A method and detection product for detecting the distribution of human cells in animals

Abstract

The present invention discloses a method and a detection product for detecting the distribution of human cells in animals. With the human-specific gene LTR6 as the target, detection primers are designed, and the specific gene sequence of human mesenchymal stem cells in the DNA of tissue and organ samples can be detected by the method of fluorescent quantitative PCR to obtain the distribution of human mesenchymal stem cells in animals, with a minimum detection limit of 5.12 copies / μL. The detection method disclosed in the present invention is simple and rapid, has good specificity, high sensitivity, and a wide detection range. It can be used to trace the directional distribution of human cells implanted in animals, and can process a large number of samples in a short time, providing data support for the selection of stem cell products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, and specifically relates to a method and a detection product for detecting the distribution of human cells in an animal body. Background Art

[0002] Stem cell therapy is an advanced medical treatment technology. Stem cell therapy is to inject healthy stem cells into the patient's body to repair damaged cells or tissues, thereby achieving the purpose of curing the disease. Its treatment range is wide, including nervous system diseases, such as spinal cord injury, motor neuron disease, sequelae of cerebral infarction, sequelae of brain trauma, etc., immune system diseases, such as diabetes, dermatomyositis, myasthenia, vascular disease, and other internal and external diseases, such as cirrhosis, femoral head necrosis, arthritis, male erectile dysfunction caused by diabetes, etc. For example, mesenchymal stem cell products are a "living drug". After administration into the body, great attention should be paid to their retention time and tissue distribution. Studies have shown that different routes of administration will lead to different retention time and distribution of mesenchymal stem cells in the body. Most mesenchymal stem cells can be quickly retained in the lungs after intravenous injection, and only a part of them will home to other tissues, such as the liver, spleen, and inflammation or injury sites. The vast majority of mesenchymal stem cells administered by local injection reside at the site of administration, and are rarely distributed in the blood and other tissues. Therefore, it is necessary to monitor the in vivo distribution and action time of mesenchymal stem cells administered by different routes of administration. Animals are often used as models to detect the distribution of stem cells in the body and understand their distribution patterns in the body. Monkeys and mice are the most commonly used animal models. Therefore, it is necessary to establish a method to detect the distribution of human cells in animals such as monkeys or mice.

[0003] In the prior art, flow cytometry is often used to detect the distribution of cells in the body. By identifying the specific proteins carried by the cells, the cells are counted by flow cytometry to determine the number of cells in the body. However, flow cytometry detection generally uses blood, and it is time-consuming and laborious to process tissue samples, and there is a problem of missed detection. Another commonly used method is to extract the total RNA of the sample, determine the mRNA sequence of a specific protein gene of the cell, reverse transcribe the mRNA sequence into cDNA, and then measure it by real-time fluorescence quantitative PCR method, and calculate the cell content by Ct value. However, in order to accurately analyze the gene expression amount, this method must meet the prerequisite that only cDNA can be detected as a template. It is necessary to extract RNA and then reverse transcribe it into cDNA. In addition, the extracted total RNA is often mixed with genomic gDNA, and can be directly amplified as a template for PCR reaction, so it will cause inaccurate results.

[0004] In addition, there are also reports of using human-specific gene fragments as targets and genomic DNA of the sample to be tested as a template for detection. In CN116355995A, LIME1 gene is used as the target gene for detection, and in CN114438176A, GAPDH gene is used as the target gene for detection. Detection primers are designed to directly extract genomic DNA of the sample to be tested, and the distribution of human genomic DNA in the sample in the animal inoculated with the cell product is detected by fluorescent quantitative PCR. The biggest difficulty of this type of technology is that since humans and monkeys, mice, especially monkeys, have a high degree of similarity in genome sequence and a high degree of similarity in genome background, it is difficult to achieve the purpose of detection specifically and sensitively, including the selection of detection targets, and the design of amplification primers and systems, which are crucial influencing factors.

