Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K11/00—Marking of animals
  • A01K11/001—Ear-tags
  • A01K11/003—Ear-tags with means for taking tissue samples, e.g. for DNA analysis
• • A—HUMAN NECESSITIES
  • A22—BUTCHERING; MEAT TREATMENT; PROCESSING POULTRY OR FISH
  • A22B—SLAUGHTERING
  • A22B5/00—Accessories for use during or after slaughtering
  • A22B5/0064—Accessories for use during or after slaughtering for classifying or grading carcasses; for measuring back fat
• • A—HUMAN NECESSITIES
  • A22—BUTCHERING; MEAT TREATMENT; PROCESSING POULTRY OR FISH
  • A22B—SLAUGHTERING
  • A22B5/00—Accessories for use during or after slaughtering
  • A22B5/20—Splitting instruments
  • A22B5/201—Removing the spinal cord or other nervous tissues
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
  • G01N1/08—Devices for withdrawing samples in the solid state, e.g. by cutting involving an extracting tool, e.g. core bit
• • 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
  • G01N33/02—Food
  • G01N33/12—Meat; Fish

Definitions

• the present invention relates to a device and a method for the extraction and first preparation of tissue / blood or other samples with nucleus or DNA-containing cells or cell components for molecular genetic analysis.
• the invention further relates to the use of the device described here for typing animal populations.
• tissue or DNA samples must be obtained for various research and application programs. Under certain circumstances, information must be obtained about entire (farm animal) populations or regional or specially characterized animal populations (e.g. biodynamic animal production).
• these ear tags are not tamper-proof and can be exchanged by manipulation, so that the implemented system of labeling can be avoided. The consumer or middleman cannot therefore ensure whether the animal really comes from the specified company.
• each subject can be identified as an individual by examining certain DNA variants (“genetic fingerprint”).
• genetic fingerprint DNA variants
• forensics for example the determination of a criminal, and in the descent of
• a sample of the subject is usually obtained from the blood by the doctor and taken for analysis.
• this is too expensive for larger populations, especially in the case of large animal populations, and cannot be carried out from an economic point of view in terms of the associated costs.
• Another object of the invention is to provide a method with which tissue and / or blood samples can be obtained from subjects in a simple manner and fed to an analytical method.
• a device for the recovery and initial preparation of samples containing DNA-containing cells which comprises a sample storage container and means for collecting the sample, which is inserted into the sample storage container after sealing of the sample and seals it, the sample storage container has a base part and side walls, is closed with an easily penetrable lid and has means for fixing the introduced sample collection agent in a region of the container side walls which is remote from the base, means for protecting against DNA-degrading enzymes being provided in the container, the means for Winning the sample is designed such that it is fixed in place when inserted into the sample receptacle with the means for fixing provided on the sample receptacle and divides the sample receptacle into at least one sample space that passes through the bottom and the Side walls of the sample container and the front end of the sample collection agent is limited.
• the above object is further achieved by a method in which, using the device according to the invention, a suitable sample is obtained with the front end of the sample collection means and this is introduced into the sample receiving container, so that a through the bottom and the side walls of the sample receiving container and the is formed in the front part of the sample collection means limited sample space, which is sealed off from the environment.
• Fig. 1 shows a cross section of an embodiment of the sample container 1, in which a projection 8 is formed on the bottom 2.
• the sample collection means 4 is located in the sample receiving container 1 and seals it.
• a recess 12 is formed for receiving a pin.
• Fig. 2 shows a cross section of an arrangement of mandrel plate 10 with mandrel 10a, perforated plate 11 with a sample receiving container 1 formed with a tab 9, which is closed with the sample collection means 4, as it appears after attaching an ear tag 10, 11 and simultaneously obtaining a sample .
• FIG 3 shows a side view of an ear tag 10, 11 consisting of a mandrel plate 10 and a perforated plate 11 and a sample receptacle 1 provided with a tab 9 before being brought together by a suitable device.
• Fig. 4 shows schematically a DNA individual typing in cattle.
• the device according to the invention contains a sample receptacle 1 and a sample extraction means 2.
• the sample container 1 has a bottom 2 and side walls 3 and is closed with an easily penetrable cover, such as a film or a membrane.
• the sample receptacle 1 can itself be subdivided by further membranes, so that two or more components are present in the sample receptacle 1 separately from one another until use and only come into contact with one another and with the sample by introducing the sample recovery agent 4 into the container 1.
