IL139890A - Device and method for collection and initial preparation of tissue samples for molecular genetic diagnostics - Google Patents

Description

139890/2 g and Initially Preparing Tissue Samples r Genetic Diagnostics 139890/2 I Those portions of the specification which describe material beyond the scope of the allowed claims are not part of the invention.

This invention relates to a device and a method for collecting and initially preparing tissue, blood or other samples of nucleated or DNA-containing cells or cell components for molecular genetic testing. The invention also relates to the use of the device described here for the typing of animal populations.

The collection of a large number of tissue- or DNA-samples is necessary for diverse research and applied programs. Under certain circumstances information about entire farm animal populations (e.g. biologically dynamic animal production) or regionally or specially characterized animal populations must be collected.

In this way, for example, triggered by the BSE scandal and the ass'ociated problems of ensuring that meat and products originate from unaffected farms, EU-wide compulsory identification of farm animals was introduced, in which - at or shortly after birth -animals are provided with ear tags for their identification. These have an individual number which, identifies each farm animal. Thus, by checking the number, it is for example possible to determine at a later date, in the abattoir, which farm the animal comes from.

However these ear tags are not safe from forgery and can be exchanged by manipulation, so that the identification system introduced can be evaded. The consumer or. the middle man cannot ensure if the animal really comes from the farm stated.

It would therefore be desirable to have a simple analytical method available which provides for. the - independent confirmation of the information from the producers, processors and marketers.

As is well known, each animal can be individually identified by testing certain DNA variants ("genetic fingerprint"). In forensic science and in. the parentage testing of breeding animals these innovative molecular genetic tests are already being used for analysis; the sample from the animal is usually collected as a blood sample by the veterinarian and analyzed. However, for larger animal populations this is too labor-intensive and not practicable from an economic point of view.

With the help of modern detection methods (PCR, sequencing, etc) it can be determined, even from very small tissue samples, whether or not they derive from a particular individual. The test can be carried out relatively easily, with a reliability of over 99.9%.

However, at present the expense in terms of finance and labor to carry out separate, targeted collection, preservation, cataloguing, and analysis of such numbers of samples is enormous.

One function of the present invention is to provide a device with which collection of DNA-containing samples from subjects can be carried out easily and cost-effectively.

A further function of the invention is to supply a method with which tissue or blood samples can easily be collected from subjects for analytic investigation.

This function is carried out through a device for the collection and initial preparation of samples of DNA-containing cells which includes a sample receiving container and tool for collecting the sample. After collecting the sample, the sample collection tool is introduced into the sample receiving container and tightly seals it. The sample receiving container has a base and side walls and is closed with an easily penetrable lid. In an area of the side walls away from the base are means to secure the introduced sample collecting tool, and substances in the container protect against DNA-degrading enzymes. The tool to collect the samples is formed in such a way that on introduction into the sample receiving container, the means to secure this tool (provided in the sample receiving container) fasten it securely in place and the tool divides the sample receiving container into at least one sample space limited by the base and side walls of the sample receiving container and the front end of the sample collecting tool.

In addition, the aforesaid function is carried out by a procedure in which with the use of the device of the present invention a suitable sample is collected with the front end of the sample collecting tool and this is introduced into the sample receiving container, so that through the base and the side walls of the sample receiving container and the front part of the sample collecting tool a limited sample space is formed, which is closed to the environment.

The invention will now be explained with the help of the accompanying diagrams, in which: Fig. 1 shows a cross-section of a model of the sample receiving container 1, which has a projection 8 on its base 2. The sample collection tool 4 is in the sample receiving container 1, and tightly seals this. On the side of the sample collection tool 4, away from the sample space 6,6a is a hollow 12 to allow for the insertion of a rod.

Fig. 2 shows a cross-section of an arrangement of spike plate 10 with spike 10a, aperture plate 1 1 with sample receiving container 1 with attached tongue 9. The sample receiving container 1 is closed with the sample collection tool 4, depicting the situation after application of an ear tag 10,1 1 and simultaneous collection of a sample.

Fig. 3 shows a side view of an ear tag 10,1 1 consisting of spike plate 10 and aperture plate 1 1, and a sample receiving container 1 complete with tongue 9, before being put together using a suitable device.

Fig. 4 shows a diagram of individual DNA typing in cattle.

