CN113462757B

CN113462757B - Quantitative detection method of human cells in pharmacokinetics research

Info

Publication number: CN113462757B
Application number: CN202110800291.2A
Authority: CN
Inventors: 张月辉; 陈涛涛; 王嘉显
Original assignee: Help Stem Cell Innovations Co ltd
Current assignee: Help Stem Cell Innovations Co ltd
Priority date: 2021-07-15
Filing date: 2021-07-15
Publication date: 2024-01-30
Anticipated expiration: 2041-07-15
Also published as: CN113462757A

Abstract

The invention belongs to the field of biotechnology, and relates to a quantitative detection method of human cells in pharmacokinetic research, which relies on primers specific to mitochondrial DNA in human cells to quantitatively detect human cells in experimental animals, tissues of experimental animals or animal cell cultures by qPCR. The technical scheme of the application promotes quantitative analysis of cell medicines in the animal experiment process, and the quantitative detection method of the humanized cells in the pharmacokinetics research provided by the embodiment of the invention has higher precision.

Description

Quantitative detection method of human cells in pharmacokinetics research

Technical Field

The invention belongs to the field of biological medicine, and relates to a quantitative detection method of human cells in pharmacokinetics research.

Background

The existing medicines cannot be completely cured, and a huge clinical pain point is formed by the functional loss diseases and degenerative diseases caused by external injury or human tissue degeneration, such as heart failure, parkinsonism, infantile cerebral palsy and the like. Studies have shown that repair of damaged or degenerated tissue by cell transplantation is an effective means of treating the above-mentioned diseases. During the drug development process, research on metabolism and movement of drugs in vivo is a necessary item, and in order to study metabolism and movement of cellular drugs in vivo, human cells must be transplanted into experimental animals and tracked through animal experiments. After transplantation, human cells may migrate from the site of transplantation with blood flow or by other means to other organs or tissues, so the assay must be able to detect human cells in different organs and tissues of the experimental animal, including blood. The existing drug metabolism and drug movement detection means mainly aim at traditional chemical drugs, and the method for in-vivo tracing of cell drugs is required to be capable of detecting human cells in an experimental animal body with high specificity and quantifying the human cells. At present, the quantitative research method of cell medicines in animals is not perfect, and development is needed.

Disclosure of Invention

The present invention aims to provide a quantitative detection method of human cells in pharmacokinetic studies, which quantitatively detects human cells transplanted or injected into experimental animals or incorporated into animal tissues or cell cultures by qPCR.

In order to achieve the above purpose, the present invention proposes the following technical scheme: quantitative detection methods of human cells in pharmacokinetic studies rely on primers specific for mitochondrial DNA within human cells and employ qPCR to quantitatively detect human cells in experimental animals, in experimental animal tissues, or in animal cell cultures.

As an improved technical scheme of the application, the primer with specificity to the mitochondrial DNA in the humanized cell is designed based on any section of DNA fragment interval of the specificity of the humanized mitochondrial DNA relative to the non-human animal DNA; wherein, the sequence fragment of the human mitochondrial DNA relative to the DNA fragment interval specific to the non-human animal DNA is more than 100bp; the length of the primer amplified fragment with specificity to mitochondrial DNA in human cells is between 100bp and 200 bp.

As an improved technical scheme of the application, the primer with specificity to the mitochondrial DNA in the human cell is designed into SEQ ID NO.4 based on the following gene sequences; and the length of the primer amplified fragment with specificity to mitochondrial DNA in human cells is between 100bp and 200 bp.

As an improved technical scheme of the application, the primer with specificity to the mitochondrial DNA in the human cell comprises a forward primer F which is SEQ ID NO.7 and a reverse primer R which is SEQ ID NO.8; or the forward primer F is SEQ ID NO.9, and the reverse primer R is SEQ ID NO.10; or the forward primer F is SEQ ID NO.11, and the reverse primer R is SEQ ID NO.12.

As an improved technical scheme of the application, the human-derived cells comprise induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells or cells derived from induced pluripotent stem cells.

As an improved technical scheme of the application, the method specifically comprises the following steps:

step one, designing a qPCR reaction system according to the primer with specificity to mitochondrial DNA in a human cell;

step two, verifying the applicability of the qPCR reaction system in detecting human cells in an experimental animal body, an experimental animal tissue or an animal cell culture;

and thirdly, quantitatively detecting the human cells in the experimental animal body, the experimental animal tissue or the animal cell culture by adopting qPCR.

As an improved technical solution of the present application, the second step includes:

standard curves were prepared with primers specific for mitochondrial DNA within human cells: adopting the primer with specificity to the mitochondrial DNA in the human cells, taking a plurality of groups of the mitochondrial DNA in the human cells with gradient concentration as a sample to perform qPCR amplification, and preparing a standard curve with the Ct mean value as a Y axis and the concentration of the mitochondrial DNA in the human cells as an X axis for the primer with specificity to the mitochondrial DNA in the human cells;

repeatability detection: the method comprises the steps of adopting a mixture of human cell DNA and non-human animal cell DNA as a test sample, wherein the concentration of the human cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is the other one of the gradient concentrations; qPCR amplification is carried out by adopting the primer with specificity to the mitochondrial DNA in the human cells, and the consistency of the Ct value and the standard curve of the primer with specificity to the mitochondrial DNA in the human cells is detected.

