Sigma), adjusted to pH 5.5 with IM HCl.

2. Bactrace affinity purified antibody to salmonella common structural antigen (CSA-I; Kirkgaard and Perry Laboratories, KPL) was dissolved in PBS (0.01 M, pH 5.5; Sigma) to a concentration of 100 μg/ml, and 0.5 ml added to the washed beads.

3. EDC coupling reagent was prepared by dissolving 0.1 g EDC (Sigma E7750) in 10 ml distilled water.

4. 0.1 ml of the EDC coupling reagent was added to the antibody and bead mixture, and incubated for 24 h at room temperature with gentle mixing. 5. Excess antibody was removed by washing three times with PBS (pH 7.4; 1 ml) IMS of Salmonella from pure culture

1. An overnight enrichment of Salmonella Enteritidis (SA029) at ~10⁸ cfu/ml was diluted 1 : 100 in BPW to give an intermediate stock at ~10⁶ cfu/ml.

2. 1 ml of the ~10⁶ cfu/ml intermediate stock was added to 100 ml of BPW to give a final working concentration of ~10⁴ cfu/ml Salmonella.

3. The spiked 100 ml broth was poured into a 2 L plastic reservoir with an outlet attached to a steel wool column. 20 μl of anti-Salmonella bead, prepared as described above was added and the total volume incubated at room temperature (20-22⁰C) for 15 minutes with occasional mixing. 4. The pipettes (12) were placed in a hole (14) in a solid block (15) containing rows of similar holes. The neck of each pipette (12) engaged with an annular lip (16) in the base of the block (15). Interposed between each row of holes (14) were permanent magnets (17).

5. The sample was passed through the steel wool column by gravity feed, with the column sitting in a magnetic block (Magnet Sales) (15) ,

6. While still held within the block (15), the column was washed with 20 ml MRD to remove the unbound cells.

7. The column was removed from the magnet and the bound beads eluted in 1 ml of MRD. The recovered Salmonella were enumerated on XLD.

IMS of Salmonella from turkey mince enrichment broth

1. 25g of turkey mince was added to 225 ml BPW and incubated for 24 h at 37°C.

2. A 10 ml aliquot of the enriched food broth was spiked with ~10⁴ cfu/ml Salmonella.

3. The 10 ml sample was passed through a food filter to remove the gross food particulates.

4. 20 μl of anti-Salmonella bead prepared as described above was added and the total volume incubated at room temperature (20-22⁰C) for 15 min with occasional mixing.

5. Each sample was passed through the steel wool column by gravity feed, with the column sitting in the magnetic block as described steps 4 and 5 above . 6. While still in the block and therefore under the influence of the magnets, the columns were washed with 20 ml MRD to remove the unbound cells.

7. The column was removed from the magnet and the bound beads eluted in 1 ml of MRD. The recovered Salmonella and total background flora were enumerated on XLD and NA.

Results Pure culture

Turkey mince Broth

The recovery levels of Salmonella from pure culture enrichment broths using the steel wool column and beads as described above was dependent on sample volume, typically -40% and -10% from 10 ml and 100 ml samples, respectively. However, this indicates that high volumes such as used in food sampling will provide better results. The non-specific retention of Salmonella on the column without the bead was low (≤l%). Using the steel wool column and beads as described above in a minced turkey food broth, Salmonella was recovered at -11% (i.e. -1 log reduction) with a corresponding -4 log reduction in the background total viable count.

The results indicate that this method is a useful novel flow through capture and concentration system for recovering low levels of Salmonella present in large volumes of complex sample matrices (in this case, exemplified by using a turkey mince enrichment broth).

Claims

1. A method for detecting the presence of target micro-organisms in a food sample; said method comprising the following modules: 1. removing large particulates from a liquid comprising a finely divided food sample;

2. forming a complex of magnetic solid beads of up to lOOOnm (lμm) in size and microorganism in the liquid, wherein either 2i) the complex is formed by a process comprising incubating the resultant liquid product with solid beads and the resultant complex is passed through a column comprising a ferromagnetic fluid permeable matrix material of fibrous ferromagnetic material, said column being under the influence of a magnetic field so that complex is retained thereon; or

(2ii) the magnetic solid beads are retained on a ferromagnetic fluid permeable matrix material of fibrous ferromagnetic material within a column under the influence of a magnetic field; and thereafter; 3. detecting target microorganisms retained on the column.

2. A method according to claim 1 wherein the beads used in step (2) are ferromagnetic nanobeads having an average diameter of 500nm or less.

3. A method according to claim 1 or claim 2 wherein before step 1, the finely divided food sample is subjected to a homogenisation step.

4. A method according to claim 3 wherein the step 1 removes particulates of greater than 3μm in size.

5. A method according to any one of the preceding claims wherein step 1 is carried out by filtration in a disposable graded filter syringe column.

6. A method according to any one of the preceding claims wherein the target microorganism is a bacteria selected from Salmonella, Listeria, E.coli and Campylobacter bacteria.

7. A method according to any one of the preceding claims wherein the complex in step 2 is formed by a method of 2i in which the liquid product from step 1 is incubated with a first specific binding agent which is specific for a target microorganism, and subsequently the resulting microorganism/antibody complex is incubated with beads carrying a secondary binding agent which binds the first specific binding agent.

8. A method according to any one of the preceding claims wherein the fibrous matrix ferromagnetic material used to fill the column used in step 2 is coarse steel wool.

9. A method according to any one of the preceding claims wherein the or each column is washed prior to step 3.

10. A method according to any one of the preceding claims wherein the microorganism is a bacteria and in step 3 the bacteria is detected by incubating the bacteria with a bacteriophage, which specifically infects the target bacteria and causes lysis thereof, and thereafter, detecting a cellular component released as a result of the lysis.

11. A method according to claim 10 wherein the released cellular component is adenylate kinase, and this is detected by adding an excess of ADP and detecting ATP produced, using a bioluminescent assay.

12. A kit for carrying out the method according to any one of the preceding claims, said kit comprising magnetic nanobeads coated with (i) a specific binding agent for a microorganism which is a pathogen and which may be found in food, or (ii) with a secondary binding agent, able to bind to a first binding agent, and in the case of (ii), a first binding agent such as an antibody, which is specific for said microorganism.

13. A kit according to claim 12 which further comprises one or more of: a bacteriophage which specifically infects and lyses a target bacteria; ADP; a bioluminescent system which is activated by ATP, such as luciferin and luciferase; and a filter for filtering food homogenate.

14. Apparatus for carrying out the method according to any one of claims 1 to 12, said apparatus comprising a housing holding a plurality of columns packed with fibrous ferromagnetic material, the housing having a plurality of openings therein which allow large volumes of sample to be delivered simultaneously to the columns, and means for creating a magnetic field able to fix beads and in particular nanobeads to said fibrous ferromagnetic material.

15. A combination comprising a kit according to claim 12 or claim 13 and apparatus according to claim 14.

16. A housing for use in apparatus according claim 14 which comprises a plurality of holes which are each adapted to accommodate a single column, and one or more permanent bar magnets arranged within the housing so as to exert a magnetic field on fibrous ferromagnetic material packed into a column accommodated within a said hole.

17. A filter syringe column suitable for use in step a of the method of claim 1, said column comprising a filter material of appropriate pore size, positioned between porous pads within the body of a syringe column.