AU738768C

AU738768C - Methods of labelling a material and of detecting such labelling

Info

Publication number: AU738768C
Application number: AU73478/98A
Authority: AU
Inventors: Peter Alestrom; Tom Senstad
Original assignee: CHEMTAG AS
Current assignee: CHEMTAG AS
Priority date: 1997-06-05
Filing date: 1998-06-02
Publication date: 2002-03-28
Anticipated expiration: 2018-06-02
Also published as: NO995917L; WO1998055648A1; AU738768B2; EP0985050A1; CA2292841A1; NO995917D0; AU7347898A; NO972556D0

Description

METHODS OF LABELLING A MATERIAL AND OF DETECΗNG SUCH LABELLING

FIELD OF THE INVENTION

The present invention pertains to the field of labelling materials with specific taggant substances comprising coded information. Specifically there is provided a simple and convenient method of detecting that a material has been labelled with a taggant substance bearing information which permits the identity and source of the material to be esta- blished. Such a method makes it possible to detect, using very simple detection means, whether or not a material has been labelled with the specific taggant substance, the structure and/or informational content of which can only be determined and/or decoded by staff having access to the necessary, specialized laboratory facilities and the code of the taggant substance.

TECHNICAL BACKGROUND AND PRIOR ART

Labelling or tagging of materials including liquids, substances, flowables and objects with the objective of prevent- ing or detecting theft, counterfeit, forgery, copyright infringement or unlawful or accidental pollution of the environment has attracted considerable attention and several methods for such labelling or tagging have been suggested. Specifically, labelling with nucleic acid fragments as tag- gants have been disclosed in a number of patent applications.

Thus, WO 87/06383 discloses a method of labelling an item or substance by means of a predetermined macromolecular signal compound such as a nucleic acid to which a complementary second compound is capable of binding thereby revealing the presence of the signal compound. WO 90/14441 generally discloses the labelling and tracing of materials by use of nucleic acid taggants. The detection of the taggant is preferably performed by PCR amplification of the nucleic acids used for labelling.

EP-B-408,424 discloses the marking of valuable objects by incorporating a target nucleic acid in the objects using a nucleic acid solution having a chosen degree of fluidity and, if necessary, subsequently identifying the object by detecting the target nucleic acid by means of nucleic acid probes that hybridize to the target nucleic acid.

WO 91/17265 discloses a method of monitoring the movement of a material such as a hydrocarbon by adding DNA molecule taggants to the material and detecting the presence of the taggant .

WO 94/04918 pertains to the marking of liquids with ≤ 1 ppm of non-cellular particles comprising i.a. nucleic acid tags attached thereto.

The use of nucleic acid taggants for labelling of materials involves a number of advantages. Firstly, the existence of effective molecular amplification methods such as PCR permits even trace amounts of nucleic acids to be detected and analyzed. Thus, the material which is labelled with the nucleic acid will be virtually unchanged by the addition of the label. Additionally, it is relatively simple to produce suitable nucleotide fragments. Secondly, it is only possible to amplify such a nucleic acid fragment when sufficient information concerning the sequences of the 3' and the 5' ends of the molecule is available. If such information is not at hand, it is not possible to produce useful primers in order for the amplification reaction to occur, and it will therefore not be possible for an unauthorized third party to detect the presence of the label, let alone to decipher or decode the informational content of the taggant sequence. However, the labelling of materials with nucleic acid taggants (whether these are in the form of naturally occurring materials or in the form of synthetically produced fragments) involves the serious drawback that it is required to have a specific set of primers or hybridization probes at one's disposal in order to be able to detect whether or not the taggant is present on or in the material. Moreover, are such primers or probes available, the detection procedures are relatively complicated and requires appropriate laboratory facilities and staff having the necessary technical skills to perform amplification and/or hybridization procedures. These requirements seriously limit the usefulness of methods involving the labelling of materials with nucleic acids, as the detection hereof cannot be performed in the field, i.e. on locations where a liquid, object or substance suspected of being labelled is found.

This problem has been addressed in WO 87/06383 where it is mentioned that the presence or absence of macromolecules such as DNA can be detected by what is referred to as simple chemical analytical procedures, which are referred to in that document as "YES/NO" tests which are indicative of whether or not the macromolecule is present. For example, the presence of DNA can be detected by using non-specific chemical agents which bind to DNA, such as ethidium bromide, acridine orange or bis-benzimide and detecting that such binding has occurred. However, such a detection requires access to relatively sophisticated laboratory equipment .