As an improved technical scheme of the application, the standard curve qualification conditions of the primer specific to the mitochondrial DNA in the human cell are as follows: r is R ² Not lower than 0.99, and the amplification efficiency is between 90 and 110 percent.

preparing a standard curve of a universal primer of mitochondrial DNA of human and non-human animal cells: adopting a general primer of human and non-human animal cell mitochondrial DNA, and adopting a plurality of groups of non-human animal cell DNA with gradient concentration as a sample to carry out qPCR amplification to prepare a general primer standard curve of human and non-human animal cell mitochondrial DNA with Ct mean value as Y axis and non-human animal cell DNA concentration as X axis;

repeatability detection: the method comprises the steps of taking a mixture of human cell DNA and non-human animal cell DNA as a test sample, wherein the concentration of the non-human animal cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is the other one of the gradient concentrations; qPCR amplification is carried out by using the human and non-human animal cell mitochondrial DNA universal primer, and the consistency of the Ct value and the standard curve of the human and non-human animal cell mitochondrial DNA universal primer is detected.

As an improved technical scheme of the application, the standard curve qualification conditions of the human and non-human animal cell mitochondrial DNA universal primer are as follows: r is R ² Not lower than 0.99, and the amplification efficiency is between 90 and 110 percent.

As an improved technical scheme of the application, the second step comprises performing a specificity test;

the specificity test comprises the following steps:

preparing a human cell DNA sample: simultaneously obtaining non-human animal cell DNA in experimental animal body tissues, and carrying out gradient dilution on the human cell DNA by using the non-human animal cell DNA to obtain a specific supply; the concentration of the human cell DNA after the gradient dilution is within the range of the gradient concentration of the standard curve for preparing the primer with specificity to the mitochondrial DNA in the human cell;

qPCR amplification is carried out on a plurality of groups of specific supplies by using the primers with specificity to mitochondrial DNA in the humanized cells, and a specificity curve with Ct average value as Y axis and humanized cell DNA concentration as X axis is prepared; after the Ct mean value RSD value is calculated to reach the standard, comparing the consistency of the specificity curve with the standard curve of the specific primer for the mitochondrial DNA in the humanized cell;

qPCR (quantitative polymerase chain reaction) amplification is carried out on a plurality of groups of specific supplies by adopting universal primers of mitochondrial DNA of human and non-human animal cells, and a specific curve which takes Ct average value as Y axis and takes the DNA concentration of human cells as X axis is prepared; after the computed Ct mean value RSD value reaches the standard, comparing the consistency of the specificity curve with the standard curve of the human and non-human animal cell mitochondrial DNA universal primer.

As an improved technical scheme of the application, the universal primer of human and non-human animal cell mitochondrial DNA is designed based on any section of human mitochondrial DNA in a conserved DNA fragment interval relative to mitochondrial DNA of other species, and the length of an amplified fragment is between 100bp and 200 bp; and the sequence fragment of the human mitochondrial DNA relative to the conserved DNA fragment interval of mitochondrial DNA of other species is more than 100bp.

As an improved technical scheme of the application, the human and non-human animal cell mitochondrial DNA universal primer is designed based on the following sequences: SEQ ID NO.13; and the sequence fragment of the human mitochondrial DNA relative to the conserved DNA fragment interval of mitochondrial DNA of other species is more than 100bp.

As an improved technical scheme of the application, the human and non-human animal cell mitochondrial DNA universal primer comprises: forward primer F: SEQ ID NO.17, reverse primer R: SEQ ID NO.18; or forward primer F: SEQ ID NO.19, reverse primer R: SEQ ID NO.20; or forward primer F: SEQ ID NO.21, reverse primer: SEQ ID NO.22.

The beneficial effects are that:

according to the technical scheme, the novel method for detecting the human cells in the animal body is provided, quantitative analysis of cell medicines in the animal experiment process is promoted, and the quantitative detection method of the human cells in the pharmacokinetics research provided by the embodiment of the invention has higher precision.

The technical scheme of the invention provides a novel method for cytopharmacokinetics research. The method has the advantages that 1) the human mitochondrial DNA is used for amplification relative to specific fragments of other species, the specificity is high, the amplification is easy, the detection sensitivity is greatly improved, the detection limit can be as low as 0.025%, and the requirement of pharmacokinetic study on the detection limit of cell drug residue in an animal body is met; 2) Compared with the traditional PCR method and flow cytometry (FACS) method, the qPCR method has the advantages that the repeatability in experiments and among experiments is greatly improved, the reliability of experimental results is ensured, the accuracy requirement of pharmacokinetic study on determination of cell drug metabolism curves in animals is met, and 3) the technical scheme provided by the study is suitable for human cell pharmacokinetic study in mice, rats and monkeys, almost covers all experimental animal species, and has wide application prospect.