[0005] Therefore, the development of efficient, simple and accurate qPCR methods for detecting the distribution of human cell products in animals focuses on exploring more detection targets and designing detection systems. Summary of the invention

[0006] The present invention aims to provide detection targets that can specifically and sensitively detect the distribution of human cells in animals (especially monkeys and mice), and to develop detection methods and detection products based on the targets.

[0007] The first object of the present invention is to provide the use of the LTR6 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.11 as a target for detecting the tissue distribution of human cells in animals.

[0008] The second object of the present invention is to provide a primer set for detecting the tissue distribution of human cells in animals.

[0009] The third object of the present invention is to provide a kit for detecting the tissue distribution of human cells in animals.

[0010] The fourth object of the present invention is to provide a method for detecting the distribution of human cells in animal tissues.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] After extensive exploration, the present invention has obtained a detection target gene that can specifically and sensitively detect human cells in animal (especially monkey and mouse) tissues, namely, a human-specific gene LTR6 specific fragment (sequence as shown in SEQ ID NO.1), using this as a target, and preferably using the fragment shown in SEQ ID NO.11 as an amplification target, designing detection primers, and using the fluorescent quantitative PCR method to detect human mesenchymal stem cells in the DNA of animal organ tissue samples to obtain the distribution of human mesenchymal stem cells in the animal body. The detection specificity is good and the sensitivity is high. Therefore, the present invention requests protection for:

[0013] Application of the LTR6 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.11 in detecting the tissue distribution of human cells in animals, or in preparing a product for detecting the tissue distribution of human cells in animals.

[0014] Application of the LTR6 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.11 as a target for detecting the tissue distribution of human cells in animals.

[0015] Use of a reagent for detecting the LTR6 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.11 in detecting the tissue distribution of human cells in animals, or in preparing a product for detecting the tissue distribution of human cells in animals.

[0016] Based on this, the present invention also provides a primer set for detecting the tissue distribution of human cells in animals, including a primer pair and a probe capable of specifically amplifying the LTR6 gene fragment shown in SEQ ID NO.1 or SEQ ID NO.11.

[0017] As a preferred embodiment, the primer set includes a primer pair as shown in SEQ ID NO.8 and SEQ ID NO.9, and a probe as shown in SEQ ID NO.10.

[0018] The use of the primer set in detecting the tissue distribution of human cells in animals, or in preparing products for detecting the tissue distribution of human cells in animals, and a kit for detecting the tissue distribution of human cells in animals containing the primer set should also be within the scope of protection of the present invention.

[0019] The present invention also provides a method for detecting the distribution of human cells in animal tissues, using the DNA of the animal tissue to be tested as a template, performing fluorescent quantitative PCR detection using the kit described in claim 7, and judging whether there are human cells in the animal tissue to be tested based on the PCR results.

[0020] If the PCR result is positive, there are no human cells in the animal tissue to be tested, otherwise there are no human cells in the animal tissue to be tested.

[0021] Preferably, the reaction system of the fluorescent quantitative PCR is:

[0022]

[0023] Preferably, the reaction procedure of the fluorescent quantitative PCR is:

[0024]

[0025]

[0026] Preferably, the animal is a monkey or a mouse.

[0027] More preferably, the animal is a rat or a cynomolgus monkey.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses gene LTR6 as a human-specific target, especially the fragment shown in SEQ ID NO.11 as an amplification target, and designs detection primers. It can detect whether there is a specific gene sequence of human mesenchymal stem cells in the DNA of organ tissue samples by fluorescent quantitative PCR to obtain the tissue distribution of human mesenchymal stem cells in animals, and the minimum detection limit is 5.12 copies / μL. The detection method disclosed in the present invention is simple and rapid, has good specificity, high sensitivity, and a wide detection range. It can be used to trace the directional distribution of human cells implanted in animals, and can process a large number of samples in a short time, so as to provide data support for the selection of cell products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : amplification curves of primer set 1 and primer set 2 in the lungs of nude rats and the peripheral blood of cynomolgus monkeys after injection of human umbilical cord mesenchymal stem cells.