WO 94/04918 describes a method of labelling a liquid such as a hydrocarbon oil with non-biological particles that comprise what is referred to as signal means to aid detection of their presence, which signal means are invisible in the liquid to the naked eye (a first, non-specific marker) . Examples of such non-specific markers include (i) that the particles are magnetic and can be separated using an appropriate apparatus for separation, (ii) that the particles have a known size or shape distribution to allow a particular batch to be iden- tified by determining the frequency of particles of one size or shape relative to the other, e.g. by means of a Coulter counter, (iii) that the particles are coloured, (iv) that the particles are provided with a detectable label such as a fluorescent label, an enzyme or a radiolabel. In this method, the particles may also be provided with a second "unique marker" e.g. in the form of a nucleic acid to permit authorities to more specifically identify the identity or source of the labelled liquid.

WO 96/17954 discloses a method for chemically labelling an object, comprising adding to the object a first, unique or specific chemical tag and a second, non-specific chemical tag, of which the first tag comprises an informational content which is not divulged to the public, which can be amplified by use of molecular amplification and the presence of which specifically establishes the identity and/or origin of the object and the second tag indicates that the object is labelled with the first tag and is "easily detectable". According to this disclosure, the second tag is preferably a nucleic acid sequence which can be detected by hybridization and/or PCR amplification by means of probes or primers that are publicly available, thus permitting anyone in possession of such detection means and the necessary facilities and skills to detect that an item is labelled with the first tag.

It is evident that the above "YES/NO" tests require that individuals or authorities who are responsible for monitoring whether or not a material including a liquid, an object, a substance and a flowable material or any other matter has been labelled with a specific taggant comprising non- divulged, coded information, which when decoded, establishes the identity and/or source of the material , must have at their disposal relatively complicated testing equipment and facilities and skilled staff who e.g. knows how to handle toxic reagents and complicated measuring instruments. A major objective of the present invention is therefore to provide a simple method for detecting whether or not a material is labelled with a taggant carrying more or less complex information on the source and origin of the item. The method only requires a microscope of a type which can easily be operated by individuals not having the skills of professional laboratory staff. Thus, the present method can be used as a rapid, cost-effective indicative test for the presence or absence on or in the material of coded taggant substances such as predetermined, specific nucleic acid sequences .

SUMMARY OF THE INVENTION

Accordingly, the invention relates in a first aspect to a method of detecting whether or not a material has been labelled with a specific taggant, the method comprising associating a material , that is labelled with at least one specific taggant substance comprising an informational content which is not divulged to the public and the presence of which specifically establishes the identity and/or origin of the material, with a microscopically detectable amount of cellular particles, the presence of which in or on the labelled material can be detected by simple microscopical detection means, collecting a sample of the material that is suspected of being labelled with the specific taggant sub- stance and testing the sample for presence of the cellular particles, subject to the limitation that, when the specific taggant substance is integrated in the cellular particles, it is not a substance that occurs naturally in said particles.

In a further aspect, the invention provides a method of labelling a material and subsequently detecting that it has been labelled, the method comprising the steps of (i) labelling the material with at least one specific taggant substance comprising an informational content which is not divulged to the public and the presence of which specifically establishes the identity and/or origin of the material and (ii) associating the material with a microscopically detectable amount of cellular particles, the presence of which in or on the labelled material can be detected by simple micro- scopical detection means, subject to the limitation that when the specific taggant substance is integrated in the cellular particles, it is not a substance that occurs naturally in said particles.

In a still further aspect, the invention pertains to a material that is labelled with a specific taggant substance comprising an informational content which is not divulged to the public and the presence of which specifically establishes the identity and/or origin of the material and associated with it, a microscopically detectable amount of cellular particles, the presence of which in or on the labelled material can be detected by simple microscopical detection means .

