WO2021136559A1 - 一种基于荧光定量pcr技术区分人源dna的方法 - Google Patents

一种基于荧光定量pcr技术区分人源dna的方法 Download PDF

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WO2021136559A1
WO2021136559A1 PCT/CN2021/073569 CN2021073569W WO2021136559A1 WO 2021136559 A1 WO2021136559 A1 WO 2021136559A1 CN 2021073569 W CN2021073569 W CN 2021073569W WO 2021136559 A1 WO2021136559 A1 WO 2021136559A1
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dna
human
seq
sequence
human animal
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French (fr)
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冯丽
冷晓燕
刘辉
王云娟
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江苏艾尔康生物医药科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to a method for distinguishing human DNA based on fluorescence quantitative PCR technology, as well as detection primers and probes.
  • experimental monkeys are a very important experimental animal in the field of new drug research. Because of its high similarity with humans, experimental monkeys are considered to be an important tool for drug evaluation. Before cell drugs are applied to humans, it is necessary to conduct a comparatively systematic evaluation in animals that are more closely related to humans.
  • the current methods for studying the distribution of cells in experimental monkeys mainly include animal in vivo imaging, fluorescent protein labeling, immunohistochemistry, qPCR and so on.
  • Each method has its advantages and disadvantages.
  • nuclear magnetic resonance (MRI) MRI can reflect the distribution of cells in the body.
  • This method requires labeling cells in vitro and detecting the cells injected into the body through imaging to determine the survival of the cells and the fate of being eliminated.
  • This method High sensitivity and long marking time.
  • certain labeling methods may have an impact on cell viability. It has been reported that the differentiation ability of bone marrow mesenchymal stem cells is impaired after MRI labeling. Moreover, the sensitivity of MRI is relatively insufficient.
  • iron oxide particles released after cell death will cause non-specific imaging and false positives.
  • green fluorescent protein labeling GFP
  • this method uses genetic modification to enable cells to express fluorescent protein, and the green fluorescent signal can be directly observed through a fluorescence microscope.
  • This method has the advantage of being easy to detect.
  • the genetic material of the cell is changed, and this change cannot be guaranteed to be 100% stable.
  • Any method of labeling cells may cause changes to cells and affect the differentiation of progeny cells.
  • the method of immunohistochemistry does not require labeling cells, but this method requires analysis of a large number of sections and microscopic observations. At the same time, due to the similarity between humans and monkeys, many antigens cross between humans and monkeys, and this method is difficult to standardize. Can get semi-quantitative results.
  • the qPCR method is a method with high sensitivity and relatively simple operation, and it is expected to realize quantitative analysis.
  • the first step is to find sequences and primers that can distinguish human and animal model DNA.
  • the experimental cells may differentiate into cells that no longer express the specific genes after being injected into monkeys. Therefore, the current differentiation between human and monkey cells There will be false negatives in genes. That is, human cells are present in monkey tissues, but they cannot be detected.
  • primers designed based on human-derived special gene fragments-Alu gene as molecular markers are expected to distinguish human and monkey DNA through qPCR methods, and ultimately achieve the purpose of detecting human cells in monkey cells or tissues.
  • these primers can only distinguish between humans and rodents.
  • Pengyue Song et al. reported in 2012 that an efficient and reproducible PCR method based on DNA-specific primers can detect xenotransplanted human cells in mouse tissues.
  • Julie et al. published an article reporting a method for measuring the number of transplanted human cells in rats and mice by qPCR.
  • few articles have reported that it is possible to distinguish between human and monkey DNA by qPCR.
  • the present invention discloses a DNA sequence, wherein the DNA sequence is selected from the group consisting of the sequence shown in SEQ ID NO:1 or a fragment thereof, and the reverse complementary sequence of SEQ ID NO:1 or a fragment thereof.
  • the DNA sequence is used to distinguish human and non-human animal DNA in a mixed human and non-human animal tissue sample.
  • sequence of SEQ ID NO:1 is:
  • the present invention provides a use of a DNA sequence in the preparation of a reagent or kit for distinguishing human and non-human animal DNA in a mixed human and non-human animal tissue sample, wherein the DNA sequence is selected from A group consisting of the sequence shown in SEQ ID NO:1 or a fragment thereof, and the reverse complementary sequence of SEQ ID NO:1 or a fragment thereof.
  • the present invention also provides the use of a reagent for detecting DNA sequence in the preparation of a reagent or kit for distinguishing human and non-human animal DNA in a mixed human and non-human animal tissue sample, wherein the DNA sequence is selected from the group consisting of: A group consisting of the sequence shown in SEQ ID NO:1 or a fragment thereof, and the reverse complementary sequence of SEQ ID NO:1 or a fragment thereof.
  • the DNA sequence is the sequence shown in SEQ ID NO:1 or its reverse complement, or a partial fragment of these full-length sequences, and the fragment is SEQ ID NO:1 or its return
  • the 5'end and/or 3'end of each complementary sequence lacks a sequence of 1-70 nucleotides, and the fragment sequence can still be used to distinguish human and non-human animal DNA in a mixed human and non-human animal tissue sample.
  • the reagents for detecting the DNA sequence are selected from primers and probes required to amplify the DNA sequence by PCR technology.
  • sequence of the probe is shown in SEQ ID NO: 10.
  • the probe has a detection label, and the detection label is preferably selected from FAM, TET, Alexa 488, Alexa 532, CF, HEX, VIC, ROX, Texas Red, QuasarFITC, cy3, cy5 , 6-joe, EDANS, rhodamine 6G, TMR, TMRITC, x-rhodamine, Texas red, biotin, and avidin.
  • the detection label is preferably selected from FAM, TET, Alexa 488, Alexa 532, CF, HEX, VIC, ROX, Texas Red, QuasarFITC, cy3, cy5 , 6-joe, EDANS, rhodamine 6G, TMR, TMRITC, x-rhodamine, Texas red, biotin, and avidin.
  • the sequence of the primer is selected from the sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3; the sequence shown in SEQ ID NO: 2 and SEQ ID NO: 5; such as SEQ The sequence shown in ID NO: 4 and SEQ ID NO: 3; the sequence shown in SEQ ID NO: 6 and SEQ ID NO: 7; and the sequence shown in SEQ ID NO: 4 and SEQ ID NO: 7 are composed Group.
  • the non-human animal is selected from the group consisting of rhesus monkeys, green monkeys, cynomolgus monkeys, rats, mice, and rabbits.
  • the mixed human and non-human animal tissues are tissues and blood samples of non-human animals such as rhesus monkeys mixed with human DNA, and the human DNA is derived from human cells.
  • the human DNA is derived from DNA in human retinal pigment epithelial cells.
  • the present invention discloses a composition comprising primers and probes, wherein the sequence of the probe is as shown in SEQ ID NO: 10, and the sequence of the primer is selected from the group consisting of SEQ ID NO: 2 and SEQ. ID NO: 3; SEQ ID NO: 2 and SEQ ID NO: 5; SEQ ID NO: 4 and SEQ ID NO: 3; SEQ ID NO: 6 and The sequence shown in SEQ ID NO: 7; and the group consisting of the sequence shown in SEQ ID NO: 4 and SEQ ID NO: 7.
  • the probe has a detection label
  • the detection label is preferably selected from FAM, TET, Alexa 488, Alexa 532, CF, HEX, VIC, ROX, Texas Red, QuasarFITC, cy3, cy5, 6- A group consisting of joe, EDANS, rhodamine 6G, TMR, TMRITC, x-rhodamine, Texas red, biotin, and avidin.
  • the present invention also provides a kit comprising the composition as described above.
  • the present invention also provides a method for distinguishing between human and non-human animal DNA sequences in mixed human and non-human animal tissues for non-diagnostic and therapeutic purposes, wherein the aforementioned composition or the aforementioned kit is used PCR amplification of DNA is performed on samples mixed with human and non-human animal tissues.
  • the steps specifically include:
  • the non-human animal is selected from the group consisting of rhesus monkeys, green monkeys, cynomolgus monkeys, rats, mice, and rabbits.
  • the mixed human and non-human animal tissues are non-human animals such as rhesus monkey tissues and blood samples mixed with human DNA
  • the human DNA is derived from human cells, preferably from retinal pigment epithelial cells.
  • the present invention finds a DNA sequence on the chromosome of the human genome, the DNA sequence is human-specific, and some primers and probes are designed on the basis of the DNA sequence, which can distinguish human DNA from multiple species. In practical applications, human-specific DNA can be detected from experimental animal DNA.
  • Figure 1 shows the amplification curve of DNA detection of different species using R1, F1 and Probe1 when targeting the DNA sequence shown in SEQ ID NO:1 of the present invention
  • 1 is human retinal pigment epithelial cell DNA
  • 2 is constant River monkey DNA
  • 3 is green monkey DNA
  • 4 is cynomolgus DNA
  • 5 is rat DNA
  • 6 is mouse DNA
  • 7 is rabbit DNA.
