Fluorescent quantitative PCR (polymerase chain reaction) detection method and detection kit for detecting differentiation potential of induced pluripotent stem cells

Abstract

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) detection method and a detection kit for detecting differentiation potential of induced pluripotent stem cells. The detection method comprises the following steps: S1, preparing an embryoid body by utilizing iPSC (induced pluripotent stem cells); s2, extracting RNA (Ribonucleic Acid) of the iPSC and the embryoid body; s3, respectively carrying out reverse transcription on the iPSC and RNA of an embryoid body to obtain cDNA; s4, establishing a fluorescent quantitative PCR reaction system by adopting the primer composition in claim 1; s5, carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) detection on the cDNA in S3; and S6, analyzing a result. The detection kit disclosed by the invention can be used for rapidly and accurately evaluating the differentiation potential of the induced pluripotent stem cells, and is suitable for large-scale cell differentiation research and clinical application.

Description

Technical Field

[0001] The present invention belongs to the field of cell and molecular biological detection, and in particular relates to a fluorescent quantitative PCR detection method and a detection kit for detecting the differentiation potential of induced pluripotent stem cells (iPSCs). Background Art

[0002] Induced pluripotent stem cells (iPSCs) are pluripotent cells that are reprogrammed by introducing specific transcription factors into terminally differentiated somatic cells. This technology was first realized by Professor Shinya Yamanaka of Kyoto University in Japan in 2006. By transferring four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into adult mouse cells through viral vectors, iPSC cells with differentiation potential were finally obtained.

[0003] iPS cells have similar properties to embryonic stem cells, and their differentiation potential is one of their most significant properties. They can be used to differentiate into various cell types including hematopoietic cells, pancreatic islet cells, cardiomyocytes, osteoblasts, adipocytes, neurons, etc., and are widely used in regenerative medicine, disease modeling, drug screening and other fields.

[0004] As pluripotent stem cells, iPS cells are able to self-renew, maintain an undifferentiated state and have multidirectional differentiation potential. Their stemness and multidirectional differentiation potential are the basis for evaluating the biological effectiveness of this type of cells, and are also the criteria for judging the biological safety of differentiated cells.

[0005] The pluripotency and differentiation potential of iPS cells can be characterized by a variety of indicators, including morphology, marker molecule expression, three-germ layer differentiation, telomerase activity, teratoma formation, etc. Among them, the embryoid body formation experiment is a common method for judging the pluripotency of iPS cells in vitro. Through suspension culture, cells can be induced to spontaneously form spherical cell clusters. By detecting the expression of representative genes of the three germ layers, endoderm, mesoderm, and ectoderm, the multidirectional differentiation potential of iPS cells can be further studied and verified. Summary of the invention

[0006] The purpose of the present invention is to provide a fluorescent quantitative PCR detection method and a detection kit for detecting the differentiation potential of induced pluripotent stem cells (iPSCs). The detection kit of the present invention can quickly and accurately evaluate the differentiation potential of induced pluripotent stem cells, which is suitable for large-scale cell differentiation research and clinical application.

[0007] In order to achieve the above purpose, this application adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a detection kit for detecting the differentiation potential of induced pluripotent stem cells (iPSCs), the detection kit comprising a primer composition for detecting the differentiation potential of iPSCs;

[0009] Inducing iPSCs to form embryoid bodies, wherein the embryoid bodies have three germ layers, namely, ectoderm, mesoderm and endoderm,

[0010] Ectodermal marker genes include CDH9, COL2A1, DLK1, DMBX1, DRD4, EN1, FABP7, HES5, LHX2, LMX1A, MAP2, MYO3B, NCAM1, NES, NOS2, NR2F1, NR2F2, OLFM3, OTX2, PAMR1, PAPLN, PAX3, PAX6, POU4F1, PRKCA, RAX, SDC2, SOX1, TFAP2A, TRPM8, WNT1, ZBTB16,

[0011] Mesoderm marker genes include ABCA4, ALOX15, APLNR, BMP10, CDH5, CDX2, COLEC10, CXCR4, DES, ESM1, FCN3, FOXF1, HAND1, HAND2, HEY1, HOPX, HOXB7, IL6ST, ISL1, MEOX1, MIXL1, NCAM1, NKX2-5, ODAM, PDGFRA, PLVAP, RGS4, SNAI2, T, TBX3, TBX6, TM4SF1,

