WO2020243978A1 - 特异性检测人源性基因组dna的引物及其应用 - Google Patents

特异性检测人源性基因组dna的引物及其应用 Download PDF

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WO2020243978A1
WO2020243978A1 PCT/CN2019/090571 CN2019090571W WO2020243978A1 WO 2020243978 A1 WO2020243978 A1 WO 2020243978A1 CN 2019090571 W CN2019090571 W CN 2019090571W WO 2020243978 A1 WO2020243978 A1 WO 2020243978A1
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human
genomic dna
sample
derived
srgap2
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French (fr)
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高歌
周安宇
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上海爱萨尔生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the invention belongs to the field of molecular biological detection, and more specifically relates to the detection of human-derived DNA.
  • Cell therapy is the transplantation of healthy stem cells into patients or themselves to achieve the purpose of repairing disease or rebuilding normal cells and tissues.
  • transplanting human-derived cells into immunodeficient mice has been widely used to study the function of stem cells in tissue repair and regeneration or cancer cell metastasis (1), and confirm cell therapy products (human-derived cells) transplantation After entering the host, its biological distribution in the host, migration or residue over time, therapeutic effect and biological safety are an important step in pre-clinical testing (2).
  • Some methods are used to detect the biodistribution of transplanted cells. For example, microscopic observation of tissue samples is widely used to detect the presence of transplanted cells.
  • Use cell labeling techniques such as cell membrane dye (DII) or nuclear dye (DAPI) to track the distribution of fluorescently labeled cells in the recipient tissue (5).
  • DII cell membrane dye
  • DAPI nuclear dye
  • this technique has its drawbacks. First, the signal may not be detected when the transplanted cell content is relatively low; secondly, the fluorescent dye is easily diluted by cell division so that it may be below the detection limit; finally, locate the cell The distribution in the body may be affected by sample errors, resulting in reduced sensitivity and reliability of the results (6).
  • genetic modification of transplanted cells can prevent the dye from being diluted, the modified cells may affect their normal function or change their original biological distribution (7). Therefore, it is particularly important to find new detection methods.
  • qPCR real-time fluorescent quantitative PCR
  • the probe of the TaqMan probe method has a 5'-end fluorescent reporter group and a 3'-end quenching group.
  • the complete probe undergoes fluorescence resonance energy transfer after being excited by the excitation light, so no signal can be detected; only when the DNA is replicated , The probe is hydrolyzed, and the reporter group and the quencher group are separated before the fluorescence can be detected. Therefore, the intensity of the fluorescence signal represents the number of templates, because the number of released fluorescent groups and the number of PCR products are a pair Therefore, this technology can accurately quantify the template.
  • the amplification efficiency of SYBR Green and TaqMan technology is not very different (9), TaqMan technology is more specific and sensitive, and is suitable for the detection and quantification of human-derived cells in xenotransplantation systems (10).
  • primers targeting highly repetitive sequences of the human genome (such as ⁇ -satellite, Alu) were used in qPCR experiments (10), but due to the highly active mobility of these sequences in the human genome, the same amount of The template DNA may contain highly variable target sequences, thus leading to inconsistent results.
  • the present invention designs and synthesizes human-specific primer probes.
  • the present invention selects the SRGAP2 gene.
  • SRGAP2 GenBank accession#: NC_000001.11
  • Slit-Robo Rho GTPase activating protein 2 which plays a role in the development of the cerebral cortex and can regulate the migration and differentiation of neurons (13). Studies have found that SRGAP2 is highly conserved in the evolution of mammals, and humans are the only lineage of its gene duplication.
  • SRGAP2 is a three- or four-copy gene, SRGAP2A (NC_000001.11: 206203556..206464443), SRGAP2B ( NC_000001.11:144887191..145095321), SRGAP2C (NC_000001.11:121184967..121392874) and SRGAP2D (NC_000001.11:143972639..144069704), the primers designed are more sensitive than FOXP2.
  • the present invention provides the application of human SRGAP2 gene in detecting human genomic DNA in samples.
  • Another aspect of the present invention provides the application of a reagent for specifically detecting human SRGAP2 gene in detecting human genomic DNA in a sample.
  • Another aspect of the present invention provides the application of a reagent for specifically detecting human SRGAP2 gene in preparing a reagent for detecting human genomic DNA in a sample.
  • the reagent for specifically detecting human SRGAP2 gene comprises a primer pair for specifically detecting SRGAP2 gene.
  • the primer pair includes a forward primer 5'-CGATACTCAGGTCAAAGGTAAGG-3' (SEQ ID NO: 1) and a reverse primer 5'-CTGCAAATCACGGTGGAAATAC-3' (SEQ ID NO: 2).
  • the reagent for specifically detecting the human SRGAP2 gene further comprises a fluorescent probe 5'-TGCAAATGCTCTGTGGACTGGTGA-3' (SEQ ID NO: 3), and the 5'end of the probe is labeled with a reporter fluorophore, 3 The'end is labeled with a quenching group.
  • the reporter fluorophore labeled at the 5'end of the fluorescent probe is FAM
  • the quenching group labeled at the 3'end is NFQ-MGB or TAMRA.
  • primers and probes that can specifically amplify and detect the human SRGAP2 gene according to the sequence of the SRGAP2 gene.
  • the detection is by qPCR detection or by digital PCR detection.
  • qPCR also known as Real-time Quantitative PCR
  • the Ct value represents the cycle threshold, that is, the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold. Since the Ct value of each template has a linear relationship with the logarithm of the initial content of the template, the more the initial copy number, the smaller the Ct value.
  • a standard curve can be made using serially diluted standards with known initial content, where the abscissa represents the logarithm of the initial content, the ordinate represents the Ct value, or the ordinate represents the logarithm of the initial content, and the abscissa represents the Ct value. .
  • the content of the sample can be calculated from the standard curve.
  • qPCR is a mature technology in the field. When using existing instruments for qPCR detection, the Ct value of the sample can be directly obtained from the output result of the instrument.
  • fluorescent probes or fluorescent dyes can be used to obtain fluorescent signals.
  • a common fluorescent probe can be, for example, a TaqMan fluorescent probe, in which a specific fluorescent probe is added while adding a pair of primers during PCR amplification.
  • the probe is an oligonucleotide, and both ends are labeled with a reporter.
  • a fluorophore and a quencher fluorophore are examples of fluorescent probes.
  • the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cleaves and degrades the probe, making the reporter fluorescent group and quencher
  • the fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescence signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescence signal is completely synchronized with the formation of the PCR product.
  • the reporter fluorophore can be, for example, FAM
  • the quencher group can be, for example, NFQ-MGB.
  • fluorescent dyes can also be used to obtain fluorescent signals.
  • an excessive amount of fluorescent dyes can be added to the PCR reaction system. After the fluorescent dyes are non-specifically incorporated into the DNA double-strand, they emit fluorescent signals without being incorporated into the strands. The dye molecule does not emit any fluorescent signal, thereby ensuring that the increase of the fluorescent signal is completely synchronized with the increase of the PCR product.
  • fluorescent dyes can be, for example, SYBR fluorescent dyes, sulforhodamine (Texas Red), fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), JOE, VIC, ROX , NED, etc.
  • Digital PCR detection is also well known to those skilled in the art.
  • digital PCR also called single-molecule PCR
  • the sample is diluted to the single-molecule level and distributed equally
  • the reaction is carried out in tens to tens of thousands of units, and the fluorescent signal of each reaction unit is collected after the amplification is completed.
  • the original concentration or content of the sample is calculated by direct counting or Poisson distribution formula.
  • Those skilled in the art know how to perform digital PCR detection.
  • Another aspect of the present invention provides a primer pair for specifically detecting human genomic DNA in a sample, which includes a forward primer and a reverse primer for specifically amplifying the human SRGAP2 gene.
  • the forward primer is 5'-CGATACTCAGGTCAAAGGTAAGG-3'
  • the reverse primer is 5'-CTGCAAATCACGGTGGAAATAC-3'.
  • Another aspect of the present invention provides a primer and probe combination for specifically detecting human-derived genomic DNA in a sample, which includes a forward primer, a reverse primer, and a fluorescent probe that specifically amplify the human SRGAP2 gene.
  • the forward primer is 5'-CGATACTCAGGTCAAAGGTAAGG-3'
  • the reverse primer is 5'-CTGCAAATCACGGTGGAAATAC-3'
  • the sequence of the probe is 5'-TGCAAATGCTCTGTGGACTGGTGA-3'.