  • Figure 2 shows the amplification curves of DNA of different species detected using R2, F2 and Probe1 when targeting the DNA sequence shown in SEQ ID NO:1 of the present invention
  • 1 human retinal pigment epithelial cell DNA
  • 2 constant River monkey DNA
  • 3 green monkey DNA
  • 4 is cynomolgus DNA
  • 5 is rat DNA
  • 6 mouse DNA
  • 7 is rabbit DNA.
  • Figure 3 shows the amplification curves of DNA detection of different species using R2, F1 and Probe1 when targeting the DNA sequence shown in SEQ ID NO:1 of the present invention
  • 1 is human retinal pigment epithelial cell DNA
  • 2 is constant River monkey DNA
  • 3 is green monkey DNA
  • 4 is cynomolgus DNA
  • 5 is rat DNA
  • 6 is mouse DNA
  • 7 is rabbit DNA.
  • Figure 4 shows the amplification curves of DNA detection of different species using R3, F3 and Probe1 when targeting the DNA sequence shown in SEQ ID NO:1 of the present invention
  • 1 is human retinal pigment epithelial cell DNA
  • 2 is constant River monkey DNA
  • 3 is green monkey DNA
  • 4 is cynomolgus DNA
  • 5 is rat DNA
  • 6 is mouse DNA
  • 7 is rabbit DNA.
  • Figure 5 shows the amplification curves of DNA of different species detected using R2, F3 and Probe1 when targeting the DNA sequence shown in SEQ ID NO:1 of the present invention
  • 1 is human retinal pigment epithelial cell DNA
  • 2 is constant River monkey DNA
  • 3 is green monkey DNA
  • 4 is cynomolgus DNA
  • 5 is rat DNA
  • 6 is mouse DNA
  • 7 is rabbit DNA.
  • Figure 6 shows the amplification curves of DNA detection of different species when targeting the gene SRGAP2 (1 human retinal pigment epithelial cell DNA; 2 rhesus monkey DNA; 3 green monkey DNA; 4 cynomolgus monkey DNA; 5 Is rat DNA; 6 is mouse DNA; 7 is rabbit DNA);
  • Figure 7 shows the amplification curves of DNA detection of different species when the gene Qhomo2 is targeted (1 human retinal pigment epithelial cell DNA; 2 rhesus monkey DNA; 3 green monkey DNA; 4 cynomolgus monkey DNA; 5 Is rat DNA; 6 is mouse DNA; 7 is rabbit DNA);
  • Figure 8 shows the amplification curves of DNA detection of different species when targeting gene Alu (1 human retinal pigment epithelial cell DNA; 2 rhesus monkey DNA; 3 green monkey DNA; 4 cynomolgus monkey DNA; 5 Is rat DNA; 6 is mouse DNA; 7 is rabbit DNA);
  • Figure 9 shows a typical standard curve for real-time fluorescent quantitative PCR to detect the target DNA sequence of human retinal pigment epithelial cells.
  • non-human animals includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians Animals, reptiles, etc.
  • the non-human animal is selected from the group consisting of rhesus monkeys, green monkeys, cynomolgus monkeys, rats, mice, and rabbits.
  • DNA sequence in the present invention refers to a DNA sequence encoding a protein, for example, but not limited to, a DNA sequence encoding a protein that exists in the genome of a cell.
  • the term "probe” in the present invention refers to an oligonucleotide molecule with a detection label.
  • Detection label in the present invention refers to a molecule or group capable of generating a detection signal. Detection labels include, but are not limited to, fluorescent molecules (for example, see European Patent EP144914), radioisotopes (for example, see US Patent Nos. 4358535 and 4446237) , Antibodies, enzymes, and oligonucleotides (e.g., oligonucleotide barcodes).
  • fluorescent molecules include but are not limited to 6-carboxyfluorescein (FAM), Tetrachlorofluorescein (TET), Alexa (e.g. Alexa 488, Alexa 532), CF, HEX, VIC, ROX, Texas Red, QuasarFITC, cy3, cy5, 6-joe , EDANS, rhodamine 6G (P6G) and its derivatives (tetramethyirhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine, Texas red, are produced by Molecular Probes, Inc., located in Eugene, Oregon, U.S.).
  • detection markers can also be found in U.S. Patent Nos. 5,723,591 and 5,928,907; WO2011066476 and WO2012149042; www.idahotech.com; Gudnason et al., Nucleic Acids Res., 35(19): e127 (2007), the entire contents of which are incorporated into the present invention by reference. .
  • the detection label can be attached to the oligonucleotide molecule through a covalent bond or a non-covalent bond.
  • Non-covalent bonds include, but are not limited to, hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic bonds.
  • the detection label may be covalently attached to the nucleotide molecule.
  • amino-allyl UTP can be incorporated in the synthesis of oligonucleotide molecules, and the resulting amino-allyl-labeled nucleic acid molecules can be combined with fluorescent molecules containing NHS-ester (NHS-ester) ( For example, Alexa 488, Alexa 594, Alexa 647 (Invitrogen) or Cy3 (GE Healthcare)) are coupled to form a covalent bond connection.
  • NHS-ester For example, Alexa 488, Alexa 594, Alexa 647 (Invitrogen) or Cy3 (GE Healthcare)
  • the detection label is selected from: FAM, Tetrachlorofluorescein (TET), Alexa 488, Alexa 532, CF, HEX, VIC, ROX, Texas Red, QuasarFITC, cy3, cy5, 6-joe, EDANS, rhodamine 6G (P6G), tetramethyirhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine, Texas red, biotin, avidin.
  • TTT Tetrachlorofluorescein
  • Alexa 488 Alexa 532
  • CF HEX
  • VIC HEX
  • ROX Texas Red
  • QuasarFITC cy3, cy5, 6-joe
  • EDANS rhodamine 6G (P6G), tetramethyirhodamine (TMR), tetramethylrhodamine isothiocyan
  • the probe also has a quenchable signal.
  • Quencher in the present invention refers to a molecule that can prevent the detection label from generating a detection signal when it is sufficiently close in space to the detection label. When the quencher is far away from the detection mark, the quencher cannot prevent the generation of the detection signal.
  • quenching molecules include but are not limited to DDQ-I, DDQ-II, Dabcyl, Eclipse, Iowa Black FQ, Iowa Black RQ, BHQ-1, BHQ-2, BHQ-3, "QSY7", “QSY-21” and “QSY33” (Molecular Probe Company), Ferrocene and its derivatives, methyl viologen, tetramethylrhodamine (TAMRA), minor groove binding non-fluorescent queueer (MGBNFQ) and N,N'-dimethyl-2,9-diazopyrenium.
  • TAMRA methyl viologen, tetramethylrhodamine
  • MGBNFQ minor groove binding non-fluorescent queueer
  • N,N'-dimethyl-2,9-diazopyrenium N,N'-dimethyl-2,9-diazopyrenium.
  • the fluorescent molecule is FAM and the quencher molecule is MGBNFQ or DDQ-I. In some embodiments, the fluorescent molecule is TAMRA, Cy3, ROX, Cy5, and the quencher molecule is DDQ-II. In some embodiments, the fluorescent molecule is FAM, HEX, ROX, JOE, and the quencher molecule is Dabcyl. In some embodiments, the probe has a fluorescent molecule FAM or VIC at the 5'end and a quencher molecule MGBNFQ at the 3'end.
  • the quencher molecule can be attached to the probe by methods well known in the art.
  • amino-allyl UTP can be incorporated in the synthesis of oligonucleotide molecules, and the resulting amino-allyl-labeled nucleic acid molecules can be used with NHS-ester-containing quenching molecules.
  • the coupling forms a covalent bond.
  • the quencher molecule can be connected to the oligonucleotide by reacting with the phosphoramidite derivative of the quencher molecule (for example, Dabcyl) at the 3'end during the synthesis of the oligonucleotide.
  • the signal when the probe is intact, the signal is quenched.
  • the detection label and quencher are attached to the 5'end and 3'end of the probe, respectively.
  • a detection label is connected to the 5'end of the probe in the non-mutation region and a quencher is connected to the 3'end, or a detection label is connected to the 3'end and a quencher is connected to the 5'end.
  • a polymerase with 5'-3' exonuclease activity is used to amplify a fragment containing SEG ID NO:1 or its complementary sequence, or with SEQ ID NO:1 sequence or its complementary sequence fragment as the Template sequence and add the probe to the reaction mixture.
  • the probe hybridizes with the template sequence, the probe will be degraded by the polymerase during the polymerization reaction, so that the fluorescent molecule on the probe is separated from the quencher molecule and produces Fluorescence signal (see US patents US5210015 and US5487972).
  • fragment refers to the 5'end and/or 3'of the DNA sequence of the present invention as shown in SEQ ID NO:1 or its reverse 100% complementary sequence.