[0031] Figure 2 The amplification curves of primer set 3 in the lungs of nude rats and the peripheral blood of cynomolgus monkeys after injection of human umbilical cord mesenchymal stem cells.

[0032] Figure 3 The standard curve (in copies / μL) of human mesenchymal stem cell concentration was determined by fluorescence quantitative PCR.

[0033] Figure 4 The figure is an amplification curve (in Copies / μL) showing the relationship between fluorescence intensity and cycle number in the fluorescence quantitative PCR assay. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0036] The human umbilical cord mesenchymal stem cells used in the following examples were provided by Guangzhou Saijun Biotechnology Co., Ltd.

[0037] The sources of experimental animals in the following examples are:

[0038] Nude rats: Nude rats, specific pathogen-free grade (SPF grade), weight 220 g to 250 g, age 7 to 9 weeks, male. Purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., Laboratory Animal Quality Certificate No.: 20231225Aazz0619000660, Laboratory Animal Production License No.: SCXK (Zhejiang) 2019-0001.

[0039] Cynomolgus monkeys: purchased from Xishan Pharmaceutical Research and Development Co., Ltd., experimental animal use license number: SYXK (Guangdong) 2022-0279), experimental animal quality certificate number: B202402270003.

[0040] Example 1 Human mesenchymal stem cell injection into rats and tissue sampling method

[0041] 1. Experimental Animals

[0042] The feeding conditions of the experimental animals, nude rats, were as follows: in IVC cages, the temperature was 20-26℃, the humidity was 40-70%, the ventilation rate was 15 times / h, the light-dark cycle was 12h / 12h, and the rats were free to eat and drink. After one week of adaptive feeding, the experiment was carried out.

[0043] The feeding conditions of the experimental animals, cynomolgus monkeys, were as follows: normal environment, single cage, national standard temperature 16-26°C, relative humidity 40-70%, ventilation 8 times / h, day and night light-dark alternation time 12h / 12h, animal illumination 100-200 / lux, free food and water. After one month of adaptive feeding, the experiment was carried out.

[0044] 2. Cell injection and sampling

[0045] Nude rats: Human umbilical cord mesenchymal stem cells (5×10^6 Cell / mL) were injected into nude rats via corpus cavernosum injection, with an injection volume of 0.1 mL / rat. The nude rats were then dissected 2 hours after administration and various organ tissues (heart, liver, spleen, lung, kidney, brain, spinal cord, testis, penis, epididymis, prostate, adrenal gland, inguinal lymph node) were taken, a total of 13 samples, for testing and use.

[0046] Cynomolgus monkeys: Human umbilical cord mesenchymal stem cells (5×10^7 Cell / mL) were obtained and injected into cynomolgus monkeys by intravenous injection, with an injection volume of 0.5 mL / monkey. Peripheral blood was then collected through the veins of the limbs 1 hour after administration, and DNA was extracted for testing.

[0047] 3. Sample DNA Extraction

[0048] According to the instructions of the magnetic bead blood / cell / tissue DNA extraction kit (DP710-T8, Tiangen Biochemical Technology (Beijing) Co., Ltd.), the samples obtained in step 2 above were subjected to DNA extraction using the fully automatic nucleic acid extraction and purification instrument TGuide S96Dex (YOSE-S616, Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0049] Example 2 Primer screening for detecting human mesenchymal stem cells

[0050] 1. Specific target selection and primer design

[0051] After investigation, comparison and analysis, we selected the sequence AC003109.1 in the LTR6 gene (as shown in SEQ ID NO: 1) as a specific gene to detect the distribution of human cells in monkeys and rats.