  • the 5'end of the probe is labeled with a reporter fluorophore, and the 3'end is labeled with a quenching group.
  • the reporter fluorophore labeled at the 5'end of the fluorescent probe is FAM
  • the quenching group labeled at the 3'end is NFQ-MGB.
  • kits for specifically detecting human-derived genomic DNA in a sample includes the aforementioned primer pair or a combination of the aforementioned primer and probe.
  • the kit also includes any one or more reagents required for qPCR detection.
  • any one or more reagents required for qPCR detection include one or more components selected from the following components: qPCR reaction solution (eg qPCR Master Mix (2X) , Including the necessary components such as enzymes required for qPCR reaction), nuclease-free high-purity water, human-derived genomic DNA standards, positive control products containing human-derived genomic DNA, and negative control products without human-derived genomic DNA.
  • qPCR reaction solution eg qPCR Master Mix (2X)
  • Including the necessary components such as enzymes required for qPCR reaction
  • the kit may optionally also include quality control products.
  • the quality control product may be, for example, a DNA sample containing human-derived genomic DNA at a specific concentration, or multiple DNA samples containing human-derived genomic DNA at different specific concentrations.
  • the negative control that does not contain human-derived genomic DNA can be genomic DNA of any animal, such as rabbit genomic DNA.
  • the human-derived genomic DNA standard may be a series of mixed samples of human-derived genomic DNA and animal genomic DNA, wherein the proportion of human-derived genomic DNA in each mixed sample is a given ratio and is set in a gradient.
  • the proportion of the human genomic DNA may be, for example, the proportion of the amount of human genomic DNA in the sample to the total amount of human genomic DNA and animal genomic DNA.
  • Another aspect of the present invention provides a method for detecting human-derived genomic DNA in a sample, which includes the following steps:
  • the human-derived genomic DNA standard may be a series of mixed samples of human-derived genomic DNA and animal-derived genomic DNA, wherein the proportion of human-derived genomic DNA in each mixed sample is a given ratio and is set in a gradient.
  • the proportion of the human genomic DNA may be, for example, the proportion of the amount of human genomic DNA in the sample to the total amount of human genomic DNA and animal genomic DNA.
  • the human-derived genomic DNA standard includes at least 6 samples with different concentrations; the concentration refers to the concentration of human-derived genomic DNA in the human-derived genomic DNA standard.
  • the animal-derived genomic DNA may be genomic DNA of a sample derived from an animal.
  • the human-derived genomic DNA may be genomic DNA derived from a human sample.
  • the highest concentration point refers to the highest limit of the concentration that can be stably detected by the above method, and can also be referred to as the upper limit of detection or the upper limit of quantification in the present invention.
  • the lowest limit of the concentration that can be stably detected is the lowest point of concentration, which can also be referred to as the lower limit of detection or the lower limit of quantification in the present invention.
  • the qPCR reaction system is PCR Master Mix (2X) 10 ⁇ l, primers/probes (20 ⁇ ) for specific amplification of SRGAP2 gene 1 ⁇ l, DNA sample + nuclease-free high-purity water 9 ⁇ l.
  • the DNA sample can be sample genomic DNA, human-derived genomic DNA standard, or other positive control, negative control or quality control.
  • the procedure of the qPCR reaction is to first activate UDG at 50°C for 2 minutes; then activate DNA polymerase at 95°C for 10 minutes; then perform 40 PCR reactions according to the following parameters: 95°C for 15 seconds; 60°C for 1 minute .
  • the qPCR reaction is completed on the Applied Biosystems ABI 7500 Real Time PCR machine.
  • the result determination may also be quantitative detection of human-derived DNA in the sample, or may further include quantitative detection of human-derived DNA in the sample.
  • the quantitative detection may include, for example, determining the concentration of human genomic DNA in the sample genomic DNA based on the Ct value of the sample genomic DNA and a fitted standard curve.
  • the combination of primers and probes that specifically detect human-derived cells or human-derived genomic DNA includes forward primers, reverse primers, and fluorescent probes that specifically amplify the SRGAP2 gene.
  • the forward primer is 5'-CGATACTCAGGTCAAAGGTAAGG-3'
  • the reverse primer is 5'-CTGCAAATCACGGTGGAAATAC-3'
  • the sequence of the probe is 5'-TGCAAATGCTCTGTGGACTGGTGA-3'.
  • the 5'end of the probe is labeled with a reporter fluorophore, and the 3'end is labeled with a quenching group.
  • the reporter fluorophore labeled at the 5'end of the fluorescent probe is FAM
  • the quenching group labeled at the 3'end is NFQ-MGB.
  • the lowest point of the concentration is 32.00 pg/5 ⁇ l.
  • the sample may be an organ, tissue, whole blood, cell or body fluid sample, such as the heart, liver, spleen and other organs.
  • the sample can be derived from any animal, such as mice, rats, rabbits, monkeys and other animals.
  • the methods of the invention are performed in vitro.
  • the methods of the invention are non-diagnostic.
  • the present invention designs and synthesizes human-specific primers/probes, and discloses a TaqMan qPCR method that can be used to detect human-derived DNA in animal organs, tissues, cells, body fluids and blood. This method is suitable for detecting cells and gene therapy products Biodistribution, metabolism and residence in treated animals.
  • the present invention provides specific primer probes that only amplify human SRGAP2 fragments.
  • TaqMan qPCR technology it can detect human-derived cells in the tissues of a variety of animals (such as New Zealand rabbits, cynomolgus monkeys, etc.), which can be used for tracking and quantification Detection of human-derived cells after allotransplantation.
  • a primer/probe-based qPCR method was used to detect human-derived genes in various animal (such as rats, mice, rabbits, monkeys) and various organs (such as heart, liver, spleen, blood, etc.).
  • the present invention can detect human-derived DNA in organ tissues and body fluids of higher mammals such as monkeys.
  • the qPCR method of the present invention can sensitively and stably detect as low as 32.00 pg of human genomic DNA.
  • the detection method of the invention has strong specificity, high precision and accuracy, and can stably detect extremely low content of human-derived cell genomic DNA.
  • Figure 1 is a simple experimental procedure of TaqMan-qPCR.
  • Figure 2 shows the FOX2A and SRGAP2 primers to detect the Ct values of samples at various concentrations.
  • Figure 3 is a standard curve diagram of SRGAP2 primer/probe specific amplification and the corresponding Ct value.
  • the 7 points on the horizontal axis from left to right represent 7 standard concentration samples.
  • the content of human genomic DNA is 32pg, 160pg, 800pg, 4000pg, 20000pg, 50000pg and 90000pg in each 5 ⁇ l sample. Repeat twice.
  • the standard curve in the figure is fitted with Ct as the ordinate (Y) and the logarithm of the concentration of the standard concentration sample as the abscissa (Y), but the abscissa shows the concentration of the standard concentration sample.
  • Figure 4 is the amplification curve diagram of the SRGAP2 qPCR method precision and accuracy verification experiment.
  • the curves from left to right in the figure represent the qPCR amplification curves when the human genomic DNA content is ULOQ (90000pg), HQC (72000pg), MQC (4000pg), LQC (80pg) and LLOQ (32pg), each There are six replicates of the concentration, and the fit of the curve represents its precision.
  • Figure 5 is the amplification curve diagram of the specific detection experiment of SRGAP2 qPCR method.
  • Figure 6 is the amplification curve diagram of the selective detection experiment of SRGAP2 qPCR method.
  • Figure 7 is the amplification curve of the dilution linear detection experiment of SRGAP2 qPCR method.
  • the specific sequence of the human gene SRGAP2 is used to design and synthesize primers and labeled probes, and a standard curve is prepared with human-derived cell genomic DNA to establish a detection method for human genomic DNA in New Zealand rabbit tissues, and perform this method Standard curve and comprehensive methodological verification of quantification range, accuracy, precision, specificity, selectivity, dilution linearity, etc. See attached figure 1 for the experimental process.
  • the 5'end of the SRGAP2 probe is labeled with the reporter fluorescent group FAM, and the 3'end is labeled with the quenching group NFQ-MGB.
  • the 5'end of the FOX2A probe is labeled with the reporter fluorescent group FAM, and the 3'end is labeled with the quenching group TAMRA.
  • Human-derived genomic DNA derived from human mesenchymal stem cells
  • Animal genomic DNA derived from New Zealand rabbit liver tissue.