  • a sequence of partial nucleotide deletions at the end for example, compared with the sequence shown in SEQ ID NO:1, the 5'end has 1-70, 2-70, 5-70, 10-70, 20-70 deletions One, 30-70, 40-70, 50-70, 60-70 nucleotide sequence, or a 3'-end deletion of 1-70, 2-70, 5-70, 10- 70, 20-70, 30-70, 40-70, 50-70, 60-70 nucleotide sequence, or 5'end and 3'end are deleted at the same time 1-70, 2 -70, 5-70, 10-70, 20-70, 30-70, 40-70, 50-70, 60-70 nucleotide sequence, this fragment is amplified by PCR Later, according to the knowledge of those skilled in the art, the same or similar lengths in the embodiments of the
  • the present invention designs multiple pairs of primers and probes on the basis of the human-specific DNA sequence Seq1 (SEQ ID NO:1).
  • SRGAP2 three groups of human-specific genes and their primers
  • Qhomo2 are applied from the literature ("Umbilical Cord Mesenchymal Stem Cells Preclinical Safety Research", Wang Youwei, Peking Union Medical College, 2013). According to reports, the primers can specifically detect human DNA.
  • the primer of SRGAP2 is quoted from the patent CN201910477468.2 "Primers for specific detection of human genomic DNA and their applications”.
  • the primers can be used from multiple species of DNA (including cynomolgus monkeys, rats, mice, and New Zealand rabbits). Detection of human-specific DNA sequences.
  • the Alu sequence of the gene amplified by primer Alu is a universal, diverse and specific short repetitive sequence in the human genome. Alu family elements can be used for individual identification in forensic DNA analysis. There are also reports in the literature that Alu sequences can be used to distinguish DNA from other species.
  • the Alu primers and probes of the present invention are applied from literature. Table 1.1 shows the sequences of all primers and probes used in the present invention. The probe has a reporter group at the 5'end and a quencher group at the 3'end.
  • tissue and cell DNA extraction kits to extract genomic DNA from cells and tissues derived from different species (human, rhesus, green monkey, cynomolgus, rat, mouse and rabbit) according to the instructions of the kit.
  • Taqman qPCR amplification reaction system a single sample is 20 ⁇ L: 10 ⁇ L 2 ⁇ SuperReal PreMix (Probe); 1 ⁇ L 50 ⁇ ROX Reference Dye; 0.6 ⁇ L primer R (10 ⁇ M); 0.6 ⁇ L primer F (10 ⁇ M); 0.4 ⁇ L Probe (10 ⁇ M); add 20ng of DNA; use RNase-Free ddH 2 O to make up the reaction system to 20 ⁇ L.
  • the primers and probes used in this step and their combinations are shown in the table below. There are 13 sets of primers and probe combinations.
  • reaction conditions for Taqman qPCR amplification are: 95°C pre-denaturation for 15 minutes; 95°C denaturation for 1 second; 62°C annealing and extension for 30 seconds for a total of 40 cycles; fluorescence signals are collected at 62°C. Obtain the CT value from the instrument after the experiment.
  • the combination of some primers and probes designed for the DNA sequence cannot distinguish between human and monkey DNA, and there are also some (such as R1, F1 plus Probe1).
  • R2, F2 plus Probe2 cannot distinguish DNA between humans and multiple species (including three monkeys, rats, mice, and rabbits).
  • Figure 1, Figure 6, Figure 7, and Figure 8 are the primer combinations R1, F1 plus Probe1, respectively.
  • Figures 2 to 5 show the amplification curves of the DNA sequence Seq1 amplified by other primer combinations.
  • the experimental results also show that the primers and probes synthesized in the present invention can detect human DNA in multiple species.
  • a standard curve sample according to the following table, where the standard is the whole genome DNA of human retinal pigment epithelial cell injection (a cell solution containing human retinal pigment epithelial cells, which can be used for subretinal injection of rhesus monkeys) (Approximate concentration: 140ng/ ⁇ L), dilute according to the table below.
  • the standard is the whole genome DNA of human retinal pigment epithelial cell injection (a cell solution containing human retinal pigment epithelial cells, which can be used for subretinal injection of rhesus monkeys) (Approximate concentration: 140ng/ ⁇ L), dilute according to the table below.
  • the standard is human retinal pigment epithelial cell whole genome DNA (concentration: 140ng/ ⁇ L). Dilute according to the table. First add a certain volume of pure water, and then add the corresponding volume of human Source retinal pigment epithelial cell injection whole genome DNA, upper limit of quantification ULOQ, high quality control HQC, middle quality control MQC, quality control C, low quality control LQC, lower limit of quantification LLOQ, and then formulated into ULOQ (100ng/ ⁇ L), HQC ( 80ng/ ⁇ L), MQC (3.2ng/ ⁇ L), C (0.8ng/ ⁇ L), LQC (0.08ng/ ⁇ L) and LLOQ (0.032ng/ ⁇ L).
  • tissue and cell DNA extraction kit to extract the genomic DNA of cells and tissues according to the instructions of the kit.
  • a single sample of Taqman qPCR amplification reaction system is 20 ⁇ L: 10 ⁇ L 2 ⁇ SuperReal PreMix (Probe); 1 ⁇ L 50 ⁇ ROX Reference Dye; 0.6 ⁇ L primer R (10 ⁇ M); 0.6 ⁇ L primer F (10 ⁇ M); 0.4 ⁇ L probe Needle Probe (10 ⁇ M); add 2 ⁇ L of DNA (DNA templates are human retinal pigment epithelial cell whole genome DNA, standard curve sample, quality control sample, test sample, blank matrix negative control sample (Neg) and pure water without template Negative control (NTC)); the reaction system was made up to 20 ⁇ L with RNase-Free ddH 2 O.
  • %RE standard deviation (SD)
  • %CV target DNA concentration
  • Sample judgment criteria 1. When the CT values of LLOQ and LOD are both smaller than the CT values of NTC and Neg: (1) The CT values of the multiple holes of the sample to be tested are all smaller than the average CT value of LLOQ, and the concentration result is issued; (2) To be tested If the CT value of the multiple holes of the sample is less than the CT value of the LOD, it means that the sample is positive; (3) If the LOD does not show a value, the batch of samples will issue a concentration result; 2.
  • CT Slope LgX 0 + y-int; where X 0 is the initial concentration of the sample, y-int is the intercept, and Slope is the slope.
  • the average relative error (RE%) within and between batches of each concentration is within -75% ⁇ 150%; the precision (CV%) within and between batches of each concentration is less than 60.00%.
  • the intra-assay accuracy %RE of each quality control sample concentration is in the range of -22.50-41.25%, and the intra-assay precision %CV is in the range of 1.37-60.00%;
  • the inter-assay accuracy %RE of each quality control sample concentration In the range of -0.91 to 15.60%, the inter-assay precision %CV is in the range of 9.28 to 35.71%, which meets the accuracy and precision requirements of intra-assay and inter-assay.
  • Table 2.5 shows that the accuracy and precision of the analysis method meet the requirements.
  • Table 2.5 The precision and accuracy of real-time fluorescent quantitative PCR in detecting the target DNA sequence of human retinal pigment epithelial cell injection
  • the sample is tested, and the relative error (RE%) of each concentration of the standard curve with blank matrix DNA added to the theoretical concentration is calculated.
  • Acceptance standard compare the two standard curves ⁇ RE% ⁇ sum of blank matrix DNA added to each tissue, and use the total amount of ⁇ RE% ⁇ smaller as the amount of template added in the reaction system when the actual sample is tested; if two sets of standards If the curve ⁇ RE% ⁇ is close to the sum, select the low concentration point on the standard curve ⁇ RE% ⁇ The total amount in this set of standard curves is the amount of template added in the reaction system when the actual sample is detected, and calculate the best detection concentration. In the actual sample detection, when the actual sample concentration is greater than 20% of the best detection concentration, the sample should be diluted to the best detection concentration. If the actual sample concentration is less than the best detection concentration, then the actual concentration will be tested.
  • the optimal detection concentration of the sample is 20ng/ ⁇ L; when the total amount of DNA in the lungs of blank rhesus monkeys is 200ng and 100ng respectively, the sum of the two standard curves ⁇ RE% ⁇ is 549.81 and 311.49 respectively, and the lungs are obtained.
  • the amount of template added in the reaction system is 100ng, and the best detection concentration of the sample is 50ng/ ⁇ L; when the total amount of liver DNA of blank rhesus monkey is 200ng and 100ng, two standard curves ⁇ RE% ⁇ The sum is 98.51 and 113.79, respectively. It is concluded that the amount of template added in the reaction system for liver sample detection is 200ng, and the optimal detection concentration of the sample is 100ng/ ⁇ L; the total amount of choroid + RPE DNA of blank rhesus monkey is 100ng respectively At 40 ng, the sum of the two standard curves ⁇ RE% ⁇ is 356.71 and 95.04, respectively.