The method comprises the following steps: the primer with specificity to the human mitochondrial DNA is designed and the use rationality of the primer is verified, so that the number of human cells in a non-human animal body can be effectively promoted to be rapidly distinguished, and the accuracy of quantitative detection is ensured.

The standard curve of qPCR amplification of the specific primer for mitochondrial DNA in the humanized cell and the standard curve of qPCR amplification of the universal primer for mitochondrial DNA of human and non-human animal cells are respectively established, repeatability and specificity verification are respectively carried out, the correctness and the accuracy of a qPCR reaction system are comprehensively verified, and the effectiveness of the qPCR system is effectively ensured.

The repeatability test is carried out on the standard curve of the specific primer for the mitochondrial DNA in the humanized cell and the standard curve of the universal primer for the mitochondrial DNA of the human and non-human animal cells respectively, and each repeatability test is provided with at least two concentrations of the test sample so as to ensure the accuracy and the effectiveness of the standard curve.

It should be understood that all combinations of the foregoing concepts, as well as additional concepts described in more detail below, may be considered a part of the inventive subject matter of the present disclosure as long as such concepts are not mutually inconsistent.

Drawings

The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. Embodiments of various aspects of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 is a standard curve for group 1 with specific primers for mitochondrial DNA in human cells.

FIG. 2 is a standard curve for group 2 with specific primers for mitochondrial DNA in human cells.

FIG. 3 is a standard curve for group 3 with specific primers for mitochondrial DNA in human cells.

FIG. 4 is a standard curve of the mitochondrial DNA universal primer for human and non-human animal cells of group 1.

FIG. 5 is a standard curve of the mitochondrial DNA universal primer for human and non-human animal cells of group 2.

FIG. 6 is a standard curve of the mitochondrial DNA universal primer for human and non-human animal cells of group 3.

FIG. 7 is a graph showing the specificity of human MSC cell DNA after dilution of mouse heart DNA.

FIG. 8 is a graph showing the specificity of human MSC cell DNA after dilution of mouse lung tissue DNA.

Detailed Description

The quantitative detection method of human cells in the pharmacokinetic study is adopted, and the quantitative detection of the human cells in an experimental animal body, an experimental animal tissue or an animal cell culture is carried out by adopting qPCR (quantitative polymerase chain reaction) depending on primers with specificity on mitochondrial DNA in the human cells. The method provides a reasonable proving mode for the effectiveness of the cell treatment technology and promotes the popularization and application of the cell treatment technology.

In order to better explain the technical solutions of the present application, the following description is made in detail in connection with specific embodiments.

Quantitative detection of human cells in non-human animal cells using qPCR is performed by:

1) Primer design

a) Primers specific for mitochondrial DNA within human cells were designed.

By comparing the whole mitochondrial DNA sequences of human, rat, mouse and monkey, a DNA fragment interval (such as sequence 5-10) of the specificity of the human mitochondrial DNA relative to mitochondrial DNA of other species is found out, and the sequence fragment is required to be more than 100bp;

sequence 5: human mitochondrial reference genome 1-570 (human mitochondrial reference genome 1-570) SEQ ID NO.1;

sequences 6,Human mitochondrial reference genome 1570-1746 (human mitochondrial reference genome 1570-1746) SEQ ID NO.2;

sequence 7,Human mitochondrial reference genome 2288-2463 (human mitochondrial reference genome 2288-2463) SEQ ID No.3;

sequence 8,Human mitochondrial reference genome 11459-12105 (human mitochondrial reference genome 11459-12105) SEQ ID NO.4;

sequences 9,Human mitochondrial reference genome 14234-14469 (human mitochondrial reference genome 14234-14469) SEQ ID NO.5;

the sequence 10,Human mitochondrial reference genome 15963-16468 (human mitochondrial reference genome 15963-16468) SEQ ID NO.6.

And designing forward and reverse primers R for qPCR amplification aiming at any section of the sequences 5-10, wherein the length of the amplified fragment is between 100bp and 200 bp.

Preferably, in order to improve qPCR detection efficiency and to better detect human cells from non-human animal cells, the present application designs qPCR amplified forward and reverse primer R for sequence 8.

In specific application, a large number of experiments are combined to verify that the primer with specificity to mitochondrial DNA in human cells is adopted, and the detection accuracy is higher. The primer with specificity to the mitochondrial DNA in the human cell comprises a forward primer F and a reverse primer R, wherein the forward primer F is SEQ ID NO.7, and the reverse primer R is SEQ ID NO.8; or the forward primer F is SEQ ID NO.9, and the reverse primer R is SEQ ID NO.10; or the forward primer F is SEQ ID NO.11, and the reverse primer R is SEQ ID NO.12.

b) Universal primer for designing mitochondrial DNA of human and non-human animal cells

By comparing the whole mitochondrial DNA sequences of human, rat, mouse and monkey, a conserved DNA fragment interval of the human mitochondrial DNA relative to mitochondrial DNA of other species is found, and the sequence fragment is required to be more than 100bp (the following sequences 11-14);

sequence 11,Human mitochondrial reference genome 2446-2606 SEQ ID NO.13.