  • the qPCR reaction system is 20 ⁇ L, including: Universal PCR Master Mix (2X) 10 ⁇ L, SRGAP2 (20x) primer/probe 1 ⁇ L, DNA sample and water total 9 ⁇ L.
  • Universal PCR Master Mix (2X) 10 ⁇ L
  • SRGAP2 20x
  • primer/probe 1 ⁇ L DNA sample and water total 9 ⁇ L.
  • genomic DNA as a template
  • Universal PCR Master Mix (2X) specific primers for human SRGAP2 and fluorescently labeled probes
  • the primers and probes of human SRGAP2 and FOX2A specific fragments were designed and synthesized.
  • the primers and probes of SRGAP2 and FOX2A were as described in Example 1.
  • the concentration of human mesenchymal stem cell genomic DNA was serially diluted, and the SRGAP2 primer probe as described in Example 1 was used for qPCR detection until the lowest limit of stable detection was 32.00 pg /5 ⁇ l, set as the lower limit of detection (or called the lower limit of quantification); the highest limit that can be stably detected, is 90000.00pg/5 ⁇ l, set as the upper limit of detection (or called the upper limit of quantification); concentrations higher than the upper limit of detection can also be detected , But there may be false positive results.
  • a standard concentration sample was prepared by mixing human mesenchymal stem cell genomic DNA and New Zealand rabbit liver genomic DNA, and using the SRGAP2 primer probe as described in Example 1 for qPCR detection.
  • the percentages of stem cell genomic DNA are 90%, 50%, 20%, 4%, 0.8%, 0.16%, and 0.032%.
  • human-derived genomic DNA diluted with animal DNA is used as a positive control (this In the example, each 5 ⁇ l contains animal DNA (99,000 pg and human-derived genomic DNA (1000 pg)), New Zealand rabbit liver genomic DNA is used as a negative control, and a quality control sample of the plate is prepared to evaluate whether the qPCR experiment is acceptable.
  • positive control substances, negative control substances and plate quality control samples see the following table (each sample is 5 ⁇ l, and the total DNA content of each sample is maintained at 100000pg):
  • HQC, MQC, and LQC are quality control samples with high, medium, and low concentrations respectively;
  • A is a transitional sample during the dilution process (to prevent the sample from being inaccurate after high-fold dilution).
  • the inter-batch accuracy is the accuracy range (lowest-highest) of the 7 standard concentration samples of the standard curve in 7 qPCR experiments.
  • LLOQ Lower limit of quantification
  • LQC Low Quality control
  • HQC High Quality control
  • MQC Middle Quality control
  • UOQ Upper limit of quantification
  • RE% (C measured- C theory or C 0 )/C theory or C 0 ⁇ 100% (the same below)
  • Table 2 The accuracy and precision of SRGAP2 qPCR method for detecting human-derived DNA in New Zealand rabbit genomic DNA
  • the amplification curve is shown in Figure 5.
  • the red curve in the figure is the genomic DNA extracted after adding human mesenchymal stem cells to New Zealand rabbit liver tissue, and the amplification curve during the qPCR reaction; while the New Zealand rabbit liver tissue adding New Zealand The genomic DNA extracted from rabbit liver tissue or DNase/RNase-free water did not detect the amplification curve during the qPCR reaction, indicating that this SRGAP2 primer can only specifically detect human genomic DNA.
  • the results are summarized in Table 3.
  • the group with human mesenchymal stem cells can detect the signal within the quantitative range, and the measured values of the other two groups are below the lower limit of detection. This indicates that the SRGAP2 primer specifically recognizes human-derived genomic DNA and can accurately detect human-derived cells or nucleic acid samples in animal tissue cells.
  • Table 3 The specificity of SRGAP2 qPCR method for detecting human-derived DNA in New Zealand rabbit genomic DNA
  • liver tissues of 6 New Zealand rabbits Take the blank liver tissues of 6 New Zealand rabbits, and take the heart, liver, lung, spleen, kidney, brain, lymph nodes, thymus, testis, uterus and other tissues of New Zealand rabbits. After extracting the genomic DNA of these tissues, they are used as diluents. Dilute the human mesenchymal stem cell genomic DNA, configure it into an LQC sample (that is, the content of human mesenchymal stem cell genomic DNA is 80pg/5 ⁇ l), use the SRGAP2 primer probe as described in Example 1 to perform TaqMan qPCR detection, and calculate its RE %value.
  • the acceptance criteria are as follows:
  • the amplification curve is shown in Figure 6, and the results are summarized in Table 4.
  • the results showed that all samples added with human mesenchymal stem cell genomic DNA can detect signals, and the detection accuracy (RE%) is -19.8% ⁇ 37.5%, while the DNA samples of various organs and tissues without human mesenchymal stem cell genomic DNA can be detected. No amplification signal of SRGAP2 gene fragment was detected. It shows that the qPCR method using SRGAP2 primers is not selective for genomic DNA extracted from different animals and different tissues/organs of animals, and can detect human-derived DNA in different organs and tissues of animals.
  • the amplification curve is shown in Figure 7.
  • the results are summarized in Table 5. The results show that the precision of all diluted samples from all sources of concentration is ⁇ 60% after recalculating the concentration, which meets the requirements, indicating that the SRGAP2 qPCR method can accurately detect human-derived DNA in animal genomic DNA.
  • Back-calculated concentration that is to say, after the qPCR test is completed, the actual values of the samples diluted 2 ⁇ , 10 ⁇ , 100 ⁇ , and 1000 ⁇ are multiplied by the respective dilution multiples.
  • the actual concentration and back-calculated concentration in the table refer to the amount of DNA contained in 5 ⁇ l of the test sample.
  • liver tissues of rats, mice, rabbits, and monkeys were mixed with human mesenchymal stem cells, and genomic DNA was extracted and configured into: a, human-derived genes accounted for 4%; b, human-derived genes accounted for 0.8%; c , Human-derived genes accounted for 0.032%; d, human-derived genes accounted for 0%.
  • the SRGAP2 primer probe as described in Example 1 was used for qPCR detection.
  • the results are shown in Table 6.
  • the results show that the genomic DNA extracted from animal tissues mixed with human mesenchymal stem cells can detect specific amplification signals, and High accuracy and high sensitivity; no amplification signal was detected in the genomic DNA extracted from the liver tissue of each animal without mixing human mesenchymal stem cells.
  • results show that human-derived gene expression can be detected in the genomic DNA of the right knee joint injected with human mesenchymal stem cells, but the signal cannot be detected in the left knee joint without the injection of cells, indicating that the SRGAP2 primer was used for The TaqMan qPCR method can sensitively and specifically detect human-derived cells transplanted in animals.
  • Table 7 SRGAP2 qPCR method to detect the genomic DNA of human mesenchymal stem cells injected into the right knee joint of New Zealand rabbits
  • the DNA content in the table refers to the amount of DNA contained in 5 ⁇ l of the test sample.
  • human-specific primers and probe SRGAP2 can also sensitively detect human-derived cells in animal blood.
  • New Zealand rabbit venous blood (whole blood) and human mesenchymal stem cells were thoroughly mixed, and whole blood not mixed with human mesenchymal stem was used as a negative control. After the genomic DNA was extracted, it was left for different time, and the SRGAP2 primer probe as described in Example 1 was used for qPCR experimental verification. The results are shown in Table 8. The results show that the genomic DNA extracted from whole blood mixed with human mesenchymal stem cells can detect the qPCR amplification signal, but the whole blood genomic DNA without human mesenchymal stem cells cannot detect the qPCR amplification signal. It shows that the human SRGAP2 primer/probe and this qPCR method can sensitively and specifically detect human-derived cells in animal blood.
  • the DNA content in the table refers to the amount of DNA contained in 5 ⁇ l of the test sample.
  • Primers and labeled probes designed and synthesized with the specific sequence of human SRGAP2 can specifically detect human-derived DNA in organs or blood of different animals (such as rabbits, monkeys, rats and mice) .
  • the experimental process is simple, convenient, accurate, sensitive and specific, and can be used to detect human-derived cells and nucleic acid preparations in preclinical research animals including cell therapy and gene therapy.
  • qPCR is one of the most effective and sensitive detection techniques. Although qPCR technology can quantitatively detect transplanted cells in recipient tissues, it is limited by some poor reproducibility (10). For this reason, we searched for and designed specific primers targeting human DNA.
  • Nguyen PK Riegler J, Wu JC. Stem cell imaging: from bench to bedside. Cell Stem Cell. 2014; 14 (4): 431-444.