  • Acceptance criteria the lowest concentration that meets the requirement of 60% sample CT value ⁇ CT value of blank mixed DNA or sensitivity sample CT has value and blank mixed DNA CT has no value is used as the detection limit of this method. In actual detection, this concentration is used as the detection limit (LOD). If the CT value of the sample to be tested is greater than the CT value of the lower limit of quantification (LLOQ), and the calculated concentration is greater than the limit of detection (LOD), the sample is defined as positive, but there is no accurate concentration.
  • LLOQ detection limit of quantification
  • LLOD limit of detection
  • the samples to be tested in the actual test include all the DNA solutions of the tissues and blood to be tested.
  • the blank matrix ie, test animal tissue and blood genome
  • the whole blood, lung, liver, and liver of rhesus monkeys were extracted. Choroid + RPE, iris DNA, and dilute it to the best detection concentration determined in the above section "4.3".

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Abstract

提供了用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA的DNA序列，以及扩增所述DNA序列所需的引物和探针，包含引物和探针的组合物或试剂盒；还提供了基于荧光定量PCR技术区分人源DNA的方法。

Description

一种基于荧光定量PCR技术区分人源DNA的方法 技术领域
本发明涉及一种基于荧光定量PCR技术区分人源DNA的方法，以及检测引物和探针。
背景技术
近年来，随着细胞类药物的快速发展，越来越多的细胞正被开发成细胞治疗药物，逐步从科研走向临床，细胞治疗是新药研发的趋势。细胞治疗类药物，研究其在体内药代动力学是判断其疗效和毒副作用不可或缺的环节。不同于传统的化学药物，对于细胞药物进入机体后的动态变化规律，包括其吸收、分布、代谢和***的过程和特征，目前国际上尚未能建立明确的细胞药物在体内药代动力学的研究方法。
非临床药代动力学研究，特别是动物实验，在新药研究开发的评价过程中起着重要作用。通过研究其在体内的动态，预测治疗中的合适计量，对降低细胞治疗的不确定性、增加疗效和减少毒副作用，都有很大的帮助。要将动物实验的结果作为一种评估细胞产品治疗效果的方法，将其与临床效果联系起来，势必需要找到一种灵敏度高且经过充分验证的方法来量化细胞产品在动物模型中的分布。与实验鼠、兔相比，实验猴在新药研究领域是一种非常重要的实验动物，因其与人的高度相似性，实验猴被认为是进行药物评价的重要工具。细胞药物在应用到人体之前，在与人亲缘关系比较相近的动物中进行比较***的评价是有必要进行的。
目前研究细胞在实验猴中分布的方法主要有动物活体成像、荧光蛋白标记、免疫组化、qPCR等。各种方法各有其优缺点。例如核磁共振(MRI)，MRI能反应细胞在体内的分布，该方法需要在体外标记细胞，通过成像的方式对注入机体内的细胞进行检测，从而判定细胞的存活及被清除的命运，该方法灵敏度高，标记时间长。但某些标记手段可能对细胞活性有影响，有报道称MRI标记骨髓间充质干细胞后，细胞分化能力受损。而且MRI灵敏度相对不足，原因是，在细 胞死亡后释放出的氧化铁颗粒(MRI标记物)，会造成非特异性显影，出现假阳性。例如绿色荧光蛋白标记(GFP)，该方法使用基因修饰，使得细胞能够表达荧光蛋白，绿色荧光信号可以通过荧光显微镜直接观察，该方法有易于检测的优点。但细胞的遗传物质被改变，无法确保这种改变百分百稳定。任何对细胞进行标记的方法都有可能对细胞造成改变，对子代细胞分化等造成影响。免疫组化的方法，不需要标记细胞，但该方法需要分析大量切片和微观观察，同时由于人与猴的相似性，很多抗原在人与猴之间存在交叉，且这种方法难以标准化，只能得到半定量结果。
qPCR的方法是一种灵敏度高，操作相对简单的方法，且有望实现定量分析。要想建立qPCR的方法，通过检测血液和组织样本中DNA来实现人源细胞在实验猴中的定量分析，首先要找到能够区分人与动物模型DNA的序列、以及引物。虽然文献中指出了一些人和猴某些特定基因表达的差异，但是由于实验用的细胞注射到猴以后，细胞可能分化成不再表达该特定基因的细胞，因此目前区分人和猴的细胞的基因会存在假阴性。即人细胞存在于猴组织中，但是其不能被检测出来。例如基于人源性特殊基因片段-Alu基因作为分子标志物设计的引物，希望通过qPCR的方法来实现人与猴DNA的区分，最终实现在猴细胞或组织中检测人细胞的目的。遗憾的是，这些引物只能区分人与啮齿动物。
例如2012年Pengyue Song等报道了，基于DNA特异性引物的高效可重复PCR方法，能够检测小鼠组织中异种移植的人细胞。2015Julie等发表文章，报道了采用qPCR测定大鼠和小鼠中移植人细胞数量的方法。但鲜少有文章报道，能通过qPCR的方式区分人与猴DNA。
发明内容
一方面，本发明公开了一种DNA序列，其中所述DNA序列选自如SEQ ID NO:1所示序列或其片段、与SEQ ID NO:1的反向互补序列或其片段组成的组，所述DNA序列用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA。
SEQ ID NO:1的序列为：
Figure PCTCN2021073569-appb-000001
Figure PCTCN2021073569-appb-000002
另一方面，本发明提供了一种DNA序列在制备用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA的试剂或试剂盒中的用途，其中所述DNA序列选自如SEQ ID NO:1所示序列或其片段、与SEQ ID NO:1的反向互补序列或其片段组成的组。
本发明还提供了一种检测DNA序列的试剂在制备用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA的试剂或试剂盒中的用途，其中所述DNA序列选自如SEQ ID NO:1所示序列或其片段、与SEQ ID NO:1的反向互补序列或其片段组成的组。
在一个具体实施例中，所述DNA序列为如SEQ ID NO:1所示序列或其反向互补序列，或这些全长序列的一部分片段，所述片段为SEQ ID NO:1或其返现个互补序列的5’端和/或3’端缺失1-70个核苷酸的序列，所述片段序列仍可用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA。
在一个具体实施例中，所述检测DNA序列的试剂选自通过PCR技术扩增所述DNA序列所需的引物和探针。
在一个具体实施例中，所述探针的序列如SEQ ID NO:10所示。
在一个具体实施例中，所述探针上带有检测标记，所述检测标记优选选自由FAM、TET、Alexa 488、Alexa 532、CF、HEX、VIC、ROX、Texas Red、QuasarFITC、cy3、cy5、6-joe、EDANS、rhodamine 6G、TMR、TMRITC、x-rhodamine、Texas red、生物素、和亲和素所组成的组。
在一个具体实施例中，所述引物的序列选自由如SEQ ID NO:2和SEQ ID NO:3所示的序列；如SEQ ID NO:2和SEQ ID NO:5所示的序列；如SEQ ID NO:4和SEQ ID NO:3所示的序列；如SEQ ID NO:6和SEQ ID NO:7所示的序列；和如SEQ ID NO:4和SEQ ID NO:7所示的序列组成的组。
在一个具体实施例中，所述非人类动物选自由恒河猴、绿猴、食蟹猴、大鼠、小鼠、兔组成的组。
在一个具体实施例中，所述混合人类与非人类动物组织为非人类动物例如恒河猴的组织及血液样本中混合了人源DNA，所述人源DNA来源于人源细胞。在一个具体实施例中，人源DNA来自于人源视网膜色素上皮细胞中的DNA。
再一方面，本发明公开了一种组合物，包含引物和探针，其中所述探针的序列如SEQ ID NO:10所示，所述引物的序列选自由如SEQ ID NO:2和SEQ ID NO:3所示的序列；如SEQ ID NO:2和SEQ ID NO:5所示的序列；如SEQ ID NO:4和SEQ ID NO:3所示的序列；如SEQ ID NO:6和SEQ ID NO:7所示的序列；和如SEQ ID NO:4和SEQ ID NO:7所示的序列组成的组。