Sequences 12,Human mitochondrial reference genome 6203-7613: SEQ ID NO.14.

Sequence 13,Human mitochondrial reference genome 9272-10341: SEQ ID NO.15.

Sequence 14,Human mitochondrial reference genome 10907-11400: SEQ ID NO.16.

The forward and reverse primers R of qPCR amplification are designed aiming at any section of the sequences 11-14, and the length of the amplified fragment is between 100bp and 200 bp.

Preferably, in order to improve qPCR detection efficiency and to better detect human cells from non-human animal cells, the present application designs qPCR amplified forward and reverse primers R for sequence segment 11.

In specific application, a large number of experiments are combined to verify that the following general primer for human and non-human animal cell mitochondrial DNA is adopted, and the detection accuracy is higher. The human and non-human animal cell mitochondrial DNA universal primer comprises a forward primer F and a reverse primer R, and is any one of the following primer pairs, wherein the forward primer F is: SEQ ID NO.17, reverse primer R: SEQ ID NO.18; or forward primer F: SEQ ID NO.19, reverse primer R: SEQ ID NO.20; or forward primer F: SEQ ID NO.21, reverse primer R: SEQ ID NO.22.

2) Obtaining a human cell, wherein the human cell comprises an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell or a cell derived from the induced pluripotent stem cell. Cells derived from the induced pluripotent stem cells include cardiomyocytes, mesenchymal stem cells, cardiac conduction cells, lymphatic cells, macrophages, keratinocytes, NK cells, nerve cells, glial cells and the like.

The experimental animal body comprises animal bodies of experimental animals such as experimental monkeys, experimental mice and the like, and experimental animal tissues or animal cell cultures. The experimental animal tissue comprises heart, lung, spleen, liver, kidney and other tissues.

3) The scheme design includes:

step one, designing a qPCR reaction system according to the primer with specificity to the mitochondrial DNA in the human cell.

Step two, verifying the applicability of the qPCR reaction system in detecting human cells in an experimental animal body, an experimental animal tissue or an animal cell culture; comprising the following steps:

1) Standard curves were prepared with primers specific for mitochondrial DNA within human cells: the primer with specificity to the mitochondrial DNA in the human cells is adopted, a plurality of groups of the mitochondrial DNA in the human cells with gradient concentration are used as samples to be subjected to qPCR amplification, and a standard curve with the Ct mean value as a Y axis and the concentration of the mitochondrial DNA in the human cells as an X axis is prepared. Meanwhile, establishing a qualified standard of a standard curve: r is R ² Not lower than 0.99, and the amplification efficiency is between 90 and 110 percent so as to ensure the effectiveness of detection.

Preferably, this step is repeated several times, each time ensuring that the concentration of non-human animal cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is another one of the gradient concentrations, each of which may not be repeated. Such as a second repeatability test: the method comprises the steps of adopting a mixture of human cell DNA and non-human animal cell DNA as a test sample, wherein the concentration of the human cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is the other one of the gradient concentrations; qPCR amplification is carried out by adopting the primer with specificity to the mitochondrial DNA in the human cells, and the consistency of the Ct value and the standard curve of the primer with specificity to the mitochondrial DNA in the human cells is detected.

2) Preparing a standard curve of a universal primer of mitochondrial DNA of human and non-human animal cells: the general primer of human and non-human animal cell mitochondrial DNA is adopted, and a plurality of groups of non-human animal cell DNA with gradient concentration are used as test products to carry out qPCR amplification to prepare human and non-human animal cell DNA with Ct average value as Y axis and non-human animal cell DNA concentration as X axisHuman animal cell mitochondrial DNA universal primer standard curve. Meanwhile, the standard curve qualification conditions of the human and non-human animal cell mitochondrial DNA universal primer are designed as follows: human and non-human animal cell mitochondrial DNA universal primer standard curve R ² Not lower than 0.99, and the amplification efficiency is between 90 and 110 percent.

Repeatability detection: the method comprises the steps of taking a mixture of human cell DNA and non-human animal cell DNA as a test sample, wherein the concentration of the non-human animal cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is the other one of the gradient concentrations; qPCR (quantitative polymerase chain reaction) amplification is carried out by using a human and non-human animal cell mitochondrial DNA universal primer, and the consistency of a Ct value and a standard curve of the human and non-human animal cell mitochondrial DNA universal primer is detected; preferably, the method further comprises repeating the step several times, each time ensuring that the concentration of the non-human animal cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is another one of the gradient concentrations. Such as a second repeatability test: the method comprises the steps of taking a mixture of human cell DNA and non-human animal cell DNA as a test sample, wherein the concentration of the non-human animal cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is the other one of the gradient concentrations; qPCR amplification is carried out by using the human and non-human animal cell mitochondrial DNA universal primer, and the consistency of the Ct value and the standard curve of the human and non-human animal cell mitochondrial DNA universal primer is detected.

