EP4031684A1 - Procédé de détermination du nombre de cellules au moyen d'un adn de référence - Google Patents

Procédé de détermination du nombre de cellules au moyen d'un adn de référence

Info

Publication number
EP4031684A1
EP4031684A1 EP20771282.9A EP20771282A EP4031684A1 EP 4031684 A1 EP4031684 A1 EP 4031684A1 EP 20771282 A EP20771282 A EP 20771282A EP 4031684 A1 EP4031684 A1 EP 4031684A1
Authority
EP
European Patent Office
Prior art keywords
dna
cell
cells
sample
reference dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20771282.9A
Other languages
German (de)
English (en)
Inventor
Wolfgang Wagner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rheinisch Westlische Technische Hochschuke RWTH
Original Assignee
Rheinisch Westlische Technische Hochschuke RWTH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rheinisch Westlische Technische Hochschuke RWTH filed Critical Rheinisch Westlische Technische Hochschuke RWTH
Publication of EP4031684A1 publication Critical patent/EP4031684A1/fr
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes

Definitions

  • the invention relates to a method for determining the cell number of eukaryotic cells in a sample, in which at least one specific region in the genome of at least one cell or cell type is identified and at least one reference DNA is provided, the number of cells being calculated using the reference DNA becomes.
  • the relative quantification of cell types is of particular importance for many applications in clinical diagnostics, for example when taking blood counts.
  • the determination of the composition of the white blood cells (leukocytes) in the blood is one of the most common diagnostic test procedures and therefore a routine examination in medical laboratory diagnostics.
  • a differential blood count can be made, for example, by microscopic examination of the blood sample and manual counting of the cells.
  • the determination of the number of cells is also necessary for many other diagnostic and cell biological processes. Numerous methods are available for this, which are based in particular on imaging or flow cytometry (impedance or scattered light methods). These automatic cell counters record, for example, the electrical impedance, the optical effects of light scattering or the intensity of fluorescence signals. Alternative measurements are based on counting chambers, automated optical cell counts, or measurements of electrical resistance (CASY).
  • the above-mentioned methods of determining the number of cells require relatively fresh material in which the cells are alive and as isolated as possible. Reliable determinations of cell counts in tissues (e.g. from punched cylinders of biopsies with defined volumes, cells in 3D tissue engineering tissue constructs or embryological developments in different organisms) or coagulated blood samples are hardly possible. As a rule, these methods also require a certain minimum volume due to the hose systems. In addition, the samples must be freshly analyzed. However, in the course of sending the sample to a large laboratory or delayed measurements, increased cell deaths can occur, so that the cell count would be estimated accordingly too low. Autolysis begins after a few hours and initially affects the granulocytes in particular.
  • Genomic areas are addressed that are basically methylated in the blood cells to be examined.
  • the method comprises the identification of a chromatin region which is reliably methylated in the different blood cells and the provision of an unmethylated reference DNA which can be produced, for example, by PCR.
  • a determination of the cell number can also be carried out on coagulated blood samples and frozen material.
  • the object is achieved by a method for determining the cell number of eukaryotic cells in a sample, which comprises:
  • At least one reference DNA which comprises at least one reference nucleotide sequence which is identical to the sequence of the specific region with the exception of at least one nucleotide exchange;
  • a reference DNA is used to determine the number of cells, regardless of epigenetic changes, which has a sequence that differs from the corresponding cellular sequence in one or more nucleotides.
  • the cells are mixed with this reference DNA in a known concentration.
  • the concentration should preferably correspond approximately to the expected number of cells.
  • the quantification allows the ratio of cellular DNA and reference DNA to be determined relatively precisely, for example by means of digital PCR, pyrosequencing or amplicon sequencing methods.
  • the cell number can then be determined from this in comparison with a reference curve.
  • the method according to the invention can in principle be implemented for determining the number of cells of the most varied of cell types and of the most varied of species. However, a corresponding reference DNA must be generated for each species.