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Abstract

本发明公开了通过特异性检测人SRGAP2基因检测人源性DNA的方法、用于特异性检测人SRGAP2基因的引物和荧光探针，以及可用于检测动物器官、组织、细胞、体液和血液中人源性DNA的TaqMan qPCR方法，此方法适用于检测任何动物源样本中的人类DNA，包括但不限于细胞和基因治疗产品在接受治疗的动物体内的生物分布、代谢和驻留。

Description

[根据细则26改正09.07.2019]　特异性检测人源性基因组DNA的引物及其应用 技术领域
本发明属于分子生物检测领域，更具体涉及人源性DNA的检测。
背景技术
细胞治疗是把健康的干细胞移植到病人或自己体内，以达到修复病变或重建功能正常的细胞和组织的目的。研究发现，将人源性细胞移植到免疫缺陷小鼠中已被广泛用于研究干细胞在组织修复和再生或癌细胞转移中的功能(1)，而确定细胞治疗产品(人源性细胞)移植到宿主体内后，其在宿主内的生物分布、随时间的迁移或残留、治疗效果及生物安全是临床应用前检测的重要一步(2)。
对细胞治疗产品来说，需进行一系列实验(如定量实验)来评估细胞移植后的效应及其在移植部分或体内的生物分布。监管指导方针规定：需考虑并检测细胞移植宿主后的生物安全性来评估细胞产品的安全性(3)。与小分子药物不同，活细胞产品的生物复杂性使之不适合使用常规的吸收、分布、代谢、排泄及药代动力学实验验证。生物分布实验是细胞治疗产品走向临床的一个必要条件，其可以提供人源性细胞在宿主体内的定位、随时间迁移及体内存活和分化的数据(4)。
一些方法被用于检测移植细胞的生物分布，如组织样品的微观观察被广泛用于检测移植细胞的存在。使用细胞标记技术，如细胞膜染料(DII)或细胞核染料(DAPI)来追踪荧光标记的细胞在受体组织内的分布(5)。然而这种技术有其缺陷，首先，在移植细胞含量比较低的情况下可能检测不到信号；其次，荧光染料易受细胞分裂而被稀释以至于其可能会低于检测限；最后，定位细胞在体内分布情况可能会受到样本错误影响，导致结果的敏感性和可靠性都降低(6)。虽然对移植细胞进行基因修饰可以避免染料被稀释的情况，但修饰后的细胞可能会影响其正常功能或改变其原本的生物分布(7)，因此，寻找新的检测方法尤为重要。
实时荧光定量PCR(qPCR)技术的发展为有效检测移植细胞提供了可靠的手段(8)。与常规PCR相比，该技术实现了PCR从定性到定量的飞跃，而且其特异性强、灵敏度高、检测方法简便快速、能有效检测出低拷贝的目的DNA片段。qPCR技术有两种方法：染料法和TaqMan探针法。染料法如SYBR Green I染料可与双链DNA的小沟结合，当被激发后可以产生荧光信号，但由于任何双链都可以与其非特异性结合产生非特异性信号，因此会造成不准确的结果。TaqMan探针法的探针具有5’端荧光报告基团和3’端淬灭基团，完整的探针受到激发光后会发生荧光共振能量转移，因此检测不到信号；只有当DNA复制时，探针被水解，报告基团和淬灭基团分离，才可以检测到荧 光，因此荧光信号的强弱就代表了模板的数量，由于被释放的荧光基团数目和PCR产物数量是一对一的关系，因此用该技术可对模板进行准确定量。虽然SYBR Green和TaqMan技术在扩增效率上差别不是很大(9)，但TaqMan技术特异性更高、敏感性更强，适用于异种移植系统中人源性细胞的检测和定量(10)。
近年来，虽然PCR技术的检测方法发展迅速，但在某些系统中受到限制。例如：靶向睾丸决定因子(SYR)或小鼠睾丸特异性的编码Y蛋白(TSPY)的特异性引物可用于检测雌性受体中的雄性细胞(11)，此方法可以检测雌性组织中0.01％的雄性细胞，但是，对于雌性受体中移植的雄性细胞，这种检测方法受到限制。将人源性和鼠源性基因组DNA在同一反应管中平行扩增去检测小鼠组织中的人源性细胞，依然只有很低的敏感性(12)。近年来，已有一些人特异性的基因(如FOX2A)被发现并用于检测人源性相关细胞的存在。但根据这些基因设计的序列因为敏感性不够而产生不准确的结果。为了增加检测的敏感性，靶向人基因组高度重复序列(如α-satellite、Alu)的引物被用于qPCR实验(10)，但由于这些序列在人类基因组中高度活跃的移动性，相同量的模板DNA可能含有高度可变量的靶序列，因此导致不一致的结果。
发明内容
为了提高qPCR实验的可重复性，本发明设计合成了人特异性的引物探针。在常见的基因如FOXP2、MYH16(人类发育过程中特异性基因)之外，本发明选择了SRGAP2基因。SRGAP2(GenBank accession#:NC_000001.11)即Slit-Robo Rho GTPase activating protein 2，在大脑皮层发育过程中起作用，可以调控神经元的迁移和分化(13)。研究发现，SRGAP2在哺乳动物进化过程中高度保守，且人类是其发生基因复制的唯一谱系；另外，SRGAP2是三或四拷贝基因，分别为SRGAP2A(NC_000001.11：206203556..206464443)、SRGAP2B(NC_000001.11：144887191..145095321)、SRGAP2C(NC_000001.11：121184967..121392874)和SRGAP2D(NC_000001.11：143972639..144069704)，其设计而成的引物比FOXP2敏感性更高。
本发明一方面提供人SRGAP2基因在检测样本中人源性基因组DNA中的应用。
本发明另一方面提供特异性检测人SRGAP2基因的试剂在检测样本中人源性基因组DNA中的应用。
本发明另一方面提供特异性检测人SRGAP2基因的试剂在制备检测样本中人源性基因组DNA的试剂中的应用。
在一些实施方案中，所述特异性检测人SRGAP2基因的试剂包含特异性检测SRGAP2基因的引物对。
在一些实施方案中，所述引物对包括正向引物5'-CGATACTCAGGTCAAAGGTAAGG-3'(SEQ ID NO:1)和反向引物5'-CTGCAAATCACGGTGGAAATAC-3'(SEQ ID NO:2)。
在一些实施方案中，所述特异性检测人SRGAP2基因的试剂还包括荧光探针5'-TGCAAATGCTCTGTGGACTGGTGA-3'(SEQ ID NO:3)，该探针的5’端标记报告荧光基团，3’端标记淬灭基团。
在一些实施方案中，所述荧光探针的5’端标记的报告荧光基团为FAM，3’端标记的淬灭基团为NFQ-MGB或TAMRA。
除上述特定的引物和探针之外，本领域技术人员还可以根据SRGAP2基因的序列设计其它能够特异性扩增和检测人SRGAP2基因的引物和探针。
在一些实施方案中，所述检测是通过qPCR检测或通过数字PCR检测。
如本领域技术人员所知，qPCR又称实时定量PCR(Real-time Quantitative PCR)，是在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测PCR进程，最后可以通过标准曲线对未知模板进行定量分析。在qPCR检测中，Ct值表示循环阈值，即每个反应管内的荧光信号达到设定阈值时所经历的循环数。由于每个模板的Ct值与该模板的起始含量的对数存在线性关系，起始拷贝数越多，Ct值越小。利用连续稀释的已知起始含量的标准品可作出标准曲线，其中横坐标代表起始含量的对数，纵坐标代Ct值，或者纵坐标代表起始含量的对数，横坐标代Ct值。只要获得未知样品的Ct值，即可从标准曲线上计算出该样品的含量。qPCR在本领域中属于成熟技术，利用现有的仪器进行qPCR检测时，可以直接从仪器的输出结果中获得样本的Ct值。
在qPCR检测中，可以使用荧光探针或荧光染料获取荧光信号。常见的荧光探针例如可以是TaqMan荧光探针，其中PCR扩增时在加入一对引物的同时加入一个特异性的荧光探针，该探针为一寡核苷酸，两端分别标记一个报告荧光基团和一个淬灭荧光基团。探针完整时，报告基团发射的荧光信号被淬灭基团吸收；PCR扩增时，Taq酶的5'－3'外切酶活性将探针酶切降解，使报告荧光基团和淬灭荧光基团分离，从而荧光监测系统可接收到荧光信号，即每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与PCR产物形成完全同步。在一些实施方案中，报告荧光基团可以是例如FAM，淬灭基团可以是例如NFQ-MGB。本领域技术人员知道其他报告荧光基因和相应的淬灭荧光基团也可以用于本发明。