DETAILED DISCLOSURE OF THE INVENTION

The above method of detecting whether or not a material has been labelled with a specific taggant substance can be applied to any material for which there is a need that it is labelled with a specific taggant so as to permit subsequent establishment of its identity and/origin. Thus, in the present context the term "material" refers to any types of matter including physical objects such as motor vehicles, antiquities, pieces of art, precious metal objects, jewe- leries, paper goods objects such as bank notes and securities; liquids such as e.g. hydrocarbon materials including petroleum and petroleum-derived liquids, pharmaceuticals, perfumes and beverages; compositions of matter including as examples pharmaceutical products, food and feed products, reagents, hydrocarbon compositions, explosives, herbicides, pesticides and environmental pollutants; and living matter such as microbial cells, animal and plant cell cultures. As used herein, the expression "specific taggant substance" refers to any substance that can be incorporated in, attached to or otherwise associated firmly with a material as defined above and which has an informational content that permits the identity and/or origin of the material with which it is associated to be recognized. In the method, any taggant including the above prior art taggants fulfilling this requirement can be used.

A presently preferred taggant substance is a nucleic acid comprising a more or less detailed information content with respect to identity or origin of the labelled material. However, the use of other macromolecules such as PNA, polypeptides and synthetic polymeric materials is also contemplated. It will be appreciated that the level of informational content of the specific taggant substance can be adapted to the particular requirements for identification. Thus, as an example, a petroleum material can be labelled with a nucleic acid, the sequence of which when determined reveals the country of origin of the material . The nucleic acid taggant may also contain further information which identifies the carrier of the material or even which tank of the carrier was used. In this manner can e.g. the source of pollution of the sea and beaches with crude oil be determined unambiguously.

The primary objective of the present method is to provide a simple "YES/NO" test which can be used by staff without any professional laboratory skills and with very simple detection means to detect whether or not a given material has been labelled with at least one specific taggant substance com- prising an informational content which is not divulged to the public, and the presence of which specifically establishes the identity and/or origin of the material. In this context, the expression "not divulged to the public" implies (i) that the structure of the taggant cannot be determined unless specific reagent substances are available and (ii) that even when the structure is determined it is required to convert the structural data into information by means of a code which is only known by authorized individuals.

The above objective is achieved by associating the material that is labelled as described above, with a microscopically detectable amount of cellular particles, the presence of which in or on the labelled material can be detected by simple microscopical detection means . It will be understood that the expression "associating with" implies that the cellular particles are linked or bound so firmly to the material that they will substantially not dissociate from the material up to the point in time where a sample of the material is examined for the presence or absence of the particles. The method of linking or binding the particles to the material will depend on the type of particles and the nature of the material. Possible methods of associating the particles with a material is to apply a suspension of the selected particles onto the outer surface of a solid material followed by drying, mixing the cellular particles into a non- solid composition of matter or, in the case of a liquid material, suspending the particles in the material. It will be appreciated that it is also possible to associate the particles with the material by using a suitable organic or inorganic ligand, optionally combined with a treatment which strengthens the ligand binding such as a heat treatment or a photochemical treatment.

As used herein the expression "cellular particles" refers to any particulate, at least initially viable cell structures that have a relatively consistent shape and structure, that have mechanical and physical characteristics permitting their shape and structure to be substantially retained during the process of their association with the labelled material and until the material is to be examined by the above method of detecting the presence or absence of the particles. In accordance with the invention, suitable cellular particles can be derived from a bacterial species, a fungal species including a yeast species, and a plant. Presently preferred cellular particles include bacterial spores and fungal spores. Several bacterial species have the capability to produce, under certain conditions such as nutrient limitation, so-called endospores which after release from the mother cell normally enter a long period of dormancy. In this state, the spores are generally highly resistant to heat, ultraviolet and ionizing radiation and toxic compounds. Bacterial endospores are generally spherical or ellipsoidal structures having a largest diameter which is typically in the range of 1-2 μm and hence, they are readily detectable by simple microscopy, optionally after staining using a conventional spore staining method. Bacterial species which have the ability to produce endospores include Bacillus spp. such as B . subtilis, B. megaterium, B. cereus and B. thuringiensis; Clostridium spp. including as examples C. perfringens and the group of butyric acid clostridia e.g. C. acetobutylicum and C. lactoacetophilum, Desulfotomaculum spp. including D. acetoxydans, Sporolactobacillus spp. and Thermoactinomyces spp. Other potentially useful cellular particles include desiccation-resistant bacterial exospores which are e.g. produced by methanotrophic bacteria including the genera Methylosinus and Methylobacterium.