其中，所述所述探针上带有检测标记，所述检测标记优选选自由FAM、TET、Alexa 488、Alexa 532、CF、HEX、VIC、ROX、Texas Red、QuasarFITC、cy3、cy5、6-joe、EDANS、rhodamine 6G、TMR、TMRITC、x-rhodamine、Texas red、生物素、和亲和素所组成的组。
又一方面，本发明还提供了一种试剂盒，包含如前所述的组合物。
进一步，本发明还提供了一种非诊断治疗目的在混合人类与非人类动物组织中区分人类和非人类动物DNA序列的方法，其中使用如前所述的组合物或如前所述的试剂盒对混合了人类和非人类动物组织的样本进行DNA的PCR扩增。
在一个具体实施例中，其步骤具体包括:
1)使用组合及细胞DNA提取试剂盒对混合了人类和非人类动物组织的样本进行DNA提取；
2)使用如前所述的组合物或如前所述的试剂盒中所述的引物和探针进行Taqman qPCR扩增；
3)收集荧光信号，计算循环阈值CT值，计算样本中人类DNA的浓度。
其中所述非人类动物选自由恒河猴、绿猴、食蟹猴、大鼠、小鼠、兔组成的组。
其中所述混合人类与非人类动物组织为非人类动物例如恒河猴的组织及血液样本中混合了人源DNA，所述人源DNA来源于人源细胞，优选来源于视网膜色素上皮细胞。
有益效果
本发明找到了人基因组染色体上的一段DNA序列，该DNA序列为人源特异性，在该DNA序列的基础上设计了一些引物及探针，能够区分人与多个物种的DNA。实际应用中可以实现从实验动物DNA中检测人源特异性DNA。
附图说明
图1示出靶向本发明的如SEQ ID NO:1所示的DNA序列时，使用R1、F1和Probe1检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)。
图2示出靶向本发明的如SEQ ID NO:1所示的DNA序列时，使用R2、F2和Probe1检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)。
图3示出靶向本发明的如SEQ ID NO:1所示的DNA序列时，使用R2、F1和Probe1检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)。
图4示出靶向本发明的如SEQ ID NO:1所示的DNA序列时，使用R3、F3和Probe1检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)。
图5示出靶向本发明的如SEQ ID NO:1所示的DNA序列时，使用R2、F3和Probe1检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)。
图6示出靶向基因SRGAP2时，检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)；
图7示出靶向基因Qhomo2时，检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)；
图8示出靶向基因Alu时，检测不同物种DNA的扩增曲线图(①为人源视网膜色素上皮细胞DNA；②为恒河猴DNA；③为绿猴DNA；④为食蟹猴DNA；⑤为大鼠DNA；⑥为小鼠DNA；⑦为兔DNA)；
图9示出实时荧光定量PCR检测人源视网膜色素上皮细胞目的DNA序列的典型标准曲线。
具体实施方式
下面将通过具体描述，对本发明作进一步的说明。
除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。
本申请中，单数形式“一个”、“该”包括复数对象，除非上下文另外清楚规定。
如本文所用，术语“非人类动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、大鼠、小鼠、两栖动物、爬行动物等。在具体实施例中，非人类动物选自由恒河猴、绿猴、食蟹猴、大鼠、小鼠、兔组成的组。
如本文所述，术语“DNA序列”本发明中是指编码蛋白的DNA序列，例如，但不限于，存在于细胞基因组中的编码蛋白的DNA序列。
如本文所述，术语“探针”在本发明中是指带有检测标记的寡核苷酸分子。“检测标记”在本发明中是指能够产生检测信号的分子或基团检测标记包括，但不限于，荧光分子(例如，参见欧洲专利EP144914)、放射性同位素(例如，参见美国专利US4358535和4446237)、抗体、酶和寡核苷酸(例如，寡核苷酸条形码)。
荧光分子的例子包括但不限于6-carboxyfluorescein(FAM)、Tetrachlorofluorescein(TET)、Alexa(例如Alexa 488,Alexa 532)、CF、HEX、VIC、ROX、Texas Red、QuasarFITC、cy3、cy5、6-joe、EDANS、rhodamine 6G(P6G)及其衍生物(tetramethyirhodamine(TMR),tetramethylrhodamine  isothiocyanate(TMRITC),x-rhodamine,Texas red,由位于美国俄勒冈州尤金市的分子探针公司(Molecular Probes,Inc.)生产的商品名为"BODJPY FL"、"BODIPY FL/C3"、"BODIPY EL/C6"、"BODIPY 5-FAM"、"BODIPY TMR"、"BODIPY TR"、"BODIPY R6G"、"BODIPY 564"、"BODIPY 581"的探针及衍生物。
检测标记的示例还可以参见美国专利号5,723,591和5,928,907；WO2011066476和WO2012149042；www.idahotech.com；Gudnason等人，NucleicAcids Res.，35(19)：e127(2007)，其全文通过引用并入本发明。
检测标记可以通过共价键或非共价键连接在寡核苷酸分子上。非共价键包括但不限于氢键、离子键、范德华力和疏水键。例如，在一些实施方式中，检测标记可以通过共价键连接在核苷酸分子上。例如，在合成寡核苷酸分子时可以掺入氨基烯丙基(amino-allyl)UTP，所产生的氨基烯丙基标记的核酸分子可以和含有NHS－酯(NHS-ester)的荧光分子(例如Alexa 488,Alexa 594,Alexa 647(Invitrogen)或Cy3(GE Healthcare))偶联形成共价键连接。
在某些实施方式中，所述检测标记选自：FAM、Tetrachlorofluorescein(TET)、Alexa 488、Alexa 532、CF、HEX、VIC、ROX、Texas Red、QuasarFITC、cy3、cy5、6-joe、EDANS、rhodamine 6G(P6G)、tetramethyirhodamine(TMR)、tetramethylrhodamine isothiocyanate(TMRITC)、x-rhodamine、Texas red、生物素、亲和素。
在某些实施方式中，所述探针还带有可淬灭信号。“淬灭剂”在本发明中是指，当与检测标记在空间上足够靠近时，能够阻止检测标记产生检测信号的分子。当淬灭剂与检测标记距离较远时，淬灭剂不能阻止检测信号的产生。
淬灭分子的例子包括但不限于DDQ-I、DDQ-II、Dabcyl、Eclipse、Iowa Black FQ、Iowa Black RQ、BHQ-1、BHQ-2、BHQ-3、"QSY7"“QSY-21”和"QSY33"(分子探针公司)、Ferrocene及其衍生物、methyl viologen、tetramethylrhodamine(TAMRA)、Minor groove binding non-fluorescent quencher(MGBNFQ)和N,N'-dimethyl-2,9-diazopyrenium。
在某些实施方式中，所述荧光分子是FAM，所述淬灭分子是MGBNFQ或DDQ-I。在某些实施方式中，所述荧光分子是TAMRA、Cy3、ROX、Cy5，所述 淬灭分子是DDQ-II。在某些实施方式中，所述荧光分子是FAM、HEX、ROX、JOE，所述淬灭分子是Dabcyl。在一些实施方式中，所述探针在5'端具有荧光分子FAM或VIC，在3'端具有淬灭分子MGBNFQ。
可以通过本领域公知的方法将淬灭分子连接在探针上。例如，在合成寡核苷酸分子时可以掺入氨基烯丙基(amino-allyl)UTP，所产生的氨基烯丙基标记的核酸分子可以和含有NHS－酯(NHS-ester)的淬灭分子偶联形成共价键连接。又例如，可以在合成寡核苷酸的过程中在3’端通过和淬灭分子(例如Dabcyl)的亚磷酰胺衍生物反应将淬灭分子连接到寡核苷酸上。
在某些实施方式中，当所述探针是完整的时，所述信号被淬灭。在某些实施方式中，检测标记和淬灭剂分别连接在探针的5’端和3’端。例如，非突变区探针的5’端连接检测标记，3’端连接淬灭剂，或者3’端连接检测标记，5’端连接淬灭剂。
在某些实施方式中，使用具有5’-3’外切酶活性的聚合酶扩增含有SEG ID NO:1或其互补序列，或与SEQ ID NO:1序列或其互补序列片段作为所述模板序列，并在反应混合物中加入所述探针。在扩增过程中，如果所述探针与模板序列杂交，所述探针将会被聚合酶在聚合反应过程中降解，从而使所述探针上的荧光分子与淬灭分子分离，并产生荧光信号(参见美国专利US5210015和US5487972)。