  • the method according to the invention can be safely used for a wide variety of human cell types.
  • the cells of other organisms can also be quantified with a corresponding reference DNA independently of epigenetic changes.
  • the samples can also be stored or shipped frozen, since only the DNA content is determined in relation to the volume.
  • a reference DNA is created which basically has the same sequence as a conserved genomic region in the cells to be tested, but which has a sequence difference in one or more bases. The optimal number of exchanges depends on the type of analysis method.
  • dPCR for example, 2-10, preferably 2-8 or 2-6, in particular 2-5, 3-5 or 2-4 exchanges in the area of the binding site of the probe have proven to be advantageous, since a single exchange may still be unspecific Allows ties.
  • the reference DNA eg approx. 100 base pairs
  • the samples can be frozen before analysis
  • - Cell numbers can be determined in tissues
  • the process according to the invention can be carried out very inexpensively.
  • two or more different reference DNAs are introduced into the sample, the reference DNAs corresponding to different specific regions.
  • a wider range of cell numbers can be reliably covered.
  • further validation and additional accuracy can be achieved through the parallel determination of different specific genomic regions.
  • the reference DNA corresponds in each case to the sequence of a specific species which is differentiated from other species. This would be advantageous in the event that human and other cells are mixed, e.g. cells on murine feeder cells.
  • the reference DNA is introduced into the sample in different concentrations.
  • the reference DNAs can, for example, be mixed beforehand in different concentrations and then, as a “ladder”, an aliquot of the sample can be added.
  • the number of cells can be calculated, for example, in such a way that different reference DNAs are added to the sample in different concentrations in order to cover a larger area. In other words, in order to broaden the measuring range, further reference DNAs can be added which have different sequence modifications.
  • a reference curve can be created in an advantageous manner, by means of which the number of cells can then be precisely determined. This also enables the concentration of the reference molecules to be determined, which roughly corresponds to the expected number of cells and thus ensures the highest possible accuracy of the measurement.
  • the number of copies of the reference DNA can also be calculated and the number of cells can thus also be determined without a reference curve.
  • the reference DNA concentration can be determined which corresponds to the same number of copies of the reference DNA and the genomic (cellular) DNA.
  • the amount of reference molecules should be around the expected number of copies of the correspond to genomic DNA, as the sensitivity is highest in this measuring range.
  • a wide range of cell numbers can be reliably covered by using different reference molecules in increasing concentrations.
  • the amount of reference DNA can be set such that the ratio of the number of reference molecules to the approximately expected number of cells is approximately 2: 1. Assuming that the cells to be analyzed each contain two copies of their DNA molecules, this ratio corresponds to the same number of copies of the reference DNA and the genomic (cellular) DNA, so that in this embodiment the highest possible sensitivity of the method according to the invention is guaranteed is.
  • two or more different reference DNAs could also be introduced into the sample, the reference DNAs corresponding to the same specific region but comprising different nucleotide exchanges. In this way, for example, the ratio of the reference DNAs to one another could be calculated.
  • the specific region comprises a conserved region without repeats and / or single nucleotide polymorphism, preferably a non-transcribed region.
  • a conserved region without repeats and single nucleotide polymorphism (SNP) should therefore be chosen in an advantageous manner, e.g. a promoter region of a gene.
  • the DNA is isolated from the sample before the quantification.
  • the entire DNA of the sample can largely be extracted.
  • the reference DNA can be added to the sample at a precisely set concentration, which is then subject to the same fluctuations in the DNA isolation during the DNA isolation. The Mixing of the reference DNA with the sample is therefore preferably carried out before the DNA isolation in order to compensate for fluctuations in the DNA isolation.
  • the ratio of cellular DNA and reference DNA can be determined relatively precisely, e.g. using quantitative PCR methods (especially digital PCR), pyrosequencing or amplicon deep sequencing methods.