The invention will now be described in more detail using the preferred model and with reference to the figures.

The invented device contains a sample receiving container 1 and sample collection tool The sample receiving container 1 has a base 2 and side walls 3, and is closed with an easily penetrable lid such as a film or membrane. The sample receiving container 1 can itself be subdivided by additional membranes, so that two or more components can be separated from each other in the sample receiving container 1 until use, and only on entry of the sample collection tool 4 into the container 1 come into contact with each other and with the sample. The sample receiving container 1 can furthermore be subdivided by a separating wall, such as a projection 8 stretching over the whole diameter of the base 2, into at least two chambers, so that on sample collection at least two completely separate samples can be collected.

In a preferred model the sample receiving container 1 receives at least the front end of the sample collection tool 4. If necessary it s base 2 can be formed so that it has a tapered projection 8; the sample collection tool 4 appropriately fits together with the projection 8. As a result, on entry of the sample collection tool 4 into the sample receiving container 1 the sample is sirongly crushed and pressed into the sample space 6, 6a limited by the base 2 and the side walls 3 of the sample receiving container 1 as well as the front part 7 of the sample collection tool 4.

The sample receiving container 1 can also possess a holding device 9 such as a flat attachment, for example a tongue with a hole to fasten this appropriately. Thus, for example, the tongue can be fastened to the pliers when pliers are being used to apply the ear tag 10,1 1 . The, tongue is also suitable for taking the identification number, which is the same as that of the ear tag. Labeling can take place by hand in the grooves provided, or the tongue can be pre-labeled e.g. imprinted, or can be printed at the same time and identical to the identification number applied onto the plate part of the ear tag 10,1 1 .

If necessary (e.g. for further processing of the samples by hand) the sample receiving container 1 can also be so formed that the area of the sample receiving container 1 which defines the sample space 6, 6a after closure with the sample collection tool 4, is connected by a screw thread to the body of the sample receiving container 1 and thus can easily be unscrewed, enabling easier access to the sample.

In a preferred model of the device the sample receiving container 1 is closed by the sample collection tool 4 in such a way that opening the sample space 6,6a formed is not possible without destroying the device. Thus it is certain that manipulation of the collected sample is not possible without it being noticed.

In the sample receiving container 1 are substances to inactivate the protein component of the tissue sample and stabilize the DNA.

The substances to inactivate the proteins (enzymes, DNAses, etc.) of the tissue sample and to stabilize the DNA can for example be selected from the group consisting of: Proteinase K (e.g. lyophilized for stabilization during storage) and (separately filled) buffer to digest proteins; Strong bases; Molecular sieve (e.g. E. Merck 0.2 nm No. 1.05704.0250, K 230045904 624, water absorption capacity > 20%), which is extremely hygroscopic and dries out the tissue sample on contact with it (and warms it) and thereby inactivates. To protect the molecular sieve from undesired uptake of moisture from the air it can, for example, be coated with the inert gas argon and closed with a film; Other components, which support the inactivation of the protein component and the stabilization of the DNA. The substances are so "formulated that they remain active for a long period of time, for example up to one year or longer, and after introduction of the sample ensure sufficient integrity of the DNA for analytic investigation for at least many months up to one year.

The sample collection tool 4 can take any form with which the sample can be collected and introduced into the sample receiving container 1, in which the sample collection tool 4 seals closed the sample receiving container 1 after introduction. This includes the form of cylinders, cones etc.

In a preferred model of the device the sample collection tool 4 consists of two parts, which before use are connected (but separable) to each other and with use separate from each other. This can for example be realized by a narrow bridge of plastic with a specified breaking point. The sample collection tool 4 can be solid or can at its rear side allow for the insertion of a rod, for example with a central hollow 12, into which a steel rod from pliers can be introduced for stabilization on use.

The sample collection tool 4 carries out several functions. The sample is collected with it, for example by simply immersing it in body fluids such as blood, lymph or urine or by scratching or punching out tissue from the subject.

With a preferred model, the ear of the subject is pierced with the invented device; the front end of the sample collection tool 4 can be formed with a sharp edge so that by pushing through the ear a small tissue sample is punched/crushed out.