Preferably, said verifying the suitability of the qPCR reaction system for detecting human cells in an experimental animal, in an experimental animal tissue or in an animal cell culture comprises performing a specificity test.

3) The specificity test comprises the following steps:

Reagents according to the examples of the present application

Table 1 reagents according to examples of the present application

Reagent name	Manufacturer(s)	Storage conditions
			DPBS	Gibco	4℃
DEPC water	Beyotime	4℃
			PowerUp™ SYBR™ Green Master Mix	Thermo Fisher Scientifics	Light protection at-20 DEG C
DNeasy Blood & Tissue Kit	Qiagen	Room temperature

Sample preparation of examples of the present application

A. Sample preparation of human cell DNA

Cell DNA extraction (dnase Blood & Tissue Kit was used in this example): human mesenchymal stem cells are human cells, after resuscitating the cells, the cells are resuspended in DPBS for cell counting and incubated after proteinase K is added, and a DNA solution is obtained in a centrifuge tube.

1 μl of cell DNA solution is taken, the DNA concentration is measured on a Nanodrop spectrophotometer, the OD260/OD280 value is 1.8-2.0 as pass, and the average value of the DNA concentration is measured for 3 times.

The DNA concentration was 5 ng/. Mu.l by dilution with DEPC water. If the sample is to be used within 48 hours, it is stored at 4 ℃. The sample can be stored for 1 month at-20deg.C.

B. Sample preparation of murine cellular DNA

The DNA of the blood of the mice is extracted, and a DNA sample stock solution with the concentration of 25 ng/mu l is prepared.

Example 1

1. qPCR reaction system is designed depending on the primer with specificity to mitochondrial DNA in human cells

1) The primer specific to mitochondrial DNA in human cells can be used in any of the following 3 groups. Group 1: the forward primer F is SEQ ID NO.7, and the reverse primer R is SEQ ID NO.8. The forward primer F of the group 2 is SEQ ID NO.9, and the reverse primer R is SEQ ID NO.10. The forward primer F of group 3 is SEQ ID NO.11, and the reverse primer R is SEQ ID NO.12.

2) Extracting human MSC cell DNA, preparing a DNA sample stock solution with the concentration of 50 ng/mu l, and carrying out gradient dilution on the DNA by using DEPC water to serve as a standard curve test sample. Taking sequence numbers 1 to 2 as an example, wherein the sequence number 1 is a sample stock solution with the DNA concentration of 50 ng/mu l, taking 5 mu l of the sample stock solution, adding 45 mu l of DEPC water, and obtaining the sequence number 2 with the concentration of 5 ng/mu l. In sequence, 6 concentrations of test samples and one negative control test sample were prepared in total, as shown in Table 2.

TABLE 2 gradient dilution of human MSC cell DNA

Serial number of test sample	1	2	3	4	5	6	7
								DNA concentration (ng/. Mu.l)	50	5	0.5	0.05	0.005	0.0005	0
DNA concentration ratio	1 x 10 ⁵	1 x 10 ⁴	1 x 10 ³	1 x 10 ²	1 x 10	1	0

3) For each group, qPCR master reaction systems were formulated according to table 3. Table 3 shows that there are 3 systems for each test, with the addition of DEPC water to control 3 systems, there is a loss in split charging of the systems, and the total coefficient is 24 for more than 3 systems.

TABLE 3 preparation of qPCR Main reaction Main System

3. qPCR was run and standard curves were prepared

After the system was configured, it was blown down uniformly and added to 384-well qPCR plates at 6 μl per well.

Standard curve samples were taken in 4 μl and added to the qPCR plate, each sample numbered 3 wells. Negative control 3 duplicate wells were added with 4 μl DEPC water. After the sample is added, a sealing film is stuck, and the qPCR plate is centrifuged at 2000rpm for 3 minutes, so that the sample can be detected on the machine. After qPCR operation is finished, data are derived, ct average value is taken as Y-Axis, log (DNA concentration ratio) is taken as X-Axis, and a standard curve with specific primers for mitochondrial DNA in human cells is prepared.

Standard curve detection limits for primers specific for mitochondrial DNA within human cells corresponding to groups 1-3: limit of detection: the concentration was 0.0005 ng/. Mu.l. Quantitative limit: the concentration was 0.0005 ng/. Mu.l.

Standard curve R of specific primer for mitochondrial DNA in human cell in combination with group 1 of figure 1 ² The amplification efficiency is 98.557% and the experiment is qualified, wherein the amplification efficiency is 0.9945.

FIG. 2, panel 2, standard Curve R, with specific primers for mitochondrial DNA in human cells ² 0.9986, the amplification efficiency is 99.557%, and the experiment is qualified.

In FIG. 3, group 2 has a standard curve of specific primers for mitochondrial DNA in human cells, the standard curve R2 is 0.9949, the reaction efficiency is 98.193%, and the experiment is qualified.