  • nucleic acid molecule which comprises at least one nucleotide sequence which is identical to the sequence of a conserved region in the genome of at least one cell or a cell type with the exception of at least one nucleotide exchange, for use in the above-described method according to the invention.
  • nucleic acid molecule which comprises at least one nucleotide sequence which is identical to the sequence of a conserved region in the genome of at least one cell or a cell type with the exception of at least one nucleotide exchange, for use in the above-described method according to the invention.
  • SEQ ID NOs: 1 to 7 according to Table 1 can be used for the method according to the invention.
  • the invention also comprises a kit which comprises at least one such nucleic acid molecule and optionally at least one oligonucleotide for amplifying and / or quantifying at least one nucleic acid molecule.
  • This kit can be used to determine the number of eukaryotic cells in a sample.
  • such a kit can comprise one or more of the reference DNA sequences according to SEQ ID NOs: 1 to 7 (Table 1) and / or one or more of the primers / probes according to SEQ ID NOs: 8 to 35 (Table 1).
  • such a kit can comprise at least one buffer solution and / or a reagent for carrying out one or more of the following methods: DNA amplification, DNA sequencing, for example pyrosequencing, digital droplet PCR, bar-coded bisulfite amplicon sequencing (BBA-seq), bar-coded amplicon sequencing, "Mass Array ® " and "SNP genotyping”.
  • DNA sequencing for example pyrosequencing, digital droplet PCR, bar-coded bisulfite amplicon sequencing (BBA-seq), bar-coded amplicon sequencing, "Mass Array ® " and "SNP genotyping.
  • the reference DNA can be multiplied, for example, by introducing the reference nucleotide sequence into at least one prokaryotic cell, increasing the nucleotide sequence in the prokayotic cell and isolating at least one DNA molecule, which comprises at least one reference nucleotide sequence, can be carried out from the prokaryotic cell.
  • the reference molecule can thus be amplified, for example, in E. coli (for example in the form of a plasmid) and then purified.
  • the plasmid DNA can be literaryized with a restriction enzyme before it is used as reference DNA.
  • the reference DNA can be amplified or synthesized in vitro, preferably by means of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the PCR product could, for example, be used as an independent DNA double strand or as cloned plasmid DNA as a standard.
  • the invention further relates to at least one artificial nucleic acid molecule which comprises at least one nucleotide sequence which is selected from the group consisting of: a) at least one nucleotide sequence which contains at least one sequence of the nucleotide sequences SEQ ID NO: 1 to SEQ ID NO: 35 (see table 1) comprises, b) at least one nucleotide sequence which is at least 90%, preferably at least 95%, identical to the nucleotide sequence according to a) and c) at least one nucleotide sequence which corresponds to the strand complementary to one of the nucleotide sequences according to a) or b) corresponds to.
  • the artificial nucleic acid molecules according to the invention can also advantageously be used to produce the kit for carrying out the method according to the invention.
  • the invention also relates to the use of the nucleic acid molecule according to the invention and / or the artificial nucleic acid molecule according to the invention and / or the kit according to the invention for determining the cell count of eukaryotic cells in a sample, ie for the absolute quantification of cells or cell types.
  • Both the method according to the invention and the nucleic acid molecules and kits according to the invention can in principle be used for determining the number of cells of the most varied of cell types in various areas of application. In particular, these are also suitable, for example, for determining the number of nucleated cells in the blood or other body fluids (liquor, urine, etc.). With the aid of the method according to the invention, for example, the number of nucleated blood cells can be reliably determined.
  • Cell counts are routinely determined in blood samples using flow cytometric measuring methods. However, the determination is not possible in all blood samples (for example due to coagulation or insufficient blood volume) and can no longer be reliably carried out on older blood samples. The samples must be freshly sent for analysis and measured promptly. In contrast to this, the determination of the cell number according to the invention can also be carried out on blood samples in which the cells are no longer isolated.
