WO2009122216A1 - Système d'essai basé sur la séparation immunomagnétique pour détecter des micro-organismes dans un échantillon d'aliments - Google Patents

Système d'essai basé sur la séparation immunomagnétique pour détecter des micro-organismes dans un échantillon d'aliments Download PDF

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Publication number
WO2009122216A1
WO2009122216A1 PCT/GB2009/050327 GB2009050327W WO2009122216A1 WO 2009122216 A1 WO2009122216 A1 WO 2009122216A1 GB 2009050327 W GB2009050327 W GB 2009050327W WO 2009122216 A1 WO2009122216 A1 WO 2009122216A1
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Prior art keywords
column
beads
complex
microorganism
bacteria
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PCT/GB2009/050327
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English (en)
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Pradip Patel
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Alaska Food Diagnostics Limited
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Publication of WO2009122216A1 publication Critical patent/WO2009122216A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/025—High gradient magnetic separators
    • B03C1/031—Component parts; Auxiliary operations
    • B03C1/033—Component parts; Auxiliary operations characterised by the magnetic circuit
    • B03C1/0332—Component parts; Auxiliary operations characterised by the magnetic circuit using permanent magnets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers

Definitions

  • 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.
  • microorganisms such as bacteria or fungi
  • 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.
  • micro-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.
  • volumes handled in the clinical field may be up to 5ml in volume.
  • 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.
  • 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.
  • 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.
  • the beads are not in direct contact with the whole volume of the enrichment broth (and consequently the target pathogens) during the capture process.
  • 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.
  • 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).
  • an overnight cultural incubation step e.g. 18 - 24h for Salmonella
  • the applicants have developed a process for the concentration of target microorganisms in food which is fast and is amenable to high-throughput operation.
  • a method for detecting the presence of target micro-organisms in a food sample comprising the following modules:
  • 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
  • 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;
  • 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.
  • step (ii) 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.
  • a method for detecting the presence of target micro-organisms in a food sample 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 37 0 C. 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 lOO ⁇ m, such as greater than 5 ⁇ m, in particular of greater than 3 ⁇ m, or for example, 2 ⁇ m or more in size are removed in the filtration step 1 or (a).
  • a particularly suitable filter arrangement comprises a filter column and in particular a graded filter syringe column which, in a particular embodiment, is disposable.
  • 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 3 ⁇ m, and in particular of about 2 ⁇ m, 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.
  • material emerging from the syringe has been clarified and may be used in the next stage of the process.
  • 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 ⁇ m 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.
  • 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.
  • adsorption includes (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.
  • an antibody binding protein such as Protein A or Protein G
  • a protein comprising an antibody binding domain is applied to the surface of the beads, and the binding antibody applied subsequently.
  • the beads 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.
  • 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.
  • step 2i or (b) the liquid is first incubated with a specific binding agent that is bound to the beads.
  • 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.
  • the matrix material is steel wool.
  • 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-
  • 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.
  • cylindrical column 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.
  • the column 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.
  • 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.
  • very high strength electromagnets are required to ensure that they are captured in many instances. These may be large, cumbersome and expensive.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • step (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.
  • the beads may be plated onto suitable nutrient medium and any resultant cultures detected and if necessary identified.
  • 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.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • LAMP loop-mediated isothermal amplification
  • MLPA multiplex ligation-dependent probe amplification
  • MLPA multiple displacement amplification
  • 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 TAQMANTM system which utilises PCR, but others may be available.
  • 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.
  • WO9406931 One method which allows for specific identification is described in WO9406931, the entire content of which is incorporated herein by reference.
  • 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.
  • 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.
  • 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.
  • the cellular component released on cell lysis which is detected is adenylate kinase.
  • This enzyme catalyses the following equilibrium reaction in cells:
  • 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.
  • 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.
  • 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.
  • ATP a bio luminescent assay
  • 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.
  • a first binding agent such as an antibody
  • kits may also comprise means for detecting the target microorganisms in step 3 or (e).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 is a schematic diagram showing an arrangement for sampling which may be used in an embodiment of the invention:
  • FIG. 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.
  • 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 37 0 C.
  • Food broths comprising food samples which may contain bacterial species are prepared in a conventional manner.
  • 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 41.5 0 C and 120rpm for 10 to 12 hours.
  • TBS+AGS broth medium
  • 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.
  • BPW Peptone Water
  • 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.
  • 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 2 ⁇ m, 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.
  • 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 ⁇ m; concentration ⁇ 20mg/ml
  • 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 ⁇ g/ml, and 0.5 ml added to the washed beads.
  • EDC coupling reagent was prepared by dissolving 0.1 g EDC (Sigma E7750) in 10 ml distilled water.
  • the spiked 100 ml broth was poured into a 2 L plastic reservoir with an outlet attached to a steel wool column.
  • 20 ⁇ l of anti-Salmonella bead, prepared as described above was added and the total volume incubated at room temperature (20-22 0 C) for 15 minutes with occasional mixing.
  • 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).
  • the column was removed from the magnet and the bound beads eluted in 1 ml of MRD.
  • the recovered Salmonella were enumerated on XLD.
  • the 10 ml sample was passed through a food filter to remove the gross food particulates.
  • 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.
  • 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%).
  • Salmonella was recovered at -11% (i.e. -1 log reduction) with a corresponding -4 log reduction in the background total viable count.

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Abstract

L'invention porte sur un procédé pour détecter la présence de microorganismes cibles dans un échantillon de produit alimentaire. Ce procédé consiste à : 1. retirer des particules de grande dimension d'un liquide incluant un échantillon de produit alimentaire finement divisé; 2.former un complexe de billes solides magnétiques d’une dimension allant jusqu'à 1 000 nm (1 µm) et de microorganisme dans le liquide, où soit (2i) le complexe est formé par un procédé comprenant l'incubation du produit liquide résultant avec des billes solides, le complexe résultant étant amené à passer à travers une colonne incluant un matériau de matrice perméable au fluide, ferromagnétique, de matériau ferromagnétique fibreux, ladite colonne étant sous l'influence d'un champ magnétique de telle sorte qu'un complexe est retenu sur celle-ci; soit (2ii) les billes solides magnétiques sont retenues sur un matériau de matrice perméable au fluide, ferromagnétique, de matériau ferromagnétique fibreux à l'intérieur d'une colonne sous l'influence d'un champ magnétique; puis 3. à détecter des microorganismes cibles retenus sur la colonne.
PCT/GB2009/050327 2008-04-04 2009-04-02 Système d'essai basé sur la séparation immunomagnétique pour détecter des micro-organismes dans un échantillon d'aliments WO2009122216A1 (fr)

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US9476812B2 (en) 2010-04-21 2016-10-25 Dna Electronics, Inc. Methods for isolating a target analyte from a heterogeneous sample
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WO2012145018A1 (fr) 2011-04-21 2012-10-26 Nanomr, Inc. Compositions pour isoler un analyte cible à partir d'un échantillon hétérogène
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US10379113B2 (en) 2012-12-19 2019-08-13 Dnae Group Holdings Limited Target detection
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CN112505117A (zh) * 2020-11-11 2021-03-16 军事科学院军事医学研究院军事兽医研究所 一种二茂铁纳米花、电化学适配体生物传感器体系及其制备方法和应用

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