• the sample receptacle 1 can also be divided into at least two areas by a partition, such as a projection 8 extending over the entire diameter of the bottom 2, so that at least two separate samples can be obtained when a sample is obtained.
• the sample receptacle 1 receives at least the front end of the sample collection means 4. It can be designed in the bottom area 2 such that a projection 8, which may have a conical shape, is present, the sample-collecting means 4 being adapted accordingly for receiving the projection 8. This leads to the fact that when the sample obtaining means 4 is introduced into the sample receiving container 1, the sample is very much crushed and crushed by pressing the two parts together and into the sample through the bottom 2 and the side walls 3 of the sample receiving container 1 and the front area 7 of the sample obtaining means 4 limited sample space 6, 6a is pressed.
• the sample receptacle 1 can also have a holding device 9, such as a flat part, for example a tab with a hole, in order to fasten it accordingly.
• a holding device 9 such as a flat part, for example a tab with a hole
• the tab can be attached to it.
• the tab is also suitable for holding the identification number that matches that of the ear tag.
• the labeling can be done by hand, entered in the pre-formed grooves provided for this purpose or by pre-labeling, e.g. Imprinting, or with a printer at the same time and identical to the application of the identification number on a plate part of the ear tag 10, 11.
• the sample receptacle 1 can optionally (for example, for further processing of the samples by hand) also be designed such that the area of the sample receptacle 1, which defines the sample space 6,6a after sealing with the sample extraction means 4, with the body of the sample receptacle 1 by a thread is connected and can therefore be easily unscrewed so that easy access to the sample is made possible.
• the sample receptacle 1 is closed with the sample extraction means 4 such that it is not possible to open the sample space 6, 6a formed without destroying the device. This can ensure that the sample once manipulated cannot be manipulated without being noticed.
• the sample receiving container 1 contains material for inactivating the protein portion of the tissue sample and for stabilizing the DNA.
• the material for inactivating proteins (enzymes, DNAsen etc.) of the tissue sample and for stabilizing the DNA can, for example, be selected from the group consisting of:
• Proteinase K e.g. for stabilization during the storage period lyophilized
• Air can e.g. covered with the noble gas argon and covered with a foil;
• the material is formulated in such a way that it remains active for a long period of time, for example up to 1 year or longer, and after the introduction of the sample allows sufficient integrity of the DNA for an analytical examination at least for several months to one year.
• the sample obtaining means 4 can take any form with which the sample can be obtained and introduced into the sample receiving container 1, the sample obtaining means 4 sealingly closing the sample receiving container 1 after insertion. This includes the shape of cylinders, cones, etc.
• the sample collection means 4 consists of two parts which are (detachably) connected to one another before use and which are in contact with one another Use separate from each other. This can be achieved, for example, via a narrow plastic bridge designed as a predetermined breaking point.
• the sample collection means 4 can be solid or have at its rear end a device for receiving a pin, for example a central cavity 12, into which a steel pin of pliers is inserted for stabilization when in use.
• the sample collection means 4 fulfills several tasks.
• the sample is obtained with it, for example by simply immersing it in body fluids, such as blood, lymph or urine, or scraping and sticking tissue out of or from the subject.
• the device according to the invention is used, for example, to remove tissue from the skin of a human, for example a tissue suspected of melanoma.
• the ear of a subject in particular that of an animal, can be pierced, the front end of the sample collection means 4 being able to be sharp-edged, so that a small tissue sample is punched / squeezed out when the ear penetrates.
• the sample collection means for pressing the sample is pressed through the ear of a subject simultaneously with an ear tag using a suitable device, such as ear pliers, so that the application of the ear tag and the collection of the sample are carried out in one operation.
• the ear tag used for the marking generally consists of two parts, a plate 10 which supports the mandrel (mandrel plate) and a perforated plate 11 which is approximately the same size as the mandrel plate 10 or can also consist of a smaller disk which is only large must be enough to prevent the dome 10a from slipping out of the ear.
• Both parts are made of food-grade and tissue-compatible plastic (e.g. polyurethane - Desmopan 795 U from Bayer) or partly made of metal (e.g. stainless steel, brass, bronze, etc.).
• a commercially available ear tag pliers can be used to pull in the ear tags 10, 11. Most of the pliers available for this purpose can, if necessary, be converted very simply by a small additional part, so that the sample receptacle 1 gets stuck on the pliers after the ear tag 10, 11 has been pulled in and can be easily absorbed.