4. First time of repeatability

Sample preparation: 6 DNA samples with the total concentration of 5 ng/mu l and 0.05 ng/mu l of humanized MSC cell DNA and 4.95 ng/mu l of mouse blood DNA are prepared and used as a repeatability verification test sample to be placed on an ice box for standby.

Preparation of qPCR main reaction system: according to Table 4, 6 parts of the repeatability test sample were prepared, 3 parts of the system was used as a negative control, and the loss of split charging of the system was found, and the total coefficient was 24.

TABLE 4 configuration of qPCR Main reaction System for first time repeat verification

4 μl of the reproducibility test sample was added to the qPCR plate, and 3 wells of each reproducibility test sample were used. Negative control 3 duplicate wells were added with 4 μl DEPC water. After the sample is added, a sealing film is stuck, and the qPCR plate is centrifuged at 2000rpm for 3 minutes, so that the sample can be detected on the machine.

And after qPCR operation is finished, data are derived, and RSD of Ct average values of 6 repeatability verification test samples are calculated, wherein the RSD is shown in table 5.

Table 53 results of first repeated experiments with primers specific for mitochondrial DNA in human cells

The reproducibility test of the primers with specificity to mitochondrial DNA in human cells in groups 1 to 3 meets the regulations and is qualified.

Second repeatability verification:

sample preparation: 6 DNA samples with the total concentration of 5 ng/mu l and 0.0005 ng/mu l of humanized MSC cell DNA and 4.9995 ng/mu l of mouse blood DNA are prepared and used as a repeatability verification test sample and placed on an ice box for standby.

Human MSC cell DNA detection qPCR main reaction system preparation: the test was conducted in accordance with Table 6, with 6 parts of the repeatability test sample per 3 systems, 3 systems for negative control, and the loss of split charging of the system, 3 systems for the number of systems, and the overall coefficient of 24.

TABLE 6 qPCR Main reaction System configuration for second repeatability verification

And after qPCR operation is finished, data are derived, and RSD of Ct average values of 6 repeatability verification test samples are calculated, wherein the RSD is shown in Table 7.

TABLE 7 results of secondary reproducibility test of human MSC cell DNA

The second repeat test of the primers specific to mitochondrial DNA in human cells of groups 1 to 3 met the specifications and passed the test.

Example 2

1. qPCR reaction system designed based on human and non-human animal cell mitochondrial DNA universal primer

1) Adopting any one of the following 3 groups of primers as general primers of human and non-human animal cell mitochondrial DNA; group 1, forward primer F: SEQ ID NO.17, reverse primer R: SEQ ID NO.18. Group 2, forward primer F: SEQ ID NO.19, reverse primer R: SEQ ID NO.20. Group 3, forward primer F: SEQ ID NO.21, reverse primer R: SEQ ID NO.22.

2) The DNA of the blood of the mice was extracted, a DNA sample stock solution was prepared at a concentration of 25 ng/. Mu.l, and the DNA was subjected to gradient dilution with DEPC water as a standard curve test piece. Taking the sample stock solution with the DNA concentration of 25 ng/. Mu.l as an example from the sequence number 1 to the sequence number 2, 5. Mu.l of the sample stock solution is taken and 45. Mu.l of DEPC water is added to obtain the sequence number 2 with the concentration of 2.5 ng/. Mu.l. Sequentially, a test sample of the concentration of 6 and a negative control test sample were prepared together. See table 8.

TABLE 8 gradient dilution of mouse cell DNA

Serial number of test sample	1	2	3	4	5	6	7
								DNA concentration (ng/. Mu.l)	25	2.5	0.25	0.025	0.0025	0.00025	0
DNA concentration ratio	1 x 10 ⁵	1 x 10 ⁴	1 x 10 ³	1 x 10 ²	1 x 10	1	0

3) For each group, a qPCR main reaction main system was formulated. Table 8 shows that there are a loss in split charging of 3 systems for each 3 systems tested, plus DEPC water control 3 systems, and a total coefficient of 24 for more than 3 systems, as shown in Table 9.

TABLE 9 qPCR Main reaction Main System formulation (Standard Curve)

3. qPCR was run and standard curves were prepared

Standard curve samples were taken in 4 μl and added to the qPCR plate, each sample numbered 3 wells. Negative control 3 duplicate wells were added with 4 μl DEPC water. After the sample is added, a sealing film is stuck, and the qPCR plate is centrifuged at 2000rpm for 3 minutes, so that the sample can be detected on the machine. And after qPCR operation is finished, data are derived, ct average value is taken as Y-Axis, log (DNA concentration ratio) is taken as X-Axis, and a human and non-human animal cell mitochondrial DNA universal primer standard curve is prepared.

Group 1 human and non-human animal cell mitochondrial DNA general primer standard curve is shown in figure 4, standard curve R ² 0.9972, the amplification efficiency is 99.934%, and the experiment is qualified.

Group 2 human and non-human animal cell mitochondrial DNA general primer standard curve is shown in figure 5, standard curve R ² 0.9953, the amplification efficiency is 99.961%, and the experiment is qualified.