Another type of cellular particles that can be used in accordance with the invention is fungal spores. A majority of fungal species including those belonging to the classes Ascomycetes , Basidomycetes and Fungi Imperfecti produce exospores which are pinched off at the tip of the hyphae. As for the above bacterial spores, fungal spores typically have a largest diameter in the range of 1-5 μm and they can there- fore be detected readily by microscopy.

A further type of cellular particles that can be applied in the above method is plant pollen grains. One advantageous feature of pollen is the fact that the wall of mature pollen grains is "sculptured" in a great variety of patterns. This feature implies that particular pollen grains which, in accordance with the invention, are associated with a labelled σ SD μ- SD hj TJ JD rt TJ JD TJ O a ra H g CQ hi Ω 3 tr H 0 IS JD TJ

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replicating in the cell from which the cellular particle is derived.

Significant advantages of integrating the taggant, e.g. a nucleic acid taggant, in the cellular particles is that the taggant will be protected effectively against adverse physical and chemical effects such as UV or other ionizing radiation, pH or extreme temperature conditions.

It will be appreciated that the labelled material preferably is associated with an amount of cellular particles that permits their ready detection by means of a simple light microscope. Thus, it is preferred that the number of particles associated with a material, into which cellular particles can be mixed such as liquids or flowable materials including powdery products, is at least 10³ per ml of the material. However, a higher number of particles is contemplated, e.g. in materials which during their normal handling may be contaminated by airborne cellular particles. Accordingly, in a further embodiment, the amount of cellular particle elements is at least 10⁴ per ml such as at least 10⁵ per ml including at least 10⁶ per ml.

In other embodiments where the cellular particles are associated with a solid material e.g. a physical object, it is preferred that the number of structures is at least 10² cellular particles per cm² including at least 10³ per cm² such as at least 10⁴ per cm², e.g. at least 10⁵ per cm² including at least 10⁶ per cm².

The present method of detecting whether or not a material is labelled with a specific taggant preferably implies that a sample of the material can be examined microscopically with- out complicated preceding steps. However, detection which includes that the cellular particles are at least partially dissociated from the material are also encompassed by the invention. Thus, when the material is a solid object, the associated cellular particles may be dissociated by rinsing the surface followed by microscopic inspection of the rinse liquid or by swabbing the surface and subsequently rinsing the swab and inspecting the rinse liquid. It is also possible to concentrate a sample or a rinse liquid prior to micro- scopy.

It will be understood that the choice of cellular particles may depend on the nature, structure and composition of the material. Thus, it is important that the particles are com- patible with the material, e.g. in the sense that they can be distributed substantially homogenous in or on the material and that this state of substantial homogeneous distribution is maintained up to the point in time where the material is to be examined for presence or absence of the particles. The fulfilment of that requirement may involve that the particles of choice be modified such that they can be associated appropriately with the labelled material or be homogeneously distributed herein. As an example, such a modification may involve that the surface of the particles are modified by linking moieties to the surface which render the particles more hydrophobic or more hydrophilic.

As it is mentioned above, the present method can be used irrespective of the type or structure of the informational content-carrying specific taggant substance. Thus, the method includes detection of labelling with a macromolecule including a nucleic acid, PNA and a polypeptide. In certain preferred embodiments, the taggant comprises the informational content in the form of an alphanumeric code e.g. as described in WO 96/17954 including such a taggant which is a nucleic acid comprising nucleotides different from A, dA, G, dG, C, dC, U, and T.

The method further includes embodiments where the material is labelled with at least two nucleic acid taggants including embodiments where the at least two nucleic acid taggants are comprised in one nucleic acid molecule or where one of said at least two nucleic acid taggants is in the form of nucleotide fragments which flank another of the at least two nucleic acid taggants, e.g. an embodiment where the nucleotide fragments which flank another of the at least two nucleic acid taggants serve as templates for primers in a PCR reaction which amplifies the informational content of the nucleic acid flanked by said nucleotide fragments.

In a still further embodiment, the method implies that at least one specific taggant substance is provided with a nonspecific label. Examples of such a non-specific label include a radiolabel, a fluorescent label, an enzyme and an immunologically detectable substance.

As it is mentioned above, the invention relates in a further aspect to a method of labelling a material and subsequently detecting that it has been labelled.