  • the method according to the invention is also useful in combination with other methods, since quantitative information is obtained without great additional effort, while otherwise only relative ratios of the hematopoietic cell types can be determined. In the high throughput process, the process according to the invention can also be carried out very inexpensively. The measuring accuracy is comparable with the accuracy of conventional methods.
  • a “conserved genomic region” within the meaning of the invention denotes a sequence segment in the genome of a cell (DNA) which comprises a nucleotide sequence which has been largely unchanged in the course of evolution, i.e. has undergone no or only minimal changes in the sequence.
  • the term also includes, in particular, non-coding DNA sequences, such as introns or promoter sequences.
  • FIG. 1 shows an exemplary image of a result of a digital droplet PCR (ddPCR).
  • X-axis reference
  • Y-axis blood
  • FIG. 2 shows a diagram for evaluating the results of the positive droplets for genomic DNA or reference DNA in order to determine the ratio of these DNA strands in the starting material.
  • 150 ⁇ l of blood were mixed with various concentrations of the reference DNAs (R2 and R3 according to Table 1).
  • X-axis dilution or reference DNA
  • Y-axis ratio of blood to reference DNA
  • Figure 3 shows a diagram for determining the ratio of blood DNA to reference DNA or the number of copies.
  • different amounts of the reference DNAs R2-R6, R8 and R9 according to Table 1 were mixed with 150 ⁇ l of blood.
  • X-axis number of copies (ratio of blood to reference)
  • FIG. 4 shows diagrams of an example of the quantification of human mesenchymal stem cells (MSC) by means of digital droplet PCR (ddPCR).
  • FIG. 5 shows diagrams of an example of the determination of the number of cells in human blood samples by means of digital droplet PCR (ddPCR).
  • X-axis log blood cells
  • automatic counter Y-axis log blood cells ddPCR
  • genomic regions were selected for the design of the reference DNAs. These sequences do not have any homologous sections in the genome and are suitable for the design of the ddPCR primers. They should preferably not be located in translated gene sequences, since an influence by RNA cannot be ruled out with certainty. Of the nine initial sequences, seven have proven successful, which are listed in Table 1. These sequences were provided, cloned into plasmid pBR322 using restriction enzymes, and amplified in E. coli. The plasmid DNA was isolated and quantified. These Reference DNAs are either used individually or combined in different concentrations as reference ladder. A certain amount of this reference DNA is added to the cell sample (eg 150 ⁇ l blood).
  • a digital droplet PCR (ddPCR) is then carried out (FIG. 1).
  • the results of the positive droplets for genomic DNA or reference DNA are evaluated using Poisson statistics in order to determine the ratio of these DNA strands in the starting material (FIG. 2).
  • the ratio of blood DNA to reference DNA was then determined in the ddPCR measurements. This allows conclusions to be drawn about the number of cells. The data show very consistent results both for the dilutions and with both reference plasmids. If necessary in the case of large fluctuations in the number of cells, or as an additional internal control, the measurement of a further reference DNA can also be included, since this additional sequence does not interfere with the method (FIG. 3). Different amounts of the reference DNAs were mixed with a certain volume of blood. The ratio of blood DNA to reference DNA was then determined. The measurements show a clear correlation without the different plasmids affecting the measurement.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détermination du nombre de cellules eucaryotes dans un échantillon, dans lequel au moins une région spécifique dans le génome d'au moins une cellule ou d'un type de cellule est identifiée et au moins un ADN de référence est fourni, le nombre de cellules étant calculé au moyen de l'ADN de référence. Selon l'invention, le nombre de cellules est déterminé à l'aide d'un ADN de référence qui a une séquence s'écartant de la séquence cellulaire correspondante d'un ou plusieurs nucléotides. Avant la quantification de l'ADN, les cellules sont mélangées avec ledit ADN de référence dans une concentration connue. Au moyen de la quantification, il est possible de déterminer le rapport entre l'ADN cellulaire et l'ADN de référence. Le nombre de cellules peut être déterminé par comparaison avec une courbe de référence.
EP20771282.9A 2019-09-16 2020-09-10 Procédé de détermination du nombre de cellules au moyen d'un adn de référence Pending EP4031684A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102019124828.0A DE102019124828A1 (de) 2019-09-16 2019-09-16 Verfahren zur Bestimmung der Zellzahl mittels einer Referenz-DNA
PCT/EP2020/075310 WO2021052854A1 (fr) 2019-09-16 2020-09-10 Procédé de détermination du nombre de cellules au moyen d'un adn de référence