在qPCR检测中，还可以使用荧光染料获取荧光信号，例如可以在PCR反应体系中，加入过量荧光染料，荧光染料非特异性地掺入DNA双链后，发射荧光信号，而不掺入链中的染料分子不会发射任何荧光信号，从而保证荧光信号的增加与PCR产物的增加完全同步。常用的荧光染料例如可以是SYBR荧光染料、磺酰罗丹明(Texas Red)、异硫氰酸荧光素(FITC)、羟基荧光素(FAM)、四氯荧光素(TET)、JOE、VIC、ROX、NED等。
数字PCR检测也是本领域技术人员公知的，简言之，数字PCR(也可称单分子PCR)包括PCR扩增和荧光信号分析，在PCR扩增阶段，将样品稀释到单分子水平并平均分配到几十至几万个单元中进行反应，在扩增结束后对每个反应单元的荧光信号进 行采集。最后通过直接计数或泊松分布公式计算得到样品的原始浓度或含量。本领域技术人员熟知如何进行数字PCR检测。
本发明的另一方面提供特异性检测样本中人源性基因组DNA的引物对，其包括特异性扩增人SRGAP2基因的正向引物和反向引物。
在一些实施方案中，所述正向引物是5'-CGATACTCAGGTCAAAGGTAAGG-3'，所述反向引物是5'-CTGCAAATCACGGTGGAAATAC-3'。
本发明的另一方面提供特异性检测样本中人源性基因组DNA的引物和探针组合，其包括特异性扩增人SRGAP2基因的正向引物、反向引物和荧光探针。
在一些实施方案中，所述正向引物是5'-CGATACTCAGGTCAAAGGTAAGG-3'，所述反向引物是5'-CTGCAAATCACGGTGGAAATAC-3'；所述探针的序列为5'-TGCAAATGCTCTGTGGACTGGTGA-3'，该探针的5’端标记报告荧光基团，3’端标记淬灭基团。
在一些实施方案中，所述荧光探针的5’端标记的报告荧光基团为FAM，3’端标记的淬灭基团为NFQ-MGB。
本发明的另一方面提供特异性检测样本中人源性基因组DNA的试剂盒，该试剂盒包括上述引物对，或包括上述引物和探针组合。
在一些实施方案中，所述试剂盒中还包括进行qPCR检测所需要的任意一种或更多种试剂。
在一些实施方案中，所述进行qPCR检测所需要的任意一种或更多种试剂包括选自下述组分的一种或更多中组分：qPCR反应液(例如qPCR Master Mix(2X)，其中包括qPCR反应所需要的酶等必须成分)、无核酸酶高纯水、人源性基因组DNA标准品、含有人源性基因组DNA的阳性对照品、不含人源性基因组DNA的阴性对照品。
所述试剂盒任选地还可以包括质控品。所述质控品例如可以是含有特定浓度的人源性基因组DNA的DNA样品，或者也可以是含有不同特定浓度的人源性基因组DNA的多个DNA样品。
不含人源性基因组DNA的阴性对照品可以是任意动物的基因组DNA，例如兔基因组DNA。
人源性基因组DNA标准品可以是一系列人源性基因组DNA和动物基因组DNA的混合样品，其中各混合样品中人源性基因组DNA的占比为给定比例且为梯度设置。所述人源性基因组DNA的占比可以是例如样品中人源性基因组DNA的量占人源性基因组DNA和动物基因组DNA的总量的比例。
本发明的另一方面提供样本中人源性基因组DNA的检测方法，包括以下步骤：
(1)提取样本基因组DNA；
(2)使用特异性扩增SRGAP2基因片段的引物和探针组合，例如上述的任意一种引物和探针组合，对人源性基因组DNA标准品和样本基因组DNA进行qPCR检测；
(3)用qPCR结果拟合标准曲线：以人源性基因组DNA标准品的Ct值为纵坐标(X)，以人源性基因组DNA标准品中人源性基因组DNA的浓度的对数为横坐标(Y)，拟合线性方程，其中R 2≥0.99；且各标准品浓度的准确度(RE％)为-75％～150％，确定可稳定检测到的浓度最低点；
(4)结果判定：如果样本的qPCR结果中出现明显的扩增曲线；且样本qPCR的Ct值小于浓度最低点的Ct值，则为阳性结果，即样本中存在人源性DNA；如果无明显扩增曲线，或者有明显扩增曲线，但Ct值大于标准曲线浓度最低点的Ct值，则为阴性结果，即样本中不存在人源性DNA。
人源性基因组DNA标准品可以是一系列人源性基因组DNA和动物源性基因组DNA的混合样品，其中各混合样品中人源性基因组DNA的占比为给定比例且为梯度设置。所述人源性基因组DNA的占比可以是例如样品中人源性基因组DNA的量占人源性基因组DNA和动物基因组DNA的总量的比例。在以上检测方法中，所述人源性基因组DNA标准品包含至少6个不同浓度的样品；所述浓度是指人源性基因组DNA标准品中人源性基因组DNA的浓度。所述动物源性基因组DNA可以是来源于动物的样本的基因组DNA。所述人源性基因组DNA可以是来源于人样本的基因组DNA。
拟合标准曲线时，如R 2和各标准品浓度的准确度不满足以上要求，可以通过重复实验，或重新制备人基因组DNA标准品、或换用不同浓度的人基因组DNA标准品，来获得满意的拟合结果。标准曲线的拟合以及获得满意拟合结果的方法属于本领域技术人员的公知技术。
浓度最高点是指用上述方法可稳定检测到的浓度的最高限，在本发明中也可称为检测上限或定量上限。可稳定检测到的浓度的最低限即为浓度最低点，在本发明中也可称为检测下限或定量下限。
判断是否出现明显扩增曲线的方法是本领域技术人员公知的，例如当ΔRn vs Cycle模式下的曲线为S形时，可确定出现明显扩增曲线。
在一些实施方案中，qPCR的反应体系为PCR Master Mix(2X)10μl，特异性扩增SRGAP2基因的引物/探针(20×)1μl，DNA样品+无核酸酶高纯水9μl。
其中DNA样品可以是样本基因组DNA、人源性基因组DNA标准品、或其它阳性对照、阴性对照或质控品。
在一些实施方案中，qPCR反应的程序为首先50℃，2min以激活UDG；其次95℃，10min激活DNA聚合酶；然后按下列参数进行40个PCR反应：95℃，15秒；60℃，1min。在一些实施方案中，在Applied Biosystems ABI 7500 Real Time PCR仪上完成qPCR反应。
在一些实施方案中，所述结果判定也可以是定量检测样品中的人源性DNA，或者可以进一步包括定量检测样品中的人源性DNA。所述定量检测可以包括例如根据样本基因组DNA的Ct值和拟合的标准曲线，确定样本基因组DNA中人源性基因组DNA的浓度。
在一些实施方案中，特异性检测人源性细胞或人源性基因组DNA的引物和探针组合包括特异性扩增SRGAP2基因的正向引物、反向引物和荧光探针。
在一些实施方案中，所述正向引物是5'-CGATACTCAGGTCAAAGGTAAGG-3'，所述反向引物是5'-CTGCAAATCACGGTGGAAATAC-3'；所述探针的序列为5'-TGCAAATGCTCTGTGGACTGGTGA-3'，该探针的5’端标记报告荧光基团，3’端标记淬灭基团。
在一些实施方案中，所述荧光探针的5’端标记的报告荧光基团为FAM，3’端标记的淬灭基团为NFQ-MGB。
在一些实施方案中，所述浓度最低点是32.00pg/5μl。
本发明中，样本可以是器官、组织、全血、细胞或体液样品，例如心脏、肝脏、脾脏等器官。
本发明中，样本可以来源于任何动物，例如小鼠、大鼠、兔、猴等动物。
在一些实施方案中，本发明的方法是在体外进行的。
在一些实施方案中，本发明的方法是非诊断性的。
本发明设计并合成人特异性的引物/探针，并公开了可用于检测动物器官、组织、细胞、体液和血液中人源性DNA的TaqMan qPCR方法，此方法适用于检测细胞和基因治疗产品在接受治疗的动物体内的生物分布、代谢和驻留。
本发明提供了只扩增人SRGAP2片段的特异性引物探针，利用TaqMan qPCR技术，可以检测多种动物(如新西兰兔、食蟹猴等)组织中的人源性细胞，可用于追踪并定量检测异体移植后的人源性细胞。实验中采用基于引物/探针的qPCR手段，能够检测多种动物(如大鼠、小鼠、兔、猴)多种器官(如心脏、肝脏、脾脏、血液等)内的人源性基因。尤其重要的是，本发明可以检测高等哺乳动物如猴的器官组织和体液中的人源性DNA。通过扩增人特异性SRGAP2序列，本发明的qPCR方法可以敏感且稳定地检测到低至32.00pg的人源性基因组DNA。本发明的检测方法特异性强、精密度和准确度高，并可以稳定检测到极低含量的人源性细胞基因组DNA。