This method comprises that the material is labelled with at least one specific taggant substance as it is defined above and which comprises an informational content which is not divulged to the public and the presence of which specifically establishes the identity and/or origin of the material and that the material is associated with a microscopically detectable amount of cellular particles as also defined above.

In a still further aspect, the invention pertains to a material that is labelled with a specific taggant substance as defined above and associated with it, a microscopically detectable amount of cellular particles as described above, the presence of which in or on the labelled material can be detected by the above method.

The invention is further illustrated in the following example and the drawings where

Fig. 1 illustrates the construction of the replacement vector pTAGl, and Fig. 2 shows the integration of a taggant sequence into Bacillus subtilis chromosome; the cloned chromosomal DNA fragment (open box) is interrupted by the taggant sequence (filled box) .

EXAMPLE 1

Preparation of spores of Bacillus subtilis comprising a DNA taggant

Two 1 kb DNA fragments homologous to a 2 kb Bacillus subtilis chromosomal sequence were produced by PCR using primers containing the recognition sites for Xbal, EcoRI, BairiHI and KpiϊL , respectively at their 5' ends. After digesting these PCR-generated fragments and the taggant fragment with the appropriate endonucleases, the three fragments were mixed and joined by ligation. The recombining cassette comprising the 2 kb B. subtilis chromosomal sequence interrupted by the taggant sequence was amplified from the ligase reaction mixture as shown i Fig. 1. The cassette was then ligated between the Xbal and Sail restriction sites of the thermosensitive plasmid pG⁺host4 (Appligene, Illkirch, France) , to give the replacement vector pTAGl.

Following introduction of the pTAGl vector into B. subtilis, a Campbell-like single crossing-over event between the chromosome and the homologous region in pTAGl will result in plasmid integration and duplication of the homologous sequence at the vector-chromosome junction regions. Subsequent recombination between these DNA repeats will lead to excision of the plasmid. Integration through a followed by excision through b (or vice versa) will result in integration of the taggant sequence (Fig. 2) .

pTAGl was introduced into B . subtilis cells by electrotrans- formation as described by McDonald et al . , J. Appl . Bacteri- ol. 79:213-218, 1995. Electroporated cells were diluted 10- fold in 2xLB medium and incubated with shaking for 2.5 hours at 30°C and subsequently mixed with 10 ml of LB containing 10 μg of erythromycin per ml. For curing of plasmid and selection for single crossing-over integrants, the temperature was raised to 37.5°C (a temperature that is non-permissive for plasmid-directed replication) and the culture was incubated overnight to obtain a population of integrants. The culture was then diluted 1:10⁵ in LB medium without antibiotic and the temperature shifted to 28°C to stimulate homologous recombination and excision of the integrated plasmid through plasmid-directed replication. After 12-15 hours of incubation at this temperature, the cell culture was plated at various cell concentrations at 37.5°C without erythromycin selection. Colonies were transferred by use of tooth sticks to plates containing 10 μg of erythromycin per ml. Erythromycin sensi- tive colonies were analyzed for the presence of taggant sequence by PCR, using PCR primers hybridizing to the chromosomal DNA sequence on each side of the taggant. Finally, the integrated state of the taggant sequence was verified by Southern hybridization.

Spores of Bacillus subtilis clones comprising integrated taggant sequences were produced as described by Evdokimova and Panikov, Microbiology 63:454-458, 1994.

Claims

1. A method of detecting whether or not a material has been labelled with a specific taggant, the method comprising associating a material, that is labelled with at least one specific taggant substance comprising an informational content which is not divulged to the public and the presence of which specifically establishes the identity and/or origin of the material, with a microscopically detectable amount of cellular particles, the presence of which in or on the labelled material can be detected by simple microscopical detection means, collecting a sample of the material that is suspected of being labelled with the specific taggant substance and testing the sample for presence of the cellular particles,

subject to the limitation that, when the specific taggant substance is integrated in the cellular particles, it is not a substance that occurs naturally in said particles.

2. A method according to claim 1 wherein the cellular particles are derived from a bacterial species, a fungal species and a plant.

3. A method according to claim 2 wherein the cellular particles are selected from the group consisting of bacterial spores and fungal spores .

4. A method according to claim 3 wherein the bacterial spores are derived from a bacterial species selected from the group consisting of Bacillus spp., Clostridium spp., Desulfotomacu - lum spp., Sporolactobacillus spp. and Thermoactinomyces spp.