[0012] Endoderm marker genes include AFP, CABP7, CDH20, CER1, CLDN1, CPLX2, CXCR4, ELAVL3, EOMES, FOXA1, FOXA2, FOXP2, GATA4, GATA6, GSC, HHEX, HMP19, HNF1B, HNF4A, KIT, KLF5, LEFTY1, LEFTY2, NODAL, PHOX2B, POU3F3, PRDM1, RXRG, SOX17, SOX7, SST,

[0013] 2-3 marker genes are selected for each germ layer, and GAPDH is selected as an internal reference gene. The primer composition includes primers for the marker genes selected for each germ layer and the internal reference gene GAPDH.

[0014] In the above technical scheme, two marker genes were selected for each germ layer, NCAM1 and PAX6 for the ectoderm, CDX2 and MEOX1 for the mesoderm, AFP and SOX17 for the endoderm, and GAPDH was selected as the internal reference gene;

[0015] The primer composition comprises

[0016] Primers for the internal reference gene GAPDH: the upstream primer is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 2;

[0017] Primers for the ectoderm target gene NCAM1: the upstream primer is shown as SEQ ID NO: 2, and the downstream primer is shown as SEQ ID NO: 4;

[0018] Primers for the ectoderm target gene PAX6: the upstream primer is shown as SEQ ID NO: 5, and the downstream primer is shown as SEQ ID NO: 6;

[0019] Primers for the mesoderm target gene CDX2: the upstream primer is shown in SEQ ID NO: 7, and the downstream primer is shown in SEQ ID NO: 8;

[0020] Primers for the mesoderm target gene MEOX1: the upstream primer is shown in SEQ ID NO: 9, and the downstream primer is shown in SEQ ID NO: 10;

[0021] Primers for endoderm target gene AFP: the upstream primer is shown as SEQ ID NO: 11, and the downstream primer is shown as SEQ ID NO: 12;

[0022] Primers for the endoderm target gene SOX17: the upstream primer is shown as SEQ ID NO: 13, and the downstream primer is shown as SEQ ID NO: 14.

[0023] In the above technical solution, the detection kit is mainly composed of reagent A and reagent B;

[0024] Reagent A is a fluorescent quantitative PCR reaction premix, comprising a highly specific qPCR reagent TB Green Premix ExTaq II, ROX Reference Dye internal reference dye, nuclease-free water, and the primer composition according to claim 1;

[0025] Reagent B is a control, containing iPSC cDNA and embryoid body cDNA.

[0026] In a second aspect, the present invention provides a fluorescent quantitative PCR detection method for detecting the differentiation potential of induced pluripotent stem cells, comprising the following steps:

[0027] S1. Preparation of embryoid bodies using iPSCs;

[0028] S2, extracting RNA from iPSCs and embryoid bodies;

[0029] S3, reverse transcribe the RNA of iPSC and embryoid body into cDNA respectively;

[0030] S4. Establishing a fluorescent quantitative PCR reaction system using the primer combination described in claim 1;

[0031] S5, performing fluorescence quantitative PCR detection on the cDNA described in S3;

[0032] S6. Result analysis.

[0033] In the above technical scheme, in S4, the fluorescent quantitative PCR reaction system is 20 μL, including 18 μL of reagent A and 2 μL of reagent B; the reagent A is a fluorescent quantitative PCR reaction premix, including highly specific qPCR reagent TB Green Premix Ex Taq II, ROX Reference Dye internal reference dye, nuclease-free water, and the primer composition described in claim 1; the reagent B is a reference substance, including iPSC cDNA and embryoid body cDNA.

[0034] In the above technical solution, in S4, the reaction conditions of the fluorescent quantitative PCR reaction system are: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 5 s, annealing at 60°C for 34 s, and 40 cycles.

[0035] In the above technical scheme, in S6, iPSC undifferentiated cells are used as a control, and the relative expression levels of genes in each germ layer are calculated through internal reference genes, and the difference multiples are calculated to determine the differentiation potential of iPSC.