附图说明
图1是TaqMan-qPCR简易实验流程。
图2显示FOX2A和SRGAP2引物检测各浓度样品的Ct值。
图3是SRGAP2引物/探针特异性扩增标准曲线图及相应Ct值。图中横轴从左至右7个点代表7个标准浓度样品，其中人源性基因组DNA的含量分别为每5μl样品中含32pg、160pg、800pg、4000pg、20000pg、50000pg及90000pg，每个样品进行两次重复。图中标准曲线以Ct值为纵坐标(Y)，以标准浓度样品的浓度的对数为横坐标(Y)拟合而成，但横坐标中显示的是标准浓度样品的浓度。
图4是SRGAP2 qPCR方法精密度和准确度验证实验的扩增曲线图。图中从左至右的曲线分别代表人源性基因组DNA含量为ULOQ(90000pg)、HQC(72000pg)、 MQC(4000pg)、LQC(80pg)和LLOQ(32pg)时的qPCR扩增曲线，每个浓度有六个重复，曲线的拟合性代表其精密度好。
图5是SRGAP2 qPCR方法特异性检测实验的扩增曲线图。
图6是SRGAP2 qPCR方法选择性检测实验的扩增曲线图。
图7是SRGAP2 qPCR方法稀释线性检测实验的扩增曲线图。
实施例
下面通过实施例，并结合附图，对本发明的技术方案作进一步详细的说明，但本发明不限于下面的实施例。
在以下实施例中，以人基因SRGAP2特异性序列设计、合成引物和标记探针，以人源性细胞基因组DNA配制标准曲线，建立新西兰兔组织中人基因组DNA的检测方法，并以该方法进行标准曲线与定量范围、准确度、精密度、特异性、选择性、稀释线性等的全面方法学验证，实验流程参见附图1。
实施例1实验方法
1、引物的设计与合成
从Genebank中获取人SRGAP2和FOXP2(即FOX2A，GenBank accession#:NC_000007.14)的基因序列，比对分析后发现能够区别其它动物序列的人特异DNA序列，设计并合成只扩增人SRGAP2和FOX2A片段的特异引物和荧光标记探针，引物和探针信息如下：
引物名称 SRGAP2 FOX2A(17)
Gene ID 23380 93986
正向引物 5'-CGATACTCAGGTCAAAGGTAAGG-3' 5'-TGGTAGTCTGGAACACCGTAAGAGT-3'
反向引物 5'-CTGCAAATCACGGTGGAAATAC-3' 5'-CATATGGCAGGCTTTAGGTACCC-3'
荧光探针 5'-TGCAAATGCTCTGTGGACTGGTGA-3' 5'-CTGGTGGGCTAAAAGGAAGAAAGAGGTC-3'
SRGAP2探针的5’端标记报告荧光基团FAM，3’端标记淬灭基团为NFQ-MGB。
FOX2A探针的5’端标记报告荧光基团FAM，3’端标记淬灭基团为TAMRA。
2、样品来源
人源性基因组DNA：来源于人间充质干细胞；
动物基因组DNA：来源于新西兰兔肝脏组织。
3、基因组DNA的提取
采用QIAGEN公司的基因组DNA提取试剂盒DNeasyBlood&Tissue Kit，按照试剂盒提供的标准程序提取动物组织或细胞基因组DNA。
取组织、全血或细胞样品，加入蛋白酶K裂解后，依次加入试剂盒内的各种缓冲液：buffer AL、无水乙醇、buffer AW1、buffer AW2等，通过DNeasy Mini spin离心柱离心富集基因组DNA，最后使用buffer AE溶解基因组DNA，用Nanodrop进行浓度和质量(A260/280)的测定后，保存于-80℃备用。
4、定量PCR方法
使用Taqman-qPCR方法，qPCR反应体系为20μL，包括：
Figure PCTCN2019090571-appb-000001
Universal PCR Master Mix(2X)10μL、SRGAP2(20x)引物/探针1μL、DNA样本和水共9μL。采用基因组DNA为模版，加入
Figure PCTCN2019090571-appb-000002
Universal PCR Master Mix(2X)、针对人SRGAP2的特异引物和荧光标记的探针，在Applied Biosystems ABI 7500 Real Time PCR仪上完成qPCR反应。首先50℃，2min以激活UDG；其次95℃，10min激活DNA聚合酶；然后按下列参数进行40个PCR反应：95℃，15秒；60℃，1min。
实施例2 SRGAP2 qPCR检测方法的灵敏度
设计合成人SRGAP2和FOX2A特异性片段的引物探针，SRGAP2以及FOX2A的引物和探针如实施例1中所述。
以人间充质干细胞(MSC)提取的基因组DNA为标准品，以100ng含量人间充质干细胞基因组DNA起始，用新西兰兔肝脏组织基因组DNA作为稀释液，5倍梯度稀释制备7个浓度样品(STD-1至STD-7，其中人源性基因组DNA的含量分别为每5μl样品中含100000pg、20000pg、4000pg、800pg、160pg、32pg、6.4pg)，分别使用SRGAP2和FOX2A的引物探针对这7个浓度样品进行qPCR实验，比较两个引物的特异性和灵敏度。结果如图2所示，其中*p<0.05；**p<0.01。结果表明，两个引物特异性都很强(都只识别人源性基因组DNA)，但人SRGAP2引物的灵敏度比FOX2A高(qPCR实验时，同一含量的DNA样品，即每一浓度DNA标准品，采用SRGAP2引物比采用FOX2A引物的qPCR实验的Ct值少2-3个cycle)。
实施例3  制定SRGAP2 qPCR检测方法的标准曲线
以新西兰兔肝脏基因组DNA为稀释液，对人间充质干细胞基因组DNA浓度进行梯度稀释，并用如实施例1中所述的SRGAP2引物探针进行qPCR检测，直至可稳定检测到的最低限为32.00pg/5μl，设为检测下限(或称定量下限)；可稳定检测到的最高限，为90000.00pg/5μl，设为检测上限(或称定量上限)；高于检测上限的浓度也可以被检测到，但有可能出现假阳性结果。
以检测上限和检测下限为限，用人间充质干细胞基因组DNA和新西兰兔肝脏基因组DNA混合配制标准浓度样品，并使用如实施例1中所述的SRGAP2引物探针进行qPCR检测，其中人间充质干细胞基因组DNA所占百分比分别为90％、50％、20％、4％、0.8％、0.16％和0.032％，另以动物DNA稀释后的不同浓度的人源性基因组DNA作为阳性对照品(本实施例中为每5μl中含动物DNA 99000 pg和人源性基因组DNA  1000 pg)，以新西兰兔肝脏基因组DNA为阴性对照品，同时配制板质控样本用于评估本次qPCR实验是否可接受。标准浓度样品、阳性对照品、阴性对照品和板质控样本配制参见下表(各样品均为5μl，并保持各样品总DNA含量为100000pg)：
标准浓度样品 动物DNA(pg) 人DNA(pg)
STD1 10000.00 90000.00
STD2 50000.00 50000.00
STD3 80000.00 20000.00
STD4 96000.00 4000.00
STD5 99200.00 800.00
STD6 99840.00 160.00
STD7 99968.00 32.00
对照
NTC 100000.00 0
阳性 99000.00 1000.00
阴性 100000.00 0
质控
HQC 28000.00 72000.00
MQC 96000.00 4000.00
A 99600.00 400.00
LQC 99920.00 80.00
备注：HQC、MQC、LQC分别为高、中、低三个浓度的质控样品；A为稀释过程中过渡样品(防止高倍稀释后，样品不准确)。
检测得到每个标准浓度样品的Ct值，以Ct值为纵坐标(Y)，以标准浓度样品的浓度的对数为横坐标(Y)，拟合成标准曲线，如图3所示；得到标准曲线回归方程及相关系数，如下表所示：