Publications (1)

Publication Number Publication Date
EP4031684A1 true EP4031684A1 (fr) 2022-07-27

Family

ID=72470378

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20771282.9A Pending EP4031684A1 (fr) 2019-09-16 2020-09-10 Procédé de détermination du nombre de cellules au moyen d'un adn de référence

Country Status (3)

Country Link
EP (1) EP4031684A1 (fr)
DE (1) DE102019124828A1 (fr)
WO (1) WO2021052854A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102017004108A1 (de) 2017-05-01 2018-11-08 Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen Verfahren zur Bestimmung der Zellzahl von eukaryotischen Zellen

Also Published As

Publication number Publication date
WO2021052854A1 (fr) 2021-03-25
DE102019124828A1 (de) 2021-03-18

Similar Documents

Publication Publication Date Title
DE69824004T2 (de) Verfahren zur quantitativen bestimmung der genexpression mit hilfe der multiplexen competitiven reversen-transkriptase polymerase kettenreaktion
DE60123448T2 (de) Verfahren zur nicht-invasiven Diagnose von Transplantationen und Transfusionen
DE102004036285A1 (de) Verfahren zum Bestimmen der Häufigkeit von Sequenzen einer Probe
DE19754482A1 (de) Verfahren zur Herstellung komplexer DNA-Methylierungs-Fingerabdrücke
DE112013004650T5 (de) Multiplexpyrosequenzierung unter Verwendung nichtinterferierender, rauschbeendender Polynukleotididentifikationstags
EP3344781A1 (fr) Utilisation de micro-arn circulant dans le sérum sanguin ou le plasma sanguin aux fins d'identification de patients tenus à une biopsie et en tant que marqueurs aux fins de diagnostic différentiel de différentes cardiomyopathies non ischémiques ou de maladies de surchage du coeur
DE102005045560B4 (de) Verfahren zur quantitativen Bestimmung der Kopienzahl einer vorbestimmten Sequenz in einer Zelle
DE69330604T2 (de) Verfahren und system zur molekularbiologischen diagnose
WO2007068305A1 (fr) Procede de determination du genotype a partir d'un echantillon biologique qui contient des acides nucleiques provenant de differents individus
EP4031684A1 (fr) Procédé de détermination du nombre de cellules au moyen d'un adn de référence
AT503721A1 (de) Alleldetektion
WO2018202669A1 (fr) Procédé pour déterminer le nombre de cellules eucaryotes
DE60025871T2 (de) Genetisches Screening Verfahren und Vorrichtung
DE102017214449B4 (de) Verfahren und Mittel zur Diagnostik von Tumoren durch Nachweis und Quantifizierung heterogen methylierter Epiallele
DE102006014000B4 (de) Verfahren zur Charakterisierung einer Mischprobe
WO2011151427A1 (fr) Stabilisation d'acides nucléiques dans des échantillons biologiques contenant de la matière cellulaire
DE60109002T2 (de) Methode zum Nachweis von transkribierten genomischen DNA-Sequenzen
WO2004027089A1 (fr) Procede d'amplification d'informations genetiques
EP1167541B1 (fr) Procédé de d'obtention de données pour le diagnostique pré-symptomatique ou pré-natal de la neurofibromatose de type 1
WO2020178134A1 (fr) Procédé de numération de types cellulaires ou de marqueurs cellulaires dans un échantillon, en particulier dans un échantillon de sang
DE102015206444B3 (de) Verfahren zum Erkennen von Mikroorganismen
WO2021122979A1 (fr) Procédé et dispositif pour déterminer le nombre de copies d'une séquence d'adn présentes dans un fluide
EP4090765A1 (fr) Procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic associé
DE102005011003B9 (de) Verfahren und Mittel zur differentiellen Diagnose von Schilddrüsentumoren
DE10030827A1 (de) Verfahren zum Nachweis von Cathepsinen, Asparaginylendopeptidase und deren Isozyme, sowie von Leukocystatin

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220317

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)