[0052] SEQ ID NO: 1 (sequence numbered AC003109.1 in the LTR6 gene)

[0053] GAATGATGTCTTTTGTTTCTAAAGTCGATATACTAGAGCGATGTGAAAATAA

[0054] TCATAAAAGCGAGATATTTCGTGGCAAAGTTATCTCGGGGTAAACGCTGCA

[0055] GCTGTAAGCACTGCCGGTGAGTATTCTCAGGACAAATGGGAAAAAGGGTT

[0056] AAAGAACCCCTAAATAAATGGAGAGAAGTAGCATGTTTGTGATTGAGAGAC

[0057] TCAGTGTCATTAATATAAAGATGTCAGTTTTGCGACTGAGCAATTGATCCAA

[0058] TGCAATTACTGTCAAAATCCCAACAAAACTATTTTTGTTTTGCTTCATTTTG

[0059] GAACTGATTCTTAAATTTAGTACAGCGAGGAGCCAGGAATCCTGGAGCCCC

[0060] TTGTATCCGCTCAGGTCCGCCAGGAGGCAGCTGCAAAAATGATGTCAGGA

[0061] GCATAAGAGGTGAAAGACAAGAGGGGGCAGAATGAGGCAGGGAAAACCT

[0062] TAGGGCCTGACACCGGTGGAAGGAAGGAGGCAGGAAGAATTGGGAGGAA

[0063] GAACTTCAGACTGACAGCACAGCTCAAAGAAAGTCTTGGCCTGTCCA CCA

[0064] GGGAACTCCTATGCAAAGATTGTTCTGGAGGACACCCTCACTGGGCAGAAG

[0065] TATTCATGAGGAGCACATGACCTCAGCTCATATACTGTGGCAGAGCAAAAG

[0066] GCACTTTAGCTGGACGCTGTCAGATAACTCACT TTCAAAGGGAGATCCGAG

[0067] CTAAGTGTTCTTTCTTCTCTCTCTCTCTCTCTCTTTCTTTCTTCCAGAGGCTT

[0068] GCTTCTTCGCCCAGGCTGGAGTGCAATGGCACGATCTCAGCTCACTGCAAC

[0069] CTCTGCCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCT

[0070] GCGATTACAGACGCATGCCAGCACGCCCGGCTAATTTTTGTATTTTTACTAG

[0071] AGACGGGGTTTCACCATGTTAGCCAGGATGGTCTTGAACTCCTGACCACAA

[0072] GTGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCC

[0073] ATTGCACCCAGCCTGGCTGCTAAGTGTTCTTAAAGGAGGCTGATGTAGGGG

[0074] AACTTACCCTATTCGATGTAAGGATATGTAGTTAAGGCCAGGCATGGTGGCT

[0075] CAGGCCTGTAATCCCAGCACTTTGGGATGCCAAGGTGGGCGGATCACGAG

[0076] GTCAAGAGATCAAGACCATTCTGGCCAACATAGTGAAACCCCCGTCTCTAC

[0077] TAAAAATACAAAAATTAGCTGGGCATGGTGGCGCACGCCTGTAGTCCCAGC

[0078] TACTACGGAGGCTGAGGCAGGAGAATTGCTTGAACCCGGGAGGCGGAAGT

[0079] TGCACTAAGCCGAGATCACACCACTGCACTCCAGCCTGGGCGACAGAGCA

[0080] AGACTCCGTCTCAAAAAAAAAAAAAAATTGTCGCTAAAGTGAGAGTATGC

[0081] TATTGGTAAAAGGATACACAAGTAGAACAGAATATAGAGCTTCAGAACAAA

[0082] CCCACACGTATGGACTTTAGCATATTTCAGAAGCAGCAAATCATTGTGGAA

[0083] AGGATGGACTATTCAAATCC

[0084] Using the above LTR6 gene fragment as a target, primers and probes as shown in Table 1 were designed for screening to determine the best primer set that can specifically detect human cells in animals.

[0085] Table 1. Primer names and sequences

[0086]

[0087] 2. Fluorescence quantitative PCR amplification

[0088] (1) PCR amplification system is shown in Table 2. 8 μL of the following PCR system and 2 μL of sample DNA were added to each reaction well.

[0089] Table 2. PCR reaction system

[0090]

[0091]

[0092] (2) The PCR amplification procedure is shown in Table 3 below.