Figure PCTCN2019090571-appb-000003
进行多次试验发现，SRGAP2引物可以稳定检测到32.000pg的人源性基因，并且标准浓度样品检测的批间准确度(RE％，即准确度，计算公式：RE％＝(C 测得-C 理论or C 0)/C 理论or C 0×100％；其中C 0为初始浓度或理论浓度，可以是已知浓度或通过其它方法测定的浓度)为-15.5％～41.4％，均在-75％～150％之间(见下表1)。
表1 SRGAP2 qPCR方法标准曲线批间准确度
Figure PCTCN2019090571-appb-000004
备注：批间准确度即7次qPCR实验中标准曲线7个标准浓度样品的准确度的范围(最低-最高)。
上述实验结果表明该SRGAP2 qPCR方法能够稳定、灵敏地检测到动物基因组DNA中的人源性DNA。
实施例4 SRGAP2 qPCR方法精密度、准确度检测
用人间充质干细胞基因组DNA和新西兰兔肝脏基因组DNA混合配制定量下限(LLOQ，Lower limit of quantification)、低浓度质控(LQC，Low Quality control)、高浓度质控(HQC，High Quality control)、中浓度质控(MQC，Middle Quality control)及定量上限(ULOQ，Upper limit of quantification)等5个浓度的样品，用如实施例1中所述的SRGAP2引物探针进行qPCR。各样品的浓度如实施例3中相应样品所述。扩增曲线如图4所示。
考察不同浓度样品的批内及批间的精密度和准确度。接受标准参考药典中生物样品定量分析方法验证指导原则(14-16)，如下：
Figure PCTCN2019090571-appb-000005
备注：
准确度即RE％，计算公式：RE％＝(C 测得-C 理论or C 0)/C 理论or C 0×100％(下同)
精密度即CV％，计算公式：CV％＝标准偏差/平均值×100％(下同)
结果表明：LLOQ和LQC的CV％值均≤70％，而MQC、HQC和ULOQ的CV％值均≤60％；所有检测项的RE％值均在-75％～150％之间(见表2)。
表2 SRGAP2 qPCR法检测新西兰兔基因组DNA中人源性DNA的准确度和精密度
Figure PCTCN2019090571-appb-000006
实施例5 SRGAP2 qPCR方法特异性检测
在新西兰兔肝脏空白组织中分别加入等体积的：①人间充质干细胞，②新西兰兔肝脏组织；③RNase/DNase-free water，提取样本总DNA，用如实施例1中所述的SRGAP2引物探针进行qPCR。接受标准如下：
Figure PCTCN2019090571-appb-000007
扩增曲线如图5所示，图中红色曲线为在新西兰兔肝脏组织中加入人间充质干细胞后提取的基因组DNA，在qPCR反应时的扩增曲线图；而在新西兰兔肝脏组织中加入新西兰兔肝脏组织或者DNase/RNase-free water后提取的基因组DNA，在qPCR反应时没有检测到扩增曲线，表明此SRGAP2引物只可以特异性的检测到人源性基因组DNA。结果总结于表3，加入人间充质干细胞的组别可以在定量范围内检测到信号，其余两组测定值均低于检测下限。这表明SRGAP2引物特异性地识别人源性基因组DNA，能够准确检测动物组织细胞中的人源性细胞或核酸样品。
表3 SRGAP2 qPCR法检测新西兰兔基因组DNA中人源性DNA的特异性
Figure PCTCN2019090571-appb-000008
实施例6 SRGAP2 qPCR方法选择性检测
取6只新西兰兔的空白肝脏组织，另取新西兰兔的心脏、肝脏、肺脏、脾脏、肾脏、脑、淋巴结、胸腺、睾丸、子宫等组织，分别提取这些组织的基因组DNA后，分别作为稀释液稀释人间充质干细胞基因组DNA，配置成LQC样品(即人间充质干细胞基因组DNA的含量为80pg/5μl)，用如实施例1中所述的SRGAP2引物探针进行TaqMan qPCR检测，并计算其RE％值。接受标准如下：
Figure PCTCN2019090571-appb-000009
扩增曲线图如图6所示，结果总结于表4。结果显示：加入人间充质干细胞基因组DNA的样品均可以检测到信号，检测准确度(RE％)为-19.8％～37.5％，而未加入人间充质干细胞基因组DNA的各器官组织DNA样品的均未检测到SRGAP2基因片段扩增信号。表明使用SRGAP2引物的qPCR方法对不同动物及动物的不同组织/器官提取的基因组DNA没有选择性，能够检测动物不同器官组织中的人源性DNA。
表4 SRGAP2 qPCR法检测新西兰兔不同器官组织基因组DNA中的人间充质干细胞DNA
Figure PCTCN2019090571-appb-000010
实施例7 SRGAP2 qPCR方法稀释线性检测
为了进一步验证SRGAP2引物及本qPCR实验方法的精确性和有效性，我们以新西兰兔肝脏基因组DNA为稀释液，将人间充质干细胞基因组DNA稀释至20.000ng/μL后，再以RNase/DNase-free water对其分别进行2倍、10倍、100倍和1000倍的梯度稀释，用如实施例1中所述的SRGAP2引物探针进行TaqMan qPCR检测，每个浓度进行六次重复。接受标准如下：
Figure PCTCN2019090571-appb-000011
扩增曲线如图7所示。结果总结于表5，结果表明所有浓度来源的稀释样品回算浓度后，其精密度均≤60％，均符合要求，说明SRGAP2 qPCR方法能够精确检测到动物基因组DNA中的人源性DNA。
表5 SRGAP2 qPCR方法检测不同稀释倍数人源性DNA实验后的回算浓度及CV％值
Figure PCTCN2019090571-appb-000012
备注：
1、回算浓度：即将2×、10×、100×、1000×稀释的样品，在qPCR检测结束后，测出的实际数值分别乘以各自的稀释倍数所得的数值。
2、表格中的实际浓度和回算浓度是指5μl检测样品中含有的DNA量。
实施例8 SRGAP2 qPCR方法对多种动物基因组DNA中人源性DNA的检测
将大鼠、小鼠、兔、猴的肝脏组织分别混合人间充质干细胞，提取基因组DNA后配置成：a，人源性基因占比4％；b，人源性基因占比0.8％；c，人源性基因占比0.032％；d，人源性基因占比0％。
用如实施例1中所述的SRGAP2引物探针进行qPCR检测，结果如表6所示，结果显示混合人间充质干细胞的动物组织所提取的基因组DNA均可以检测到特异性扩增信号，而且准确度高、灵敏性强；未混合人间充质干细胞的各动物肝脏组织所提取的基因组DNA均未检测出扩增信号。
表6 SRGAP2 qPCR方法检测不同动物基因组DNA中人间充质干DNA
大鼠 小鼠 兔 猴
准确度(RE％) -13.1～1.2 -0.4～0.4 -56.0～6.9 -23.5～13.6
精密度(CV％) 4.1～52.5 5.4～19.8 2.6～81.8 0.8～37.1
肝组织DNA 未检测到 未检测到 未检测到 未检测到
实施例9用SRGAP2 qPCR方法检测动物体内注射的人源性细胞
为了进一步验证SRGAP2引物及TaqMan qPCR方法可以检测移植到动物体内的人源性细胞，我们进行了新西兰兔右膝关节腔注射人间充质干细胞的实验。在实验中，成年新西兰兔右膝关节注射1mL的人间充质干细胞(细胞量为12×10 6)，左膝关节为对照关节，未注射细胞。细胞注射3d后，处死动物并对左、右膝关节提取基因组DNA，用如实施例1中所述的SRGAP2引物探针进行TaqMan qPCR实验。结果如表7所示，结果表明注射人间充质干细胞的右膝关节基因组DNA中可以检测到人源性基因的表达，而未注射细胞的左膝关节不能检测到信号，说明使用该SRGAP2引物进行的TaqMan qPCR方法可以敏感而特异性的检测到动物体内移植的人源性细胞。
表7 SRGAP2 qPCR方法检测注射入新西兰兔右膝关节的人间充质干细胞的基因组DNA
Figure PCTCN2019090571-appb-000013
备注：表格中的DNA含量是指5μl检测样品中含有的DNA量。