Group 3 human and non-human animal cell mitochondrial DNA general primer standard curve is shown in figure 6, standard curve R ² The amplification efficiency is 101.324% and the experiment is qualified, wherein the amplification efficiency is 0.9948%.

4. First time of repeatability

Preparation of qPCR main reaction system: 6 parts of repeatability test samples are prepared, 3 systems are used for each part, the negative control is used for 3 systems, the split charging of the systems is lost, 3 systems are added, and the total coefficient is 24, and is shown in table 10.

TABLE 10 configuration of qPCR Main reaction System for first time repeat verification

And after qPCR operation is finished, data are derived, and RSD of Ct average values of 6 repeatability verification test samples are calculated, wherein the RSD is shown in Table 11.

TABLE 11 results of first-time repeated experiments on murine blood DNA

The repeatability test of the universal primer of the mitochondrial DNA of the non-human animal cells in the groups 1 to 3 meets the rule and is qualified.

Second repeatability verification:

Human MSC cell DNA detection qPCR main reaction system preparation: 6. the number of the test samples is 3 per one number of the systems, the number of the negative control systems is 3, the loss of split charging of the systems is caused, the number of the systems is 3, and the total coefficient is 24, and the total coefficient is shown in Table 12.

TABLE 12 qPCR Main reaction System configuration for second repeatability verification

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And after qPCR operation is finished, data are derived, and RSD of Ct average values of 6 repeatability verification test samples are calculated, wherein the RSD is shown in a table 13.

TABLE 13 results of a second repeat test of murine blood DNA

And the repeatability 2 test of the murine blood DNA meets the regulation, and the test is qualified.

Example 3

1. Sample configuration

Extracting heart and lung tissue DNA of mice (lung tissue DNA of experimental cynomolgus monkey can also be adopted), respectively preparing DNA samples with the concentration of 2.5 ng/mu l, and placing the DNA samples on an ice box for standby.

Preparing a humanized MSC cell DNA sample with the concentration of 2.5 ng/mu l, and placing the humanized MSC cell DNA sample on an ice box for standby.

Human MSC DNA was subjected to gradient dilution with mouse heart or lung tissue DNA, respectively as a specific sample. Taking serial numbers 1 to 2 as an example, wherein the serial number 1 is a human DNA sample stock solution with the concentration of 0.25 ng/mu l, taking 5 mu l of the sample stock solution, adding 45 mu l of mouse heart and lung DNA, and obtaining serial number 2 with the concentration of 0.025 ng/mu l of human DNA. In sequence, 4 concentrations of test samples and one negative control test sample were prepared in total, as shown in Table 14.

Table 14 preparation of specific test samples

Serial number of test sample	1	2	3	4	5
						Human MSC DNA concentration (ng/μl)	0.25	0.025	0.0025	0.00025	0
Mouse heart or lung tissue DNA concentration (ng/μl)	2.25	2.475	2.4975	2.49975	2.5
						Human DNA concentration ratio	1 x 10 ⁴	1 x 10 ³	1 x 10 ²	1 x 10	0

2. qPCR main reaction system was designed with human MSC cell mitochondrial DNA primers, as shown in Table 15.

The total of the specific test sample (heart or lung) needs 30 parts of system, the negative control system has 3 parts, the split charging has loss, and the total coefficient is 33 by adding more than 3 parts of system.

TABLE 15 qPCR reaction System under mitochondrial DNA primer of human MSC cells

In table 15, the mitochondrial DNA primers of the human MSC cells (primers specific for mitochondrial DNA in the human cells) were used as follows: the forward primer F is SEQ ID NO.7, and the reverse primer R is SEQ ID NO.8.

After qPCR operation is finished, data are derived, and the mouse myocardial tissue DNA is used for carrying out gradient dilution on human MSC mitochondrial DNA as a result of a specific test sample, and the result is shown in a table 16 and corresponds to FIG. 3.

TABLE 16 Ct values corresponding to DNA concentration gradients in human MSC cells

Serial number of test sample	1	2	3	4	5
						DNA concentration (ng/. Mu.l)	0.25	0.025	0.0025	0.00025	0
Ct mean value	22.528	26.225	29.770	32.351	33.799

A specificity curve was prepared by taking Ct mean as Y-Axis and Log (human DNA concentration ratio) as X-Axis, as shown in FIG. 7. The standard curve R2 is 0.9937, the amplification efficiency is 100.859%, and the experiment is qualified.

The results of the serial dilutions of human MSC mitochondrial DNA with mouse lung tissue DNA as proprietary test samples are shown in table 17, corresponding to fig. 8.

TABLE 17 Ct values corresponding to DNA concentration gradients in human MSC cells

Serial number of test sample	1	2	3	4	5
						DNA concentration (ng/. Mu.l)	0.25	0.025	0.0025	0.00025	0
Ct mean value	22.693	26.407	30.070	32.864	0

3. The qPCR main reaction system was designed with human and murine blood DNA universal primers, see Table 18.

The total amount of the special test sample (lung) needs 30 parts of the system, the negative control system needs 3 parts, the split charging has loss, and the total coefficient is 33 when the number of the special test sample (lung) is 3 parts.