[0093] Table 3. PCR reaction program

[0094]

[0095] (3) According to the above PCR amplification system and procedure, the lungs of nude rats and the peripheral blood genomes of cynomolgus monkeys injected with human umbilical cord mesenchymal stem cells in Example 1 were used as templates, and the amplification results of the three primer sets were compared to find that:

[0096] like Figure 1 As shown, primer set 1 (LTR6-Forward Primer 1, LTR6-Reverse Primer 1, LTR6-Probe 1) and primer set 2 (LTR6-Forward Primer 2, LTR6-Reverse Primer 2, LTR6-Probe 2) had low amplification efficiency in nude rats and cynomolgus monkeys.

[0097] like Figure 2 As shown, primer set 3 (LTR6-Forward Primer 3, LTR6-Reverse Primer 3, LTR6-Probe 3) has a high amplification efficiency in nude rats and cynomolgus monkeys, and the amplified sequence is shown in SEQ ID NO.11, so this sequence is a key specific detection target.

[0098] SEQ ID NO.11 (Amplified sequence of LTR6-Primer 3)

[0099] CCAGGGAACTCCTATGCAAAGATTGTCTGGAGGACACCCTCACTGGGCAGAAGTATTCATGAGGAGCACATGACCTCAGCTCATATACTGTGGCAGAGCAAAAGGCACTTTAGCTGGACGCTGTCAGATAACTCACT

[0100] Example 3 Specificity detection of primers

[0101] The preferred primer set 3 (LTR6-Forward Primer 3, LTR6-Reverse Primer 3, LTR6-Probe 3) of Example 2 was specifically detected.

[0102] Using the same DNA extraction method in Example 1, DNA was extracted from liver tissues of 6 blank nude rats and 6 blank cynomolgus monkeys and human mesenchymal stem cell samples, and fluorescent quantitative PCR detection was performed using the PCR system and amplification program shown in Table 2 and Table 3 in Example 1. It was found that the preferred primer set 3 (as shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively) was amplified in human mesenchymal stem cell samples, but not in blank rat and blank cynomolgus monkey liver tissues. The results show that the preferred primer set 3 in Example 2 (as shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively) has good specificity in distinguishing human cells from animal cells (mouse and monkey), and meets the specificity requirements of the detection.

[0103] Example 4 Standard curve drawing and sample detection

[0104] 1. Standard curve drawing (using Copies / μL as the unit to make a standard curve)

[0105] Take 5 mg / tube of liver tissue of blank nude rats that have not been inoculated with human mesenchymal stem cells, for a total of 13 tubes, add a certain volume of lysis buffer to each tube for grinding, and obtain 450 μL of homogenate in each tube after grinding. Mix the 13 tubes of homogenate in a 15 mL centrifuge tube for later use.

[0106] Prepare human mesenchymal stem cell stock solution (concentration of 1.0×10^8 cells / mL) and use the blank nude rat liver tissue homogenate as a diluent to dilute the human mesenchymal stem cell stock solution to prepare the following concentrations of cell suspension (S1-S7): 2.0×10^7, 4.0×10^6, 8.0×10^5, 1.6×10^5, 3.2×10^4, 6.4×10^3, 1.28×10^3 cells / mL, take 400 μL of cell suspension at each concentration level, extract genomic DNA, elute with 100 μL enzyme-free sterile water during extraction, prepare S1~S7 standards, and add 2 μL of genomic DNA sample to each reaction for fluorescence quantitative PCR detection, that is, the theoretical concentrations of S1~S7 are 80000, 16000, 3200, 640, 128, 25.6, and 5.12 copies / μL, see Table 5 below.

[0107] Table 5. Standard curve range in Copies / μL

[0108]

[0109]

[0110] The Log value of the DNA content of the standard was used as the horizontal axis, and the Ct value was used as the vertical axis. A linear coordinate curve was fitted. The standard curve (in Copies / μL) for determining the concentration of human mesenchymal stem cells by fluorescence quantitative PCR was as follows: Figure 3 As shown, y = -3.1992x + 36.402, R 2 =0.9995, amplification efficiency E = 1.05. The amplification curve of the relationship between fluorescence intensity and cycle number of the fluorescence quantitative PCR assay (in copies / μL) is as follows Figure 4 As shown, the amplification curve showed a normal S-shape.