• the additional part is, for example, a small knob over which the hole opening of the tab 9 with the sample container 1 is drawn. After pulling the ear tag 10, 11 into the ear and, if necessary, jerkily pulling the ear tag 10, 11 out of the holders of the pliers, the knob holds the tab 9 with the sample receptacle 1 on the pliers. The tab can then be pulled very easily over the knob and thus the sample container 1 can be collected.
• the sample extraction means 4 seals the sample receiving container 1 by insertion and is fixed in place by fastening means 5, so that leakage of sample material and penetration of foreign material is prevented.
• the sample receptacle 1 and the sample extraction means 4 can be made of any suitable material, such as metal or possibly glass fiber reinforced plastic. If the sample collection means 4 is made of plastic, it can be reinforced when drawn into the ear by an internal metal pin on the pliers used.
• the holding device 9 which adjoins the sample-holding container and which takes the form of a flat tab and which can be used to label / mark the sample.
• the labeling on the mostly round surfaces of mostly cylindrical vessels e.g. Eppendorf tubes
• the labeling on the mostly round surfaces of mostly cylindrical vessels has to be applied more or less with difficulty and possibly difficult to read by hand. Reading these numbers or data and automated removal is difficult to implement.
• the system according to the invention is particularly advantageous when samples have to be obtained from material that is still in a composite, so that a sample normally has to be taken with a device (knife, sharp spoon, scalpel, etc.).
• a device usually has to be taken with a device.
• the risk of contamination is very high, since with the very sensitive analytical measures such as PCR (polymerase chain reaction) can contaminate individual cells and thus lead to false positive results.
• PCR polymerase chain reaction
• the device according to the invention can also be used to reliably and safely record and process liquid samples (blood, urine, saliva, etc.), for example.
• the liquid is applied either to the opening of the sample receptacle 1 or to the front area of the sample extraction means 4 and then pressed into the sample receptacle 1 using a suitable device such as a pair of pliers, for example.
• Samples of surfaces can also be obtained cleanly, reliably and with reliable identification using the device according to the invention by using the front area of the sample collection means 4 as a "sharp spoon" and pulling / scraping briefly over the surface.
• sample receptacle 1 can be labeled with the ear tags (mandrel plate 10, perforated plate 11) at the same time before being distributed to the users or animal owners, and thus unmistakably bear the same numbering.
• the label can e.g. done with a printer with which, in the case of a plastic ear tag, a (black) master mix is applied, which bonds so stably to the plastic that it cannot be blurred or removed during normal use.
• the corresponding parts can have an individual number stamp.
• the samples obtained with the device according to the invention are then brought to a central collection point and analyzed there.
• the collection of the sample receptacle 1 can take place without any special requirements with regard to temperature and duration of the transport.
• the sample receptacle 1 can be transported to the laboratory or archive by post, courier or collective action without any problems and without risk.
• the processing of the samples in the laboratory - taking an aliquot, analysis reaction and evaluation - can be done fully automatically with robot-controlled systems.
• the identification number of the samples is recorded and processed by a reader. By avoiding manual data entry, the error rate can be kept negligibly low.
• the sample receptacles 1 are placed in a conveyor belt in such a way that a reader can record the identity of the sample. Then a cannula is inserted through the bottom of the sample chamber 6, 6a, liquid is injected to take up the sample, and an aliquot is then removed. The puncture site can then be resealed so that the sample container can be archived. If the sample space 6, 6a has already been subdivided beforehand in a suitable manner, there are further samples that can be used for analysis in the device according to the invention.
• DNA isolation can be automated by using a pipetting robot (e.g. Biomec 2000, Beckman) and DNA cleaning using silicone particles (e.g. InstaGene ® Matrix, BIO-RAD). The analysis reactions with these samples can also be created automatically.
• a pipetting robot e.g. Biomec 2000, Beckman
• silicone particles e.g. InstaGene ® Matrix, BIO-RAD.
• the device and the method described here have the further advantage that once a sample has been packed it can no longer be changed (exchanged, falsified) without the necessary manipulation of the sample receptacle being recognizable.
• an ear tissue sample for DNA typing must be obtained from each born animal and transmitted to the typing center. There, (automated) PCR DNA fingerprints with microsatellite primers are created and thus an unmistakable pattern is recorded for each individual animal.
• This data is stored in a central file and can be accessed via:
• the fingerprint that occurred during a check-up i.e. if a "second sample” is sent in, the corresponding animal can be found after the DNA typing by comparison with the fingerprints from the "initial examination". This result can usually be provided within a day. In extreme cases e.g. for animal transports or border controls, express information can be given within three hours.