[0036] In the above technical solution, the relative expression amount is calculated by the formula:

[0037] If the marker gene expression of the control group is set to 1, the relative expression of the marker gene in the experimental group, i.e. the difference multiple, is 2. -ΔΔCt =2 -[(实验组标志基因Ct-实验组内参基因Ct)-(对照组标志基因Ct-对组照内参基因Ct)] ;

[0038] If the difference in the relative expression of the three germ layer marker genes was ≥2, the iPSCs in the experimental group were considered to have the potential for three germ layer differentiation.

[0039] In the above technical solution, in S1, the method for preparing embryoid bodies using iPSCs comprises the following steps:

[0040] S1. Use digestion solution to digest iPSC into single cells or small cell clusters;

[0041] S2. After centrifugation, resuspend the cells in induction medium and adjust the cell volume to 1×10 6 / mL and inoculated into 6cm non-tissue culture treated culture dishes;

[0042] S3: On the third day, use a polypropylene centrifuge tube to collect the embryoid bodies formed by sedimentation and transfer them to a new culture dish;

[0043] S4. Based on the growth of the embryoid bodies, the cell clusters are transferred to a tissue culture treated cell dish on the 5th to 7th day, and culture is continued for 7-14 days to obtain the embryoid bodies.

[0044] In the above technical solution, the differentiation induction medium includes DMEM / F12 medium, KnockOut serum replacement and 2-mercaptoethanol.

[0045] The beneficial effects of the present invention are:

[0046] 1. At present, the main technologies for detecting the stemness and differentiation potential of iPSCs include the embryoid body method, three-germ layer directed differentiation and teratoma formation experiment. The method of the present invention is simple to operate and convenient to culture. Compared with the single germ layer directed differentiation method, the method of the present invention can induce the multidirectional differentiation of embryoid bodies through a single culture medium, and comprehensively examine the differentiation potential of iPSCs. Compared with the teratoma experiment in animals, the experimental cycle is short, and combined with the changes in relative gene expression, it can accurately and quickly reflect the cell differentiation situation.

[0047] 2. The detection methods for embryoid body differentiation mainly include gene expression analysis, immunofluorescence staining and histological analysis. The quality of antibodies in immunofluorescence staining and the cross-reactions between different antibodies will affect the specificity of staining. At the same time, the judgment of staining results is often subjective. Histological analysis may cause antigen loss during tissue fixation and sectioning, thus affecting the staining effect. The method of the present invention mainly uses qPCR to perform gene expression analysis on the marker genes of the three germ layers, which has higher sensitivity and specificity, and can also accurately and quickly reflect the changes in gene expression during the differentiation process, so as to evaluate the ability of cells to differentiate into the exoderm, mesoderm and endoderm;

[0048] 4. The present invention selects multiple three germ layer related marker genes, which have low expression levels in undifferentiated iPSC cells. By selecting the target gene spanning intron segment sequence, the primer combination with good specificity and high sensitivity is designed and screened, and the genome removal operation is introduced to eliminate the interference of cell genome DNA;

[0049] 5. The detection kit for detecting the differentiation potential of iPSCs of the present invention tests the qPCR detection method. The primers have strong specificity. Through primer screening and method optimization, all target genes can be amplified by qPCR in the same reaction system and conditions, which can meet the requirements of detecting the expression of multiple target genes simultaneously in one program;

[0050] 6. Through repeated screening and optimization of the reaction system, the detection method of the present invention ultimately requires very little sample volume, with a minimum of only 2 μL of nucleic acid sample required for each test and still able to detect stably. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a picture of embryoid bodies on the third day of culture (40×);

[0052] Figure 2 This is a picture of embryoid bodies on the third day of culture (100×);

[0053] Figure 3 This is a picture of embryoid bodies on the third day of culture (200×);

[0054] Figure 4 This is a picture of embryoid bodies on day 14 of culture (40×);

[0055] Figure 5 This is the result of induction of PX+7 generation cells;

[0056] Figure 6 This is the result of induction of PX+12 generation cells;

[0057] Figure 7 Induces results in specificity and durability. DETAILED DESCRIPTION

[0058] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0059] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0060] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial channels).