实施例10用SRGAP2 qPCR方法检测动物血液中的人源性细胞
除动物各组织/脏器外，人特异性引物及探针SRGAP2还可以敏感性地检测到动物血液中的人源性细胞。为此，我们设计实验：将新西兰兔静脉血(全血)和人间充质干细胞充分混合，未混入人间充质干的全血作为阴性对照。提取基因组DNA后，放置不同时间，用如实施例1中所述的SRGAP2引物探针进行qPCR实验验证。结果如表8所示，结果表明混合人间充质干细胞的全血提取的基因组DNA能够检测到qPCR扩增信号，而未添加人间充质干细胞的全血基因组DNA未能检测到qPCR扩增信号，说明人SRGAP2引物/探针及此qPCR方法可以敏感而特异性的检测到动物血液中的人源性细胞。
表8 SRGAP2 qPCR方法敏感而特异性检测新西兰兔血液中的人源性基因组DNA
Figure PCTCN2019090571-appb-000014
备注：表格中的DNA含量是指5μl检测样品中含有的DNA量。
结论
以人SRGAP2特异性序列设计并合成的引物、标记探针，结合TaqMan qPCR实验技术，可以特异性检测不同动物(如兔、猴、大鼠和小鼠)器官组织或血液中的人源性DNA。实验过程简单、方便，准确度高，灵敏性和特异性强，可用于检测包括细胞治疗和基因治疗临床前研究动物体内的人源性细胞和核酸制剂。
讨论
研发的细胞治疗产品首先需要考虑病人的安全，在细胞治疗产品进入临床前，对其进行细胞类型、活性、迁移特性等的鉴定是确保产品质量和安全的有效手段。更重要的是，知道细胞的生物分布及残留是毒性评估的重要一步。尽管人间充质干细胞(MSC)已进行了临床试验验证其生物安全(14)，但仍缺乏有效的手段追踪或检测移植细胞的生物分布。
一些方法如：影像学、免疫组化和流式细胞技术等被用于评估临床前移植细胞的生物分布(15)，但这些检测方法敏感性低，且不能够定量。qPCR是最有效、最敏感的检测技术之一。虽然qPCR技术可以对受体组织内的移植细胞进行定量检测，但其受到一些重复性差的限制(10)，为此，我们寻找并设计了靶向人源性DNA的特异性引物。
在本实验中，我们设计并合成了人特异性序列SRGAP2的引物、探针，利用TaqMan qPCR技术，可以对动物体内的人源性移植细胞进行特异性检测。基于对引物和TaqMan qPCR技术进行特异性、选择性、精密度和准确度等的验证，本方法可以灵敏的检测动物(新西兰兔、食蟹猴、大鼠和小鼠)体内的人源性细胞(人间充质干细胞)的基因组DNA。研究发现：人间充质干细胞归巢缺血组织是其很重要的一个细胞特性，但这种特征会在细胞培养扩增后逐渐减少(16)，但只需要很少的细胞迁移到心脏即可检测到细胞在移植后的位置及含量。
使用我们设计合成的人SRGAP2特异性的引物及qPCR技术，我们可以检测新西兰兔和食蟹猴膝关节腔中移植的人间充质干细胞。不同于其它有不足或缺陷的PCR检测手段，我们的方法具有很高的精确度和重复性，而且灵敏度高，可以稳定检测到32.000pg的人源性基因组DNA。除此之外，我们对新西兰兔和食蟹猴不同组织(如：心脏、肝脏、脾脏、肺脏、淋巴结、脑等)基因组DNA进行了检测，都未发现交叉反应。因此，我们的SRGAP2引物及TaqMan PCR技术可以用于临床前动物模型中检测移植的人源性细胞的生物分布，并进行精确定量。
本发明的实施方式并不限于上述实施例所述，在不偏离本发明的精神和范围的情况下，本领域普通技术人员可以在形式和细节上对本发明做出各种改变和改进，而这些均被认为落入了本发明的保护范围。
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Claims (16)

  1. 人SRGAP2基因在检测样本中人源性基因组DNA中的应用。
  2. 特异性检测人SRGAP2基因的试剂在检测样本中人源性基因组DNA中的应用。
  3. 特异性检测人SRGAP2基因的试剂在制备检测样本中人源性基因组DNA的试剂中的应用。
  4. 根据权利要求2或3的应用，所述特异性检测人SRGAP2基因的试剂包含特异性检测人SRGAP2基因的引物对。
  5. 根据权利要求4的应用，所述引物对包括正向引物5'-CGATACTCAGGTCAAAGGTAAGG-3'和反向引物5'-CTGCAAATCACGGTGGAAATAC-3'。
  6. 根据权利要求4或5的应用，所述特异性检测人SRGAP2基因的试剂还包括荧光探针5'-TGCAAATGCTCTGTGGACTGGTGA-3'，该探针的5’端标记报告荧光基团，3’端标记淬灭基团；优选地，所述荧光探针的5’端标记的报告荧光基团为FAM，3’端标记的淬灭基团为NFQ-MGB或TAMRA。
  7. 特异性检测样本中人源性基因组DNA的引物对，其包括特异性扩增人SRGAP2基因的正向引物和反向引物。
  8. 根据权利要求7的引物对，其中所述正向引物是5'-CGATACTCAGGTCAAAGGTAAGG-3'，所述反向引物是5'-CTGCAAATCACGGTGGAAATAC-3'。
  9. 特异性检测人源性基因组DNA的引物和探针组合，其包括特异性扩增SRGAP2基因的正向引物、反向引物和荧光探针。
  10. 根据权利要求9的引物和探针组合，其中所述正向引物是5'-CGATACTCAGGTCAAAGGTAAGG-3'，所述反向引物是5'-CTGCAAATCACGGTGGAAATAC-3'，所述探针的序列为5'-TGCAAATGCTCTGTGGACTGGTGA-3'，该探针的5’端标记报告荧光基团，3’端标记淬灭基团；优选地，所述荧光探针的5’端标记的报告荧光基团为FAM，3’端标记的淬灭基团为NFQ-MGB。
  11. 特异性检测人源性基因组DNA的试剂盒，该试剂盒包括根据权利要求7或8的引物对，或包括根据权利要求9或10的引物和探针组合。
  12. 根据权利要求11的试剂盒，所述试剂盒还包括选自qPCR反应液、无核酸酶高纯水、人源性基因组DNA标准品、作为阳性对照品的人源性基因组DNA、不含人源性基因组DNA的阴性对照品、和质控品中的任意一种或更多种试剂。
  13. 样本中人源性基因组DNA的检测方法，其包括以下步骤：
    (1)提取样本基因组DNA；
    (2)使用根据权利要求9或10的引物和探针组合对一系列人源性基因组DNA标准品和样本基因组DNA进行qPCR检测；
    (3)用qPCR结果拟合标准曲线：以所述人源性基因组DNA标准品的Ct值为纵坐标(X)，以人源性基因组DNA标准品中人源性基因组DNA的浓度的对数为横坐标(Y)，拟合标准曲线，使得回归线性方程后，R 2≥0.99；且各标准品浓度的准确度(RE％)为-75％～150％，确定可稳定检测到的浓度最低点；
    (4)结果判定：如果样本的qPCR结果中出现明显的扩增曲线；且样本qPCR的Ct值小于浓度最低点的Ct值，则为阳性结果，即样本中存在人源性DNA；如果无明显扩增曲线，或者有明显扩增曲线，但Ct值大于标准曲线浓度最低点的Ct值，则为阴性结果，即样本中不存在人源性DNA。
  14. 根据权利要求13的检测方法，其中步骤(4)进一步包括根据样本基因组DNA的Ct值和拟合的标准曲线，确定样本基因组DNA中人源性基因组DNA的浓度。
  15. 样本中人源性基因组DNA的检测方法，其包括以下步骤：
    (1)提取样本基因组DNA；
    (2)使用根据权利要求9或10的引物和探针组合对一系列人源性基因组DNA标准品和样本基因组DNA进行qPCR检测；
    (3)用qPCR结果拟合标准曲线：以所述人源性基因组DNA标准品的Ct值为纵坐标(X)，以人源性基因组DNA标准品中人源性基因组DNA的浓度的对数为横坐标(Y)，拟合线性方程，其中R 2≥0.99；且各标准品浓度的准确度(RE％)为-75％～150％，确定可稳定检测到的浓度最低点；
    (4)结果判定：根据样本基因组DNA的Ct值和拟合的标准曲线，确定样本基因组DNA中人源性基因组DNA的浓度。
  16. 根据权利要求13-15任一项的检测方法，其中所述浓度最低点是32.00pg/5μl。
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