In a further preferred model of the device the sample collection tool for the collection of the sample with a suitable instrument, such as ear tagging pliers, is applied at the same time as an ear tag is pressed through the ear of a subject, so that the application of the ear tag and the collection of the sample takes place in one working procedure.

The ear tags used for identification usually consist of two parts, a plate carrying a spike 10 (spike plate) and an aperture plate 11, which has about the same size as the spike plate 10 or can consist of a smaller plate, which only has to be large enough to prevent the spike 10a from sliding out to the ear. Both parts are made from tissue compatible synthetic material suitable for use with food (e.g. polyurethane - Desmopan 795 U from Bayer) or in part from metal (e.g. stainless steel, brass, bronze or such like).

To insert the ear tag 10,11 commercial ear tagging pliers can be used. Most of the pliers available for this purpose can if necessary very easily be modified by the addition of a small extra part, so that the sample receiving container 1 remains hanging on the pliers after insertion of the ear tag 10,1 1 and can therefore easily be collected. The extra part is for example a small knob over which the hole in the tongue 9 with the sample receiving container 1 is drawn. After insertion of the ear tag 10,11 in the ear, if necessary the ear tag 10,1 1 is abruptly withdrawn from the hold in the pliers and the knob holds the tongue 9 with the sample receiving container 1 firmly on the pliers. Subsequently the tongue can easily be drawn over the knob and thus the sample receiving container 1 can be collected.

The sample collection tool 4 tightly closes the sample receiving container 1 on introduction, and is fixed in place by fixation means 5, so that escape of sample material and entry of foreign material is prevented.

The sample receiving container 1 as well as the sample collection tool 4 can be made from any suitable material, such as for example metal or from glass-fiber reinforced synthetic material. If the sample collection tool 4 is made from synthetic material it can be reinforced during insertion in the ear by a metal rod in the pliers used .

With the invented device it is possible to take samples from animals at the same time as the identification marking with ear tags 10,11 which is routinely performed, without much additional effort or expense. The savings resulting from this are very substantial.

A further advantage of the invented device compared with conventional sample receiving containers is the holding device 9 on the sample receiving container, which has the form of a flat tongue and can be used label/identify the sample. Usually labeling has to take place" by hand on the mostly round surface of the mostly cylindrical containers (e.g. Eppendorf tubes) with considerable effort and results which can be hard to read. It is therefore difficult to scan in numbers or data and automate collection.

The invented system is of particular advantage when samples have to be taken from material which is still part of a whole structure, so that a sample would normally have to be taken with an instrument (knife, sharp spoon, scalpel etc). With the use of reusable instruments the danger of contamination is very high, as with very sensitive tests such as PC (polymerase chain reaction) even single cells can lead to contamination and thereby to false positive results. With the use of the invented device only single use parts come into contact with the sample material.

The invented device can also, for example, be used to reliably and securely collect and process fluid samples (blood, urine, saliva and others). In this case the fluid is applied to either the opening of the sample receiving container 1 or onto the front part of the sample collection tool 4 and subsequently, for example with a suitable device such as pliers, pressed into the sample receiving container 1 . Samples from surfaces (skin, mucous membranes etc.) can also be collected, cleanly, reliably and with secure identification using the invented device in that the front part of the sample collection tool 4 is used as a "sharp spoon" and briefly drawn/scraped over the surface.

A substantial advantage of the invention is that the sample receiving container 1 can be labeled at the same time as the ear tags (spike plate 10, aperture plate 1 1 ) - thereby unmistakably having the same numbering fi before distribution to the users or owners of the animals. The labeling can, for example, be carried out with a printer, with which in the case of plastic ear tags a (black) masfermix is apblied which binds so strongly with the synthetic material that it cannot be wiped off or removed with normal use. Furthermore, the corresponding parts can have an individual number printed into them.

The samples collected with the invented device are then brought to a central collecting location and analyzed there. The collection of the sample receiving container 1 can take place without special consideration to temperature and duration of transport. The sample receiving containers 1 can be transported by post, courier, or sample collection to the laboratory or to storage, problem-free and risk-free.

The work up of the samples in the laboratory ή taking an aliquot, test reactions and analysis ή can be automated with a robot controlled system. The identification number of the samples is registered and further processed by a reading device (scanner). By avoiding the entry of data by hand the quota of mistakes can be kept negligibly low.