5. A method according to claim 2 wherein the cellular particles are plant pollen particles.

6. A method according to claim 1 wherein the labelled material is associated with different types of cellular particles.

7. A method according to claim 1 wherein the specific taggant substance is incorporated in the cellular particles.

8. A method according to claim 7 wherein the specific taggant substance is a nucleic acid that is chromosomally integrated.

9. A method according to claim 8 wherein the specific taggant substance is a nucleic acid that is integrated in an episomal element.

10. A method according to claim 1 wherein the material is labelled with a specific taggant substance that is not integrated in the cellular particles.

11. A method according to claim 1 wherein the labelled material is a liquid or flowable material that is associated with at least 10³ cellular particles per ml.

12. A method according to claim 1 wherein the labelled material is a solid material that is associated with at least 10² cellular particles per cm².

13. A method according to claim 1 wherein the specific taggant substance is a macromolecule selected from the group consisting of a nucleic acid, PNA and a polypeptide.

14. A method according to claim 13 wherein the informational content of the specific taggant is in the form of an alphanumeric code.

15. A method according to claim 1 wherein the specific taggant substance comprises a nucleic acid.

16. A method according to claim 15 wherein the nucleic acid of the taggant substance comprises nucleotides different from A, dA, G, dG, C, dC, U, and T.

17. A method according to claim 13 wherein the material is labelled with at least two nucleic acid taggants.

18. A method according to claim 17 wherein the at least two nucleic acid taggants are comprised in one nucleic acid molecule.

19. A method according to claim 18 wherein one of said at least two nucleic acid taggants is in the form of nucleotide fragments which flank another of the at least two nucleic acid taggants.

20. A method according to claim 19 wherein the nucleotide fragments which flank another of the at least two nucleic acid taggants serve as templates for primers in a PCR reaction which amplifies the informational content of the nucleic acid flanked by said nucleotide fragments.

21. A method according to claim 1 wherein at least one specific taggant substance is provided with a non-specific label.

22. A method according to claim 1 wherein the cellular particles are modified such that they can be associated with the labelled material.

23. A method according to claim 1 wherein the labelled material is selected from the group consisting of a non- viable material and a viable material.

24. A method according to claim 23 wherein the non-living material is selected from the group consisting of an industrial product, a work of art, an antiquity, an environmental pollutant, an air pollutant, a hydrocarbon, an aromatic compound, an explosive, a food product, a food additive, an animal feed, a medicament, an ink, a paper goods including securities such as bank notes and bonds.

25. A method according to claim 23 wherein the viable material is selected from the group consisting of a virus, a prokaryotic organism, an eukaryotic organism, a multicellular organism and a cell culture derived from a multicellular organism.

26. A method of labelling a material and subsequently detecting that it has been labelled, the method comprising the steps of (i) labelling the material with at least one specific taggant substance comprising an informational content which is not divulged to the public and the presence of which specifically establishes the identity and/or origin of the material and (ii) associating the material with a micro- scopically detectable amount of cellular particles, the presence of which in or on the labelled material can be detected by simple microscopical detection means,

subject to the limitation that when the specific taggant substance is integrated in the cellular particles, it is not a substance that occurs naturally in said particles.

27. A method according to claim 26 wherein the cellular particles are derived from a bacterial species, a fungal species and a plant.

28. A method according to claim 27 wherein the cellular particles are selected from the group consisting of bacterial spores and fungal spore.

29. A method according to claim 28 wherein the bacterial spores are derived from a bacterial species selected from the group consisting of Bacillus spp., Clostridium spp., Desulfo - toiTiaculum spp., Sporolactobacillus spp. and Thermoactinomyces spp.

30. A method according to claim 27 wherein the cellular particles are plant pollen particles.

31. A method according to claim 26 wherein the material being labelled is associated with different types of cellular particles.

32. A method according to claim 26 wherein the material being labelled is a liquid or flowable material that is associated with at least 10³ cellular particles per ml.

33. A method according to claim 26 wherein the material being labelled material is a solid material that is associated with at least 10² cellular particles per cm².

34. A material that is labelled with a specific taggant substance comprising an informational content which is not divulged to the public and the presence of which specifically establishes the identity and/or origin of the material and associated with it, a microscopically detectable amount of cellular particles, the presence of which in or on the labelled material can be detected by simple microscopical detection means.