TABLE 18 qPCR reaction System under human & murine blood DNA Universal primers

In Table eleven, human & murine blood DNA universal primers (human and non-human animal cell mitochondrial DNA universal primers), forward primer F: SEQ ID NO.17, reverse primer R: SEQ ID NO.18.

And (3) after qPCR operation is finished, deriving data, and preparing a specificity curve by taking Ct average value as Y-Axis and Log (human DNA concentration ratio) as XAxis. Meanwhile, calculating a Ct mean value RSD value and a heart Ct mean value of the specific test sample (heart) and (lung). The mean cardiac Ct values are shown in Table 19.

TABLE 19 results of tests for specificity of mitochondrial DNA of human & murine origin

The average value RSD of the whole Ct of the human and mouse blood DNA specificity test is 0.383, meets the regulations and is qualified.

The cardiopulmonary Ct mean is shown in Table 20.

TABLE 20 results of specificity test of mitochondrial DNA of human & murine origin

The average value RSD of the whole Ct of the human and mouse blood DNA specificity test is 0.704%, meets the regulations and is qualified.

Example 4

Using the same qPCR reaction system as in example 3, after adding a known concentration of cynomolgus monkey DNA to a sample of myocardial cell DNA derived from human iPSC, the detection was carried out by the procedure of example 3, and the results were not affected.

Sequence listing

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Claims

1. A quantitative detection method of human cells in pharmacokinetic studies, characterized in that the quantitative detection of human cells in an experimental animal, in an experimental animal tissue or in an animal cell culture is performed by qPCR, depending on primers specific for mitochondrial DNA in human cells; the primer with specificity to the mitochondrial DNA in the human cell comprises a forward primer F which is SEQ ID NO.7 and a reverse primer R which is SEQ ID NO.8; or the forward primer F is SEQ ID NO.9, and the reverse primer R is SEQ ID NO.10; or the forward primer F is SEQ ID NO.11, and the reverse primer R is SEQ ID NO.12.

2. The method of quantitative determination of human cells in pharmacokinetic studies according to claim 1, wherein the human cells comprise induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells or cells derived from induced pluripotent stem cells.

3. The method for quantitatively detecting human cells in pharmacokinetic studies according to claim 1, comprising the steps of:

4. The method for quantitative detection of human cells in pharmacokinetic studies according to claim 3, wherein the second step comprises:

5. The method for quantitative determination of human cells in pharmacokinetic studies according to claim 4, wherein the standard curve qualification conditions for the primers specific for mitochondrial DNA in human cells are: r is R ² Not less than 0.99, amplificationThe efficiency is between 90% and 110%.

6. The method for quantitative detection of human cells in pharmacokinetic studies according to claim 3, wherein the second step comprises:

repeatability detection: the method comprises the steps of taking a mixture of human cell DNA and non-human animal cell DNA as a test sample, wherein the concentration of the non-human animal cell DNA in the test sample is any one of the gradient concentrations, and the total DNA concentration in the test sample is the other one of the gradient concentrations; qPCR (quantitative polymerase chain reaction) amplification is carried out by using a human and non-human animal cell mitochondrial DNA universal primer, and the consistency of a Ct value and a standard curve of the human and non-human animal cell mitochondrial DNA universal primer is detected;

the human and non-human animal cell mitochondrial DNA universal primer comprises: forward primer F: SEQ ID NO.17, reverse primer R: SEQ ID NO.18; or forward primer F: SEQ ID NO.19, reverse primer R: SEQ ID NO.20; or forward primer F: SEQ ID NO.21, reverse primer: SEQ ID NO.22.

7. The method for quantitatively detecting human cells in pharmacokinetic studies according to claim 6, wherein the standard curve qualification conditions of human and non-human animal cell mitochondrial DNA are: r is R ² Not lower than 0.99, and the amplification efficiency is between 90 and 110 percent.

8. The method for quantitative detection of human cells in pharmacokinetic studies according to claim 3, wherein step two comprises performing a specificity test;

the specificity test comprises the following steps:

preparing a human cell DNA sample: simultaneously obtaining non-human animal cell DNA in experimental animal body tissues, and carrying out gradient dilution on the human cell DNA by using the non-human animal cell DNA to obtain a specific supply; the concentration of the human cell DNA after the gradient dilution is in the range of the gradient concentration of a standard curve with specific primers for the mitochondrial DNA in the human cell;

qPCR (quantitative polymerase chain reaction) amplification is carried out on a plurality of groups of specific supplies by adopting universal primers of mitochondrial DNA of human and non-human animal cells, and a specific curve which takes Ct average value as Y axis and takes the DNA concentration of human cells as X axis is prepared; after the computed Ct mean value RSD value reaches the standard, comparing the consistency of the specificity curve with the standard curve of the human and non-human animal cell mitochondrial DNA universal primer;