To process or analyze the DNA from the samples collected with the invented devices, the sample receiving containers 1 are put onto a conveyor belt so that a scanner can record the identity of the sample. Subsequently a canula is inserted through the floor of the sample space 6,6a and fluid is injected for the uptake of the sample and then an aliquot is taken. The point of insertion can then be sealed again so that the sample receiving container can be stored. If the sample space 6,6a was divided in a suitable way to begin with, then there are further samples available for analysis in the invented device.

DNA isolation can be automated through the use of a pipetting robot (Biomec2000, Beckman) and the DNA purification using silicon particles (e.g. InstaGene Matrix, BIO-RAD). Test reactions with these samples can also be prepared by automation.

The system described here for the easy collection of samples and automated analysis has the following advantages: Forgery-free proof of origin and identity for all cattle and meat from these cattle, in all stages of processing up to consumption and thereby proof of origin from BSE free area's. Enables control and with this stabilization of targeted sales programs such as animals kept on organic farms or kept on national parks or with special grade identification. Verification and monitoring of the transport routes and times and distances for all cattle transport. Efficient border control and follow up of cattle within the EU and on international export. Unmistakable identification of all breeding cattle sold with confirmation of identity possible at any time e.g. at auctions, exhibitions etc. Complete proof of origin for all cattle born and detection of mistaken or falsified papers. Completely reliable forensic evidence (also from already consumed beef from the stomach contents by the medical examiner). Completely reliable monitoring of (culling) and epidemic control. Determination of the mix of beef in all (processed) foodstuffs tested, including in pet food. Detection of producers from abattoirs where in the course of random testing treatment with banned hormones or growth promoters have been carried out. Proof of origin for other products from cattle production such as hides, bones, horns, hooves, organ preparations, blood etc. Proof and testing of the animal and stock origin with directly sold milk and milk products. Exact registration of all cattle herds and routes of sale. Samples for detailed analysis of DNA polymorphisms, measurements of genetic distance and genetic screening programs would be available.

Optimization of breeding programs. Measurement of genetic distance. Investigation of genealogical and breeding history of animal breeds. Analyses as part of marker assisted selection programs (MAS). Exact evidence of the presence and frequency of particular genetic defects. From a forensic point of view the device described here and the procedure described here has the further advantage that the collected sample cannot be changed (exchanged, falsified) without the necessary manipulation of the sample receiving container being recognizable.

By collecting and cataloguing the samples taken further typing, testing of origin, genetic defect screening, population analyses, mutation detection, as well as screening of foodstuffs, pathogen diagnostics, transgenic diagnostics, monitoring of hygiene in food processing operations etc are possible without great expense.

With the system described here testing the genetic information, which is in all animals and all products made from animals until consumption, is possible. With the device described here the costs normally associated with this are reduced manifold.

Even in intensively processed food of animal origin (sausages, processed meat, "Leberkas" (finely ground meat with spices and other ingredients, to be baked in the oven or available ready-baked), schnitzel etc) species and individual specific DNA can be established. It is thereby possible to detect contamination of food with foreign meat components. Of course it is also possible with the help of this method to prove beyond doubt that a particular foodstuff derives from a particular animal - as stated - if on the original farm a suitable (first) sample was taken (fig.3).

For this an ear tissue sample must be taken from every new-born farm animal for DNA typing and transported to the typing center. There using (automated) PCR DNA fingerprints from microsatellite primers are made and an unmistakable pattern for each animal is registered.

These data are saved in a central file and made accessible through. A fingerprint from a routine test i.e. on sending in a second sample after DNA typing on comparison with the fingerprints in the first analysis the matching animal can be found. This result is normally available within one day. In extreme cases e.g. in the transport of animals or border control an express result can be available within three hours. The number of the animal i.e. the DNA fingerprint and all other data can be called up for every animal registered. The owner of the animal i.e. all the animals sold by one owner or from one herd can be pooled. The parents of the animal i.e. after several years of registration of all cattle born the parentage and also the number of offspring of each parent can be looked up via this file. Individual typing of a complete population of farm animals is novel. Every consumer, guest, customer, dealer, butcher, processor, owner or inspector can check on the identity and thereby the origin of an animal or animal product (fig. 4). For this test it is merely necessary to take a second sample, for example from the abattoir, the supermarket or even from the already prepared meal (for example in a restaurant from a schnitzel already on the table).