[0111] 2. Sample testing

[0112] Samples: According to the method of Example 1, 13 organ tissues including heart, liver, spleen, lung, kidney, brain, prostate, adrenal gland, penis, spinal cord, testis, epididymis, and inguinal lymph node were obtained from nude rats 2 hours after injection of human mesenchymal stem cells into the corpus cavernosum of the penis; peripheral blood of cynomolgus monkeys was obtained 1 hour after intravenous injection of human mesenchymal stem cells.

[0113] The unknown concentration sample was homogenized and DNA was extracted using the same method as the standard curve sample. The Ct value of the unknown concentration sample was obtained by the fluorescent quantitative PCR determination method of Example 2, and the Ct value was then substituted into the standard curve to obtain the concentration of the unknown sample.

[0114] 3. Test results

[0115] 2 hours after the nude rats were injected with human mesenchymal stem cells into the corpus cavernosum of the penis, 13 organs and tissues were detected, including the heart, liver, spleen, lung, kidney, brain, prostate, adrenal gland, penis, spinal cord, testis, epididymis, and inguinal lymph nodes. The results are shown in Table 6 below. Human mesenchymal stem cells were detected in the penis (the site where human mesenchymal stem cells were injected) and lung, and no other organs and tissues were detected. This result is consistent with the detection result of the method disclosed in Chinese patent CN114196764B, indicating that this method can be used to analyze the distribution and content of human cells in rat tissues.

[0116] Table 6. Distribution of human mesenchymal stem cells detected in 13 organs of nude rats

[0117]

[0118]

[0119] One hour after intravenous injection of human mesenchymal stem cells into cynomolgus monkeys, human mesenchymal stem cell DNA was detected in the peripheral blood. The results are shown in Table 7 below. Human mesenchymal stem cells were detected in the peripheral blood. This result is consistent with the detection result using the method disclosed in Chinese patent CN114196764B, indicating that this method can be used to analyze the distribution and content of human cells in the peripheral blood of cynomolgus monkeys.

[0120] Table 7. Distribution of human mesenchymal stem cells detected in peripheral blood of cynomolgus monkeys

[0121]

[0122] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of the LTR6 gene sequence shown in SEQ ID NO.11 in detecting the tissue distribution of human cells in animals, or in preparing products for detecting the tissue distribution of human cells in animals.

2. Application of the LTR6 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.11 as a target for detecting the tissue distribution of human cells in animals.

3. Use of a reagent for detecting the LTR6 gene sequence shown in SEQ ID NO.11 in detecting the tissue distribution of human cells in animals, or in preparing a product for detecting the tissue distribution of human cells in animals.

4. A primer composition for detecting the tissue distribution of human cells in an animal body, characterized in that: It comprises a primer pair and a probe capable of specifically amplifying the LTR6 gene fragment shown in SEQ ID NO.

11.

5. The primer composition according to claim 4, characterized in that It includes a primer pair as shown in SEQ ID NO.8 and SEQ ID NO.9, and a probe as shown in SEQ ID NO.

10.

6. Use of the primer composition according to any one of claims 4 or 5 in detecting the tissue distribution of human cells in animals, or in preparing a product for detecting the tissue distribution of human cells in animals.

7. A kit for detecting the tissue distribution of human cells in an animal, characterized in that: A primer composition comprising the primer composition according to any one of claims 4 or 5.

8. A method for detecting the distribution of human cells in animal tissues, characterized in that: Using the DNA of the animal tissue to be tested as a template, the kit described in claim 7 is used to perform fluorescent quantitative PCR detection, and whether there are human cells in the animal tissue to be tested is determined based on the PCR results.

9. The method according to claim 8, characterized in that The reaction system of the fluorescent quantitative PCR is: The reaction system of the fluorescent quantitative PCR is: The reaction procedure of the fluorescent quantitative PCR is: 。 10. The method according to claim 8, characterized in that The animal is a monkey or a mouse.

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