[0061] Experimental materials used in the examples of the present invention:

[0062] DMEM / F12 medium was purchased from Gibco, catalog number: 11320033;

[0063] KnockOut serum replacement was purchased from Gibco, catalog number: 10828010;

[0064] 2-Mercaptoethanol was purchased from Gibco, catalog number: 31350010;

[0065] RNA extraction kit was purchased from Tiangen, catalog number: DP419;

[0066] RNA reverse transcriptase kit was purchased from TaKaRa, catalog number: RR047Q;

[0067] Highly specific qPCR reagent TB Green Premix Ex Taq II (Tli RNaseH Plus) was purchased from TaKaRa (containing ROX Reference Dye internal reference dye), catalog number: RR820A;

[0068] Nuclease-free water was purchased from Invitrogen, catalog number: 10977-015.

[0069] Example 1 Induction of iPSCs into embryoid bodies

[0070] The induction medium was prepared with the following composition: DMEM / F12+20% KnockOut serum replacement+0.1 mM 2-mercaptoethanol.

[0071] Take iPS cells with good growth status, wash them with DPBS 1-2 times, choose appropriate digestion solution to digest them into single cells or small cell clusters, resuspend the cells in induction medium after centrifugation, test cell viability and cell number, and adjust the cell density to 10 6 The cells were inoculated into 6 cm non-tissue culture treated culture dishes and cultured in a 37°C, 5% CO2 cell culture incubator.

[0072] On the third day, circular or quasi-circular three-dimensional structures with clear boundaries were observed under a microscope. The culture medium with the cell clusters was transferred to a polypropylene centrifuge tube using a pipette and allowed to stand for 5-10 minutes until the embryoid bodies formed by aggregation settled to the bottom of the centrifuge tube. The supernatant and cell debris were removed by aspiration, and the cell clusters were resuspended in fresh culture medium and transferred to a new non-tissue culture treated culture dish for continued culture. During this period, the cell growth was observed every day and the culture medium was replaced every two days.

[0073] Depending on the growth of embryoid bodies, the cell clusters were transferred to tissue culture treated cell dishes on days 5-7 and cultured for another 7-14 days.

[0074] Cell culture Figure 1-4 As shown, Figure 1 This is a photo of the cell cluster formed on the third day of culture at 100 times magnification. Figure 2 This is a photo magnified 200 times. Figure 3 For 200 times magnified photos, Figure 4 This is a photo taken after the cells were transferred to tissue culture treated cell dishes and cultured for another 7 days. It can be seen that the embryoid bodies grow adherently and differentiated cells of different morphologies can be clearly observed around the cell clusters.

[0075] Example 2 Sample RNA extraction and reverse transcription

[0076] Uninduced iPSCs and embryoid bodies prepared in Example 1 were collected, centrifuged and the supernatant was discarded, 1 ml of lysis solution RZ was added, 200 μl of chloroform was added, shaken for 15 seconds, and placed at room temperature for 3 minutes.

[0077] Centrifuge at 4℃12,000rpm for 10min. The sample will separate into three layers: yellow organic phase, middle layer and colorless aqueous phase. Transfer the aqueous phase to a new centrifuge tube. Slowly add 0.5 times volume of anhydrous ethanol and mix well. Transfer the obtained solution and precipitate to the adsorption column and centrifuge at 4℃12,000rpm for 30sec.

[0078] Add 500 μL of deproteinization solution RD, centrifuge at 4℃12,000rpm for 30 seconds, discard the waste liquid, and place the adsorption column in a collection tube. Add 500 μL of rinse solution, let stand at room temperature for 2 minutes, centrifuge at 4℃12,000rpm for 30 seconds, discard the waste liquid, and repeat the wash once. Centrifuge at 4℃12,000rpm for 2 minutes to remove the residual liquid.

[0079] Transfer the adsorption column to a new 1.5 ml centrifuge tube, add 30-100 μl RNase-Free ddH2O, place at room temperature for 2 min, and centrifuge at 12,000 rpm at 4°C for 2 min to obtain RNA from iPSC and embryoid body samples. Measure the concentration and store at -80°C.