With live animals e.g*. in checking that animals have been transported in an approved manner (distance) typing would similarly be possible. This degree of certainty for the test results will, with a sufficiently high testing frequency, automatically lead to fraud being reduced to a minimum due to fear of reliable and legally incontestable evidence and risk of detection.

All orders which may be made in the future to monitor particular groups of animals, or herds for reasons of risk prevention, epidemic control or testing for illegal residues (hormones, growth promoters etc.) in animals, could be immediately undertaken using the DNA data already collected.

If individual typing with the system described here is consistently carried out in a country, one will at all times be in the position to immediately, i.e. on the same day, react to problems which arise (e.g. suspicion of BSE in an animal, outbreak of swine fever on a farm etc.) thereby providing for the consumer unparalleled protection and unparalleled testing possibilities for monitoring.

In particular it would be possible, for example, through individual typing to completely and beyond doubt prove that animals and products from "alternative" production really derive from there. That means that every customer could arrange testing of the information accompanying the meat, even after lengthy transport and sale of the meat, by sending in samples (second sample).

The invention will now be explained in more detail using examples, which are not to limit the invention described in the claims.

Example 1 Individual typing in cattle A cow has been marked with a conventional ear tag. On the spike of the spike plate the sample collection tool was attached, which has the form of a cone with the point at the front and a cylindrical base and a cavity in the* base for accepting the spike of the spike plate.

On the pliers the sample receiving container, made in one piece with the tongue, was positioned under the position provided for the aperture plate in such a way that the lid of the sample receiving container fi closed with a lid of synthetic film n was lined up with the hole of the aperture plate.

In a location away from the sample receiving container the tongue had a hole for securing it in place on the pliers.

By pressing the spike provided with the sample collection tool of the spike plate, through the ear of the cow, the sample of the ear punched out was pressed into the sample receiving container. After entry the sample collection tool was fixed in place by the projections on the inside walls of the sample receiving container, whereby the sample space formed was sealed closed.

In the sample receiving container was a molecular sieve (Merck, 0.2nm Nr. 1.05704.0250), which protects the DNA of the collected sample from degradation.

DNA footprints and PC analyses of known genes could be undertaken problem free on the genetic material present in the sample receiving container after half a year.

Example 2 Screen-out of functional mutants for the breeding of special lines (e.g. of 1.3Cal/Gal3 negative pigs for xenotransplantation).

Due to the natural mutation rate, in a population of sufficient size in single individuals every gene present in the genome contains not just various silent mutations, but rather with a certain probability also mutations which result in an inactivation of the allele. In the majority of these cases this involves recessive rather than dominant mutations. According to the Hardy-Weinberg law the predominant number of such chafiges in genotype caused by mutations is masked by heterozygote individuals.

In a screening procedure it is critical to discover using suitable molecular genetic analyses of the appropriate locus in the available pig population, a (single) heterozygous carrier animal which has the mutation. Then a homozygous negative line can be built up with this animal, which has the same desired and needed gene defect as a line generated through gene knock-out. It would in no way be inferior to this.

The suggested approach represents to a certain degree the screening of a stem cell line after recombination with a suitable construct which contains no marker. The difference is the following: all cells of a stem cell line have the same genotype with the exception of those, which have a change or recombination after the single mutation event. In the screening of pig populations all the analyzed genotypes are different. They have not be mutagen ized in a targeted way but carry mutations, which in the course of evolution and work of breeders have accumulated (as long as in the heterozygote state they were under no negative selection pressure).

It is known that there are quantitative differences in the expression of 1 .3Gal/Gal3. As the mutation analysis is aimed at finding already present mutations in diverse populations, breeding animals will be analyzed. If one ignores new mutations, then production pigs can only carry mutations which were already present in the breeding parents. For practical completion the preservation and collection of tissue samples with the Typi-fix system is a critical factor for success or financial realization. In the analysis to find mutation carriers it must be assumed that possibly up to 100,000 genotypes will have to be tested in order to find potential animals with a 1.3Gal/Gal3 deficiency. For the costly collection of individual samples from 100,000 pigs the invented system is the method of choice. The samples need only to be transported into the laboratory (by mail or sample collection) and further worked up there.