本文所述的术语“片段”是指在与本发明中所述DNA序列如SEQ ID NO:1所示序列或其反向100％互补序列的片段相比，其5’端和/或3’端缺失部分核苷酸的序列，例如与SEQ ID NO:1所示序列相比，5’端缺失了1-70个、2-70个、5-70个、10-70个、20-70个、30-70个、40-70个、50-70个、60-70个核苷酸的序列，或者3’端缺失了1-70个、2-70个、5-70个、10-70个、20-70个、30-70个、40-70个、50-70个、60-70个核苷酸的序列，或者5’端和3’端同时缺失了1-70个、2-70个、5-70个、10-70个、20-70个、30-70个、40-70个、50-70个、60-70个核苷酸的序列，这个片段通过PCR扩增后按照本领域技术人员的认识依然可以扩增出本发明实施例中相同或者相似的长度，同样达到用于检测和区别人类和非人类组织DNA的目的。本领域技术人员应该了解，针对SEQ ID NO:1的PCR，通过引物探针最终获得的PCR产物片段的长度可以是SEQ ID NO:1全长，也可以是其部分。
实施例
通过以下实施例进一步说明本发明。提供实施例仅用于说明目的，并且不应被解释为以任何方式限制本发明的范围或内容。
实施例1：检测方法特异性验证
1.引物的设计与合成：
本发明在人源特异性DNA序列Seq1(SEQ ID NO:1)的基础上设计了多对引物及探针，同时从文献及专利中找到了3组人源特异性性基因及其引物(SRGAP2、Qhomo2及Alu)作为对照。其中Qhomo2的引物应用自文献(《脐带间充质干细胞临床前安全性研究》，王有为，北京协和医学院,2013年)，根据报道该引物可以特异性检测人源DNA。SRGAP2的引物引用自专利CN201910477468.2《特异性检测人源性基因组DNA引物及其应用》，根据报道该引物能从多个物种DNA(包括食蟹猴、大鼠、小鼠、新西兰兔)中检测人源特异性DNA序列。而引物Alu扩增的基因Alu序列是人类基因组中具有普遍性、多样性和特异性的短重复序列，Alu家族元件在法医DNA分析中能被用来进行个体识别。也有文献报道Alu序列能用于人与其他物种DNA的区分，本发明的Alu引物及其探针应用自文献。表1.1为本发明中使用的的所有引物和探针的序列，其中探针(Probe)的5’端带有报告基团，3’端带有淬灭基团。
表1.1人源特异性DNA检测引物序列表
Figure PCTCN2021073569-appb-000003
Figure PCTCN2021073569-appb-000004
2.DNA提取
使用组织及细胞DNA提取试剂盒，按照试剂盒的说明书提取不同物种(人、恒河猴、绿猴、食蟹猴、大鼠、小鼠和兔)来源的细胞及组织的基因组DNA。
3.Taqman qPCR步骤
(1)Taqman qPCR扩增的反应体系，单个样本为20μL：10μL 2×SuperReal PreMix(Probe)；1μL 50×ROX Reference Dye；0.6μL引物R(10μM)；0.6μL引物F(10μM)；0.4μL探针Probe(10μM)；加入20ng的DNA；用RNase-Free ddH 2O将反应体系补足20μL。该步骤使用的引物及探针及其组合见下表，一共13组引物及探针组合。
表1.2人源特异性DNA检测引物组合表
Figure PCTCN2021073569-appb-000005
(2)Taqman qPCR扩增的反应条件为：95℃预变性15分钟；95℃变性1秒；62℃退火延伸30秒，共进行40个循环；在62℃处收集荧光信号。实验结束后从仪器中获取CT值。
(3)根据(2)的实验得到的CT值判定上述引物和探针检测人源DNA的效果， 有CT值说明有扩增，无CT说明无扩增。若某一组引物及探针的组合，在其他物种中无扩增，只在人DNA样本中有扩增，说明该组合能扩增人源特异性DNA序列。
4.实验结果
表1.3人源特异性DNA检测结果统计表
Figure PCTCN2021073569-appb-000006
表1.3中为实验结果：其中“+”表示有扩增，“-”表示无扩增；“/”表示未进行实验。SRGAP2、Qhomo2及Alu为文献或专利中报道过的人源特异性基因，探针及引物均来自报道，实际在我们的实验中验证后表明，在区分人与实验猴DNA的实验中仅仅只有Qhomo2能区分人与绿猴DNA，SRGAP2、Alu区分不了人与三种实验猴(绿猴、食蟹猴及恒河猴)DNA。本发明中发现的DNA序列Seq1，针对该DNA序列设计的部分引物及探针的组合(例如R2、F2加上Probe2)无法实现人与猴DNA的区分，也有一部分(例如R1、F1加上Probe1)可以实现人与 多个物种(包括三种猴、大鼠、小鼠及兔)之间DNA的区分，图1、图6、图7、图8分别为引物组合R1、F1加上Probe1、SRGAP2、Qhomo2、Alu的扩增曲线图。图2至5示出了其他引物组合扩增DNA序列Seq1的扩增曲线图。实验结果也表明，本发明中合成的引物和探针，可以实现在多个物种中检测人源DNA。
表1.4引物的位置
Figure PCTCN2021073569-appb-000007
表1.5扩增片段长度
Figure PCTCN2021073569-appb-000008
实施例2:定量方法的开发及方法学验证
5组可以实现人与三种猴DNA区分的引物及探针的组合R1(SEQ ID NO:2)、 F1(SEQ ID NO:3)加上Probe1(SEQ ID NO:10)；R1(SEQ ID NO:2)、F2(SEQ ID NO:5)加上Probe1(SEQ ID NO:10)；R2(SEQ ID NO:4)、F1(SEQ ID NO:3)加上Probe1(SEQ ID NO:10)；R3(SEQ ID NO:6)、F3(SEQ ID NO:7)加上Probe1(SEQ ID NO:10)；R2(SEQ ID NO:4)、F3(SEQ ID NO:7)加上Probe1(SEQ ID NO:10)。以下仅以其中一对引物及探针的组合(R1、F1加上Probe1)为例进行定量方法的开发及方法学验证。本领域技术人员应理解，其他引物因为可以区别人和其他物种，具有较好的特异性，因此应该也具有相似的方法学验证的效果。
1.标准曲线和质控样本的配制
1.1标准曲线样本的配制
根据下表配制标准曲线样本，其中标准品为人源视网膜色素上皮细胞注射液(含有人源视网膜色素上皮细胞的细胞溶液，该溶液可用于向恒河猴的双眼视网膜下注射治疗)的全基因组DNA(浓度约为：140ng/μL)，按下表进行稀释先加入一定量体积的纯水，再加入相应体积人源视网膜色素上皮细胞注射液全基因组DNA、STD1～STD6到离心管中，涡旋混匀备用。
表2.1标准曲线样本的配制
Figure PCTCN2021073569-appb-000009
注：以上配制可根据实际需要按比例放大或缩小。
1.2质控样本的配制
根据下表配制质控样本，其中标准品为人源视网膜色素上皮细胞全基因组DNA(浓度约为：140ng/μL)，按下表进行稀释先加入一定量体积的纯水，再加入相应体积的人源视网膜色素上皮细胞注射液全基因组DNA、定量上限ULOQ、高质控HQC、中质控MQC、质控C、低质控LQC、定量下限LLOQ，进而配制成ULOQ(100ng/μL)、HQC(80ng/μL)、MQC(3.2ng/μL)、C(0.8ng/μL)、LQC(0.08ng/μL)及LLOQ(0.032ng/μL)。
表2.2质控品样本的配制
Figure PCTCN2021073569-appb-000010
注：以上配制可根据实际需要按比例放大或缩小。
2.检测步骤
2.1DNA提取
使用组织及细胞DNA提取试剂盒，按照试剂盒的说明书提取细胞及组织的基因组DNA。
2.2Taqman qPCR步骤
(1)Taqman qPCR扩增的反应体系单个样本为20μL：10μL 2×SuperReal PreMix(Probe)；1μL 50×ROX Reference Dye；0.6μL引物R(10μM)；0.6μL引物F(10μM)；0.4μL探针Probe(10μM)；加入2μL的DNA(DNA模板分别为人源视网膜色素上皮细胞全基因组DNA、标准曲线样本、质控品样本、待测样本、空白基质阴性对照样本(Neg)和纯水无模板阴性对照(NTC))；用RNase-Free ddH 2O将反应体系补足20μL。
(2)Taqman qPCR扩增的反应条件为：95℃预变性15分钟；95℃变性1秒；62℃退火延伸30秒，共进行40个循环；在62℃处收集荧光信号。实验结束后从仪器中获得CT值，扩增效率(Efficiency)，标准曲线的R 2及标准曲线方程的Slope和截距。
3.数据处理
从获取的标准品CT值，扩增效率(Efficiency)，R 2及标准曲线方程的Slope和截距，计算标准曲线样本、HQC、MQC、LQC样本及待测样本的目的片段等的DNA浓度，Conc.＝10 (CT值-y-int)/Slope，浓度数据四舍五入后保留小数点后3位，％RE(相对误差)，％CV(变异系数)保留小数点后2位。
报告中所用％RE、标准偏差(SD)、％CV以及目的DNA浓度等值均由Office  Excel 2010(美国Microsoft公司)软件计算得出。计算公式如下：
平均值：
Figure PCTCN2021073569-appb-000011
(C t为实测浓度)；
相对误差百分比：
Figure PCTCN2021073569-appb-000012
(C t为实测浓度，C n为理论浓度)；
变异系数百分比：
Figure PCTCN2021073569-appb-000013
(
Figure PCTCN2021073569-appb-000014
为检测浓度平均值)；
标准偏差：
Figure PCTCN2021073569-appb-000015
(
Figure PCTCN2021073569-appb-000016