• the animal number i.e. the DNA fingerprint and all other data can be called up for each recorded animal
• the pet owner i.e. all animals marketed by an owner or herd can be pooled
• the parents of the animal i.e. After recording all born cattle for several years, the file can automatically check parenthood and the number of offspring per parent.
• a cattle was marked with conventional rakes.
• the sample collection agent was placed, which was in the form of a cone tapering towards the front end with a cylindrical base part and a recess in the base part for receiving the dome of the mandrel plate.
• the sample receptacle formed in one piece with a tab was also arranged under the position provided for the perforated plate in such a way that the lid of the sample container made of a plastic film was aligned with the hole in the perforated plate.
• the tab had a through hole at a location remote from the location of the sample container for the positionally stable attachment of the tab to the pliers.
• the tissue sample punched out of the ear was pressed into the sample receptacle by pushing the dome of the mandrel plate provided with the sample extraction agent through the ear of the cow. After insertion, the sample collection agent was fixed on the inner walls of the projections provided in the sample receiving container, the sample space formed being sealed off.
• a molecular sieve (Merck 0.2 ⁇ m No. 1.05704.0250) was located in the sample container, which protects the DNA of the tissue sample taken from degradation.
• each gene present in the genome due to the known natural mutation rate, contains not only different silent mutations in individual individuals, but also, with a certain probability, mutations that result in inactivity of the allele. In the majority of these cases, these are not dominant but recessive mutations. According to the Hardy-Weinberg rule, the majority of these mutation-related changes in the genotype of heterozygous individuals are masked.
• the proposed procedure corresponds to a degree to screening a stem cell line after recombination with a suitable construct that does not contain a selective marker.
• the difference is as follows: All cells of a stem cell line have the same genotype with the exception of those that show a change or recombination after the one-time mutation event.
• all genotypes analyzed are different. They have not been specifically mutagenized, but they do have mutations that have accumulated in the course of evolution and breeding (provided that they were not exposed to negative selective pressure in the heterozygous state).

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Citations (7)

• 1998
  • 1998-05-25 UA UA2000116630A patent/UA66854C2/uk unknown
  • 1998-05-25 YU YU73700A patent/YU73700A/sh unknown
  • 1998-05-25 PL PL98344256A patent/PL187288B1/pl unknown
  • 1998-05-25 DK DK98928317T patent/DK1088212T3/da active
  • 1998-05-25 CN CNB988141647A patent/CN1196920C/zh not_active Expired - Fee Related
  • 1998-05-25 WO PCT/EP1998/003075 patent/WO1999061882A1/de not_active Ceased
  • 1998-05-25 EP EP98928317A patent/EP1088212B1/de not_active Expired - Lifetime
  • 1998-05-25 NZ NZ507811A patent/NZ507811A/xx not_active IP Right Cessation
  • 1998-05-25 HU HU0101861A patent/HUP0101861A3/hu unknown
  • 1998-05-25 KR KR1020007013183A patent/KR100586106B1/ko not_active Expired - Lifetime
  • 1998-05-25 DE DE59802771T patent/DE59802771D1/de not_active Expired - Lifetime
  • 1998-05-25 IL IL13989098A patent/IL139890A0/xx active IP Right Grant
  • 1998-05-25 BR BRPI9815866-0A patent/BR9815866B1/pt not_active IP Right Cessation
  • 1998-05-25 TR TR2000/03503T patent/TR200003503T2/xx unknown
  • 1998-05-25 DE DE29824186U patent/DE29824186U1/de not_active Expired - Lifetime
  • 1998-05-25 AU AU80201/98A patent/AU765204B2/en not_active Expired
  • 1998-05-25 EA EA200001073A patent/EA002605B1/ru not_active IP Right Cessation
  • 1998-05-25 AT AT98928317T patent/ATE209342T1/de active
  • 1998-05-25 JP JP2000551232A patent/JP3836676B2/ja not_active Expired - Lifetime
  • 1998-05-25 ES ES98928317T patent/ES2168765T3/es not_active Expired - Lifetime
• 1999
  • 1999-05-26 AR ARP990102480A patent/AR019576A1/es active IP Right Grant
• 2000
  • 2000-11-22 BG BG104976A patent/BG64014B1/bg unknown
  • 2000-11-23 IL IL139890A patent/IL139890A/en not_active IP Right Cessation

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