Claims (17)

139890/2 15 Claims

1. A device for collecting and initially preparing samples comprising DNA containing cells from an animal, comprising a sample receiving container (1), and sample collecting means (4), which means enters the sample receiving container after collecting the sample and tightly seals it, wherein the sample receiving container (1) has a base (2) and side walls (3), has on the container's side walls, in an area removed from the base, means (5) for securing the introduced sample collecting means, and is provided with an easily penetrable lid, and provided in the container are means to protect from DNA-degrating enzymes, and wherein the sample collecting means (4) is made in such a way that in one step the sample is collected and introduced into the sample receiving container, and that it on entry into the sample receiving container is fixed in place by the means for securing (5) and divides the sample receiving container into at least one sample space (6, 6a), which is limited by the base (2) and the side walls (3) of the sample receiving container (1) and the front end of the sample collecting means (7), and wherein the sample receiving container (1) is associated with an ear tag, which is provided with a means of verifying identify and comprising an apertures plate (11) and a spike plate (10), and which may be used for the identification of an animal, wherein the sample collection container (1) is releaseable connected with the aperture plate (11) of the ear tag and is provided with the same means of verifying identify as the ear tag, and wherein the sample collecting means (4) is releasable connected with the spike plate (10) of the ear tag.

2. The device according to claim 1, characterized in that the sample collecting means (4) is placed on the spike (10a) of the spike plate.

3. The device according to any of the claims 1 or 2, characterized in that the tag is an identification number.

4. The device according to any of the preceding claims, characterized in that the sample collecting means (4) has at least one sharp edge at the end facing the base of the sample receiving container.

5. The device according to any of the preceding claims, characterized in that the sample collecting means (4) tapers at the end facing the base of the sample receiving container. 139890/2 16

6. The device according to any of the preceding claims, wherein the base (2) of the sample receiving container (1) has a projection (8) into the container's internal space and the sample collecting means (4) on its end (7) facing the base of the sample receiving container is suited to accept the projection, so that the sample material introduced by the sample collecting means (4) is crushed between the projection (8) and the sample collection means (4).

7. The device according to claim 6, wherein the projection (8) is connected in such a way with the side walls (3) of the sample receiving container (1), that after introduction of a suitable sample collecting means (4) the sample receiving container (1) is divided into 2 sample spaces (6, 6a).

8. The device according to claim 6 or 7, wherein the projection (8) comprises a cone form.

9. The device according to any of the claims 6 to 8, wherein the projection (8) is a wall dividing the sample receiving container into 2 areas.

10. The device according to any of the preceding claims, wherein the sample receiving container (1) is attached to a holding device (9).

11. 1 1. The device according to claim 10, wherein the holding device (9) is a tongue.

12. The device according to any of the preceding claims, wherein the sample collecting means is formed at the end opposite to the base of the sample receiving container such as to allow for the insertion of a rod at its rear side.

13. The device according to any of the preceding claims, wherein the means protecting from DNA degrading enzyme is alkali, proteinase K or a molecular sieve.

14. The device according to claim 13, wherein the proteinase K is separated from a suitable buffer by a membrane, which is destroyed on entry of the sample-collecting means, so that the proteinase K is brought into contact with the buffer.

15. A process for collecting a sample comprising DNA-containing cells from an animal by means of a device according to any of the claims 1 to 14, comprising a sample receiving container (1) and a sample collecting means (4), wherein in one step and at the same time as performing the ear tagging of the animal with an ear tag, with the end of the sample collecting means (7) facing the base of the sample receiving container a suitable sample is collected by pressing the sample collecting means (4) with an appropriate device through the ear of an animal and introducing it into the sample receiving container (1) and securing it there, such that a sample space (6, 6a) is formed, limited by the base (2) and the side walls (3) of the sample receiving container (1) and the end (7) of the 139890/2 17 sample collecting means (4) facing the base of the sample receiving container, which is tightly sealed from the environment, and wherein, for identifying the animal, the sample receiving container (1) is provided with the same tag as the ear tag.

16. Use of a device according to any of the claims 1 to 14 in a process for typing animal populations, wherein samples are collected with the device and are analytically examined and classified in a laboratory.

17. Use of a device according to any of the claims 1 to 14 for the collection of a sample comprising DNA containing cells from an animal. Jabotinsky Street Ramat Gan 52520