为检测浓度平均值)。
样本判断标准：1、当LLOQ和LOD的CT值均小于NTC和Neg的CT值：(1)待测样本的复孔CT值均小于LLOQ的平均CT值，出具浓度结果；(2)待测样本的复孔CT值均小于LOD的CT值，表示该样本为阳性；(3)如果LOD未出值，该批样本出具浓度结果；2、当Neg和NTC的CT值有一个小于LOD的CT值或者LOD未出值，则只有当待测样本的复孔CT值均小于LLOQ的平均CT值，出具浓度结果，NTC最小CT值与LLOQ之间为阳性样本；3、如果Neg和NTC的CT值有一个小于LLOQ的CT值，则只有当待测样本的复孔CT值均小于Neg和NTC的中最小的CT值，出具浓度结果。
4.方法学验证结果
4.1标准曲线与定量下限
按试验方案1.1配制标准曲线样本，上机检测后获取标准品CT值、扩增效率(Efficiency)、R 2及标准曲线方程的斜率(Slope)和截距，进而获得标准曲线。确定方法的线性范围和定量下限(标准曲线的最低点)。至少2人分至少2天进行至少6个分析批的验证，统计各分析批内各浓度与理论浓度的相对误差(RE％)及所有分析批间各浓度的平均相对误差(RE％)和精密度(CV％)。
接受标准：各分析批内、批间的标准曲线样本浓度满足相对误差(RE％)在-75％～150％之间；批间精密度(CV％)≤60.00％；所有分析批需满足R 2≥0.980。
结果显示：qPCR检测人源视网膜色素上皮细胞注射液目的片段的标准曲线的线性范围为：0.032～100.000ng/μL，定量下限为：0.032ng/μL。R 2在0.991～0.999范围内；标准曲线各浓度点的批内准确度％RE在-16.75～43.75％范围内；各浓度点的批间准确度％RE在-4.53～12.50％范围内，精密度％CV在5.42～13.89％范围内；满足标准曲线准确度、精密度要求。具体结果见表2.3，典型标准曲线见图9，标 准曲线拟合参数汇总见表2.4。
表2.3实时荧光定量PCR检测人源视网膜色素上皮细胞注射液目的DNA序列的标准曲线结果
Figure PCTCN2021073569-appb-000017
备注：“/”表示无计算数据。
表2.4标准曲线拟合参数汇总
Figure PCTCN2021073569-appb-000018
备注：标准曲线拟合公式：CT＝Slope LgX 0+y-int；其中X 0为样本起始浓度，y-int为截距，Slope为斜率。
4.2精密度与准确度
为了验证此方法的批内、批间精密度(Precision)和相对误差(Accuracy)，在同一分析批内按表2.2项配制成ULOQ、HQC、MQC、LQC、LLOQ 5个浓度的质控样本各3套，至少2人分至少2天进行至少6个分析批的验证。统计各质控样本浓度批内的精密度(CV％)、平均相对误差(％RE)以及批间总的精密度 (％CV)、平均相对误差(RE％)。
接受标准：各浓度批内、批间的平均相对误差(RE％)在-75％～150％以内；各浓度批内、批间的精密度(CV％)≤60.00％。
结果显示，各质控样本浓度的批内准确度％RE在-22.50～41.25％范围内，批内精密度％CV在1.37～60.00％范围内；各质控样本浓度的批间准确度％RE在-0.91～15.60％范围内，批间精密度％CV在9.28～35.71％范围内，满足批内、批间准确度、精密度要求。以上数据表明该分析方法的准确度和精密度符合要求，具体结果见表2.5。
表2.5实时荧光定量PCR检测人源视网膜色素上皮细胞注射液目的DNA序列的精密度与准确度
Figure PCTCN2021073569-appb-000019
Figure PCTCN2021073569-appb-000020
4.3不同空白基质基因组质量及浓度对目的DNA序列检测的影响
提取空白恒河猴的全血、肺、肝脏、脉络膜+RPE(视网膜色素上皮细胞)、虹膜DNA作为干扰基质。将标准曲线样本加入分别含有空白肺、肝脏DNA总量为200ng、100ng的反应体系中；将标准曲线样本加入分别含有空白脉络膜+RPE、虹膜、全血DNA总量为100ng、40ng的反应体系中(当空白基质DNA不足以补充到相对应的DNA总量时，以实际总量进行配制)。对样本进行检测，计算添加空白基质DNA的标准曲线各浓度与理论浓度的相对误差(RE％)。
接受标准：比较每个组织添加空白基质DNA的两个标准曲线∣RE％∣总和，并将∣RE％∣总和小的总量作为实际样本检测时反应体系中模板的添加量；如果两套标准曲线∣RE％∣总和接近，则选择标准曲线上低浓度点∣RE％∣较小的这套标准曲线中总量作为实际样本检测时反应体系中模板的添加量，并计算出 最佳的检测浓度。在实际样本检测时，当实际样本浓度大于最佳检测浓度20％时，要将样本稀释为最佳检测浓度，如果实际样本浓度小于最佳检测浓度，则按实际浓度进行检测。
结果显示，空白恒河猴的全血DNA的总量分别为100ng、40ng时，两个标准曲线∣RE％∣总和分别为207.11、125.32，得出全血样本检测时反应体系中模板的添加量为40ng，样本的最佳的检测浓度为20ng/μL；空白恒河猴的肺脏DNA的总量分别为200ng、100ng时，两个标准曲线∣RE％∣总和分别为549.81、311.49，得出肺脏样本检测时反应体系中模板的添加量为100ng，样本的最佳的检测浓度为50ng/μL；空白恒河猴的肝脏DNA的总量分别为200ng、100ng时，两个标准曲线∣RE％∣总和分别为98.51、113.79，得出肝脏样本检测时反应体系中模板的添加量为200ng，样本的最佳的检测浓度为100ng/μL；空白恒河猴的脉络膜+RPE DNA的总量分别为100ng、40ng时，两个标准曲线∣RE％∣总和分别为356.71、95.04，得出脉络膜+RPE样本检测时反应体系中模板的添加量为40ng，样本的最佳的检测浓度为20ng/μL；空白恒河猴的虹膜DNA的总量分别为100ng、40ng时，两个标准曲线∣RE％∣总和分别为576.19、218.31，得出虹膜样本检测时反应体系中模板的添加量为40ng，样本的最佳的检测浓度为20ng/μL。具体结果见表2.6。
表2.6恒河猴的不同空白基质基因组质量及浓度对目的DNA序列检测的影响
Figure PCTCN2021073569-appb-000021
Figure PCTCN2021073569-appb-000022
Figure PCTCN2021073569-appb-000023
备注：“*”表示未出值，实际浓度以0计算。
4.4检测限(LOD)
用纯水将LLOQ稀释成浓度为S1(0.016ng/μL)、S2(0.008ng/μL)、S3(0.004ng/μL)、S4(0.002ng/μL)的样本，每个浓度16个单孔进行检测确定方法的灵敏度，同时以空白恒河猴的全血、肺、肝脏、脉络膜+RPE、虹膜混合DNA作为阴性对照,进行检测确定方法的检出限(LOD)，若某一浓度的样本已经达不到接受标准，则不进行比该浓度低的样本的检测。
接受标准：以满足60％样本的CT值<空白混合DNA的CT值或者灵敏度样本CT有值而空白混合DNA CT无值的最低浓度作为该方法的检测限，实际检测中以此浓度作为检测限(LOD)。若待测样本CT值大于定量下限(LLOQ)的CT值，计算出的浓度大于检测限(LOD)，则定义该样本为阳性，但无准确浓度。
结果显示，当浓度为S1(0.016ng/μL)时，62.50％的S1样本的CT出值， 空白混合DNA未出值；当浓度为S2(0.008ng/μL)时，31.25％的S2样本的CT值<空白混合DNA的CT值，得出该方法的检出限为S1(0.016ng/μL)。具体结果见表2.7和表2.8。
表2.7实时荧光定量PCR检测人源视网膜色素上皮细胞注射液目的DNA序列的检测限测定结果
(一)
Figure PCTCN2021073569-appb-000024
备注：“NaN”表示无CT值。
表2.8实时荧光定量PCR检测人源视网膜色素上皮细胞注射液目的DNA序列的检测限测定结果
(二)
Figure PCTCN2021073569-appb-000025
Figure PCTCN2021073569-appb-000026
备注：“NaN”表示无CT值。
4.5选择性
实际检测中的待检样本包含各待检组织及血液所有的DNA溶液，为了评价空白基质(即试验动物组织及血液基因组)对于样本检测的影响，分别提取恒河猴全血、肺、肝脏、脉络膜+RPE、虹膜的DNA，并将其稀释为上述“4.3”部分中确定的最佳检测浓度，检测空白基质的CT，需没有明显的内源性DNA干扰影响样本阴阳性判定。
接受标准：恒河猴空白恒河猴全血、肺、肝脏、脉络膜+RPE、虹膜的DNA及纯水(NTC)的每个孔的CT值＞LOD复孔中大的CT(或空白基质显示无CT)。
结果显示，恒河猴空白全血、肺、脉络膜+RPE、虹膜的DNA及纯水(NTC)的CT值均显示无CT，只有肝脏的复孔中有一个CT为39.54，大于LOD复孔中大的CT值，符合要求，表明没有明显的内源性DNA干扰影响样本阴阳性判定。具体结果见表2.9。
表2.9实时荧光定量PCR检测人源视网膜色素上皮细胞注射液目的DNA序列的选择性测定结果
Figure PCTCN2021073569-appb-000027
备注：“NaN”表示无CT
5.结论
以上验证结果表明，用实时荧光定量PCR检测恒河猴体内人源DNA(人源视网膜色素上皮细胞DNA)的方法的线性范围为：0.032～100.000ng/μL，定量下限为0.032ng/μL，检测限为0.016ng/μL；精密度及准确度符合要求，无明显的内源性DNA干扰影响样本阴阳性判定，选择性良好，可用于恒河猴组织及血液样本中人源视网膜色素上皮细胞目的DNA序列的浓度检测。
前述实施例被认为是说明性的，而不是对本文所述的本发明的限制。本发明的范围由所附权利要求书而不是由前述说明书表示，并且意在将落入权利要求书的等同物的含义和范围内的所有改变包括在其中。

Claims (16)

  1. 一种DNA序列，其中所述DNA序列选自如SEQ ID NO:1所示序列或其片段、与SEQ ID NO:1的反向互补序列或其片段组成的组，所述DNA序列用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA。
  2. 一种DNA序列在制备用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA的试剂或试剂盒中的用途，其中所述DNA序列选自如SEQ ID NO:1所示序列或其片段、与SEQ ID NO:1的反向互补序列或其片段组成的组。
  3. 一种检测DNA序列的试剂在制备用于在混有人类与非人类动物组织样本中区分人类和非人类动物DNA的试剂或试剂盒中的用途，其中所述DNA序列选自如SEQ ID NO:1所示序列或其片段、与SEQ ID NO:1的反向互补序列或其片段组成的组。
  4. 如权利要求3所述的用途，其中所述检测DNA序列的试剂选自通过PCR技术扩增所述DNA序列所需的引物和探针。
  5. 如权利要求4所述的用途，其中所述探针的序列如SEQ ID NO:10所示。
  6. 如权利要求5所述的用途，其中所述探针上带有检测标记，所述检测标记优选选自由FAM、TET、Alexa 488、Alexa 532、CF、HEX、VIC、ROX、Texas Red、QuasarFITC、cy3、cy5、6-joe、EDANS、rhodamine 6G、TMR、TMRITC、x-rhodamine、Texas red、生物素、和亲和素所组成的组。
  7. 如权利要求4所述的用途，其中所述引物的序列选自由如SEQ ID NO:2和SEQ ID NO:3所示的序列；如SEQ ID NO:2和SEQ ID NO:5所示的序列；如SEQ ID NO:4和SEQ ID NO:3所示的序列；如SEQ ID NO:6和SEQ ID NO:7所示的序列；和如SEQ ID NO:4和SEQ ID NO:7所示的序列组成的组。
  8. 如权利要求1所述的DNA序列和如2至7中任一项所述的用途，其中所述非人类动物选自由恒河猴、绿猴、食蟹猴、大鼠、小鼠、兔组成的组。
  9. 如权利要求1所述的DNA序列和如2至7中任一项所述的用途，其中所述混合人类与非人类动物组织为非人类动物例如恒河猴的组织及血液样本中混合了人源DNA，所述人源DNA来源于人源细胞，优选来源于视网膜色素上皮细胞。
  10. 一种组合物，包含引物和探针，其中所述探针的序列如SEQ ID NO:10所示，其中所述引物的序列选自由如SEQ ID NO:2和SEQ ID NO:3所示的序列；如SEQ ID NO:2和SEQ ID NO:5所示的序列；如SEQ ID NO:4和SEQ ID NO:3所示的序列；如SEQ ID NO:6和SEQ ID NO:7所示的序列；和如SEQ ID NO:4和SEQ ID NO:7所示的序列组成的组。
  11. 如权利要求10所述的组合物，其中所述探针上带有检测标记，所述检测标记优选选自由FAM、TET、Alexa 488、Alexa 532、CF、HEX、VIC、ROX、Texas Red、QuasarFITC、cy3、cy5、6-joe、EDANS、rhodamine 6G、TMR、TMRITC、x-rhodamine、Texas red、生物素、和亲和素所组成的组。
  12. 一种试剂盒，包含如权利要求10或11所述的组合物。
  13. 一种非诊断治疗目的在混合人类与非人类动物组织中区分人类和非人类动物DNA序列的方法，其中使用如权利要求10或11所述的组合物或如权利要求12所述的试剂盒对混合了人类和非人类动物组织的样本进行DNA的PCR扩增。
  14. 如权利要求13所述的方法，其步骤具体包括:
    1)使用组合及细胞DNA提取试剂盒对混合了人类和非人类动物组织的样本进行DNA提取；
    2)使用如权利要求10或11所述的组合物或如权利要求12所述的试剂盒中所述的引物和探针进行Taqman qPCR扩增；
    3)收集荧光信号，计算循环阈值CT值，计算样本中人类DNA的浓度。
  15. 如权利要求13或14所述的方法，其中所述非人类动物选自由恒河猴、绿猴、食蟹猴、大鼠、小鼠、兔组成的组。
  16. 如权利要求13或14所述的方法，其中所述混合人类与非人类动物组织为非人类动物例如恒河猴的组织及血液样本中混合了人源DNA，所述人源DNA来源于人源细胞，优选来源于视网膜色素上皮细胞。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7537889B2 (en) * 2003-09-30 2009-05-26 Life Genetics Lab, Llc. Assay for quantitation of human DNA using Alu elements
CN101988123A (zh) * 2010-08-24 2011-03-23 中国医学科学院医学实验动物研究所 人源细胞的人线粒体dna特异片段扩增检测法
WO2018101375A1 (ja) * 2016-11-30 2018-06-07 国立大学法人秋田大学 ヒトゲノムｄｎａ検出方法
CN108384842A (zh) * 2018-02-27 2018-08-10 宁波海尔施基因科技有限公司 一种对疑是人源的未知生物样本进行物种及人源个体识别鉴定的复合pcr扩增方法
CN110218770A (zh) * 2019-06-03 2019-09-10 上海爱萨尔生物科技有限公司 特异性检测人源性基因组dna引物及其应用
CN111041106A (zh) * 2019-12-30 2020-04-21 江苏艾尔康生物医药科技有限公司 一种基于荧光定量pcr技术区分人源dna的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2016281193B2 (en) * 2015-06-15 2022-06-16 Murdoch Childrens Research Institute Method of measuring chimerism

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7537889B2 (en) * 2003-09-30 2009-05-26 Life Genetics Lab, Llc. Assay for quantitation of human DNA using Alu elements
CN101988123A (zh) * 2010-08-24 2011-03-23 中国医学科学院医学实验动物研究所 人源细胞的人线粒体dna特异片段扩增检测法
WO2018101375A1 (ja) * 2016-11-30 2018-06-07 国立大学法人秋田大学 ヒトゲノムｄｎａ検出方法
CN108384842A (zh) * 2018-02-27 2018-08-10 宁波海尔施基因科技有限公司 一种对疑是人源的未知生物样本进行物种及人源个体识别鉴定的复合pcr扩增方法
CN110218770A (zh) * 2019-06-03 2019-09-10 上海爱萨尔生物科技有限公司 特异性检测人源性基因组dna引物及其应用
CN111041106A (zh) * 2019-12-30 2020-04-21 江苏艾尔康生物医药科技有限公司 一种基于荧光定量pcr技术区分人源dna的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUANG JICHENG, HUANG HUI-MIN;LIU YAN;LI XIAO-BO;ZHENG KUI;SHI YONG-XIA: "Development of a real -time fluorescence PCR detecting method for human genes in imported biological materials", CHINESE FRONTIER HEALTH QUARANTINE, vol. 39, no. 1, 1 February 2016 (2016-02-01), pages 1 - 4, XP055826778, ISSN: 1004-9770, DOI: 10.16408/j.1004-9770.2016.01.001 *
JERILYN A. WALKER, GAIL E. KILROY, JINCHUAN XING, JAIPRAKASH SHEWALE, SUDHIR K. SINHA , MARK A. BATZER: "Human DNA quantitation using Alu element-based polymerase chain reaction.", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, AMSTERDAM, NL, vol. 315, no. 1, 1 April 2003 (2003-04-01), Amsterdam, NL, pages 122 - 128, XP002627311, ISSN: 0003-2697, DOI: 10.1016/S0003-2697(03)00081-2 *

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