[0080] Use the RNA reverse transcriptase kit to prepare a genomic DNA removal system as shown in Table 1, and the reaction procedure is shown in Table 2:

[0081] Table 1 Genomic DNA removal system

[0082] Components Addition amount 5×gDNAEraserBuffer 4.00μL gDNAEraser 2.00μL Total RNA 2.00 μg <![CDATA[RNaseFreedH2O]]> Refill to 20.00 μL

[0083] Table 2 Genomic DNA removal procedures

[0084] step temperature Duration Stage 1 42℃ 2min Stage2 4℃ ∞

[0085] After the reaction is completed, the RNA reverse transcription system is prepared as shown in Table 3, and the reverse transcription procedure is shown in Table 4:

[0086] Table 3 RNA reverse transcription system preparation

[0087]

[0088]

[0089] Table 4 Reverse transcription procedure

[0090] step temperature Duration Stage 1 37℃ 15min Stage2 85℃ 5s Stage3 4℃ ∞

[0091] Example 3 Design of primers for internal reference genes and germ layer genes

[0092] Ectodermal marker genes include CDH9, COL2A1, DLK1, DMBX1, DRD4, EN1, FABP7, HES5, LHX2, LMX1A, MAP2, MYO3B, NCAM1, NES, NOS2, NR2F1, NR2F2, OLFM3, OTX2, PAMR1, PAPLN, PAX3, PAX6, POU4F1, PRKCA, RAX, SDC2, SOX1, TFAP2A, TRPM8, WNT1, and ZBTB16.

[0093] Mesoderm marker genes include ABCA4, ALOX15, APLNR, BMP10, CDH5, CDX2, COLEC10, CXCR4, DES, ESM1, FCN3, FOXF1, HAND1, HAND2, HEY1, HOPX, HOXB7, IL6ST, ISL1, MEOX1, MIXL1, NCAM1, NKX2-5, ODAM, PDGFRA, PLVAP, RGS4, SNAI2, T, TBX3, TBX6, and TM4SF1.

[0094] Endoderm marker genes include AFP, CABP7, CDH20, CER1, CLDN1, CPLX2, CXCR4, ELAVL3, EOMES, FOXA1, FOXA2, FOXP2, GATA4, GATA6, GSC, HHEX, HMP19, HNF1B, HNF4A, KIT, KLF5, LEFTY1, LEFTY2, NODAL, PHOX2B, POU3F3, PRDM1, RXRG, SOX17, SOX7, and SST.

[0095] 2-3 marker genes can be selected for each germ layer. In this embodiment, 2 marker genes are selected for each germ layer. NCAM1 and PAX6 are selected as ectoderm marker genes; CDX2 and MEOX1 are selected as mesoderm marker genes; AFP and SOX17 are selected as endoderm marker genes, and GAPDH is selected as the internal reference gene.

[0096] By consulting references and comparing with the NCBI database, the target gene mRNA sequence information was confirmed, representative sequences spanning the intron region were selected, and primers were designed and tested. The relevant primer information is shown in Table 5.

[0097] Table 5 Primer probe sequence information

[0098]

[0099] Example 4 qPCR detection method

[0100] iPSC undifferentiated cells and differentiated cell samples were selected for primer screening and testing. The configuration of the qPCR reaction premix is ​​shown in Table 6 below:

[0101] Table 6 qPCR reaction system

[0102]

[0103]

[0104] Use ABI7500PCR instrument, set the qPCR reaction program according to Table 7, and collect signals at 60°C.

[0105] Table 7 qPCR reaction procedure

[0106]

[0107] Example 5 A gene detection kit for detecting the differentiation potential of iPSCs

[0108] iPSCs of different generations (PX+7, PX+12) were selected for induction experiments. After 14 days of induction, cells were collected for RNA extraction, inversion and qPCR detection. The test results are shown in Tables 8-9. Figure 5-Figure 6 As shown:

[0109] Table 8 iPSC-1 cell detection results

[0110]

[0111]

[0112] Table 9 iPSC-2 cell detection results

[0113]

[0114] According to Table 8-9 and Figure 5-Figure 6 The results show that after 14 days of induction, the expression levels of the three germ layer marker genes of iPSC cells of different generations were upregulated to varying degrees compared with undifferentiated iPSCs. By comparing with the internal reference gene GAPDH data, the relative expression levels and fold changes were calculated and it was found that the fold changes of all marker genes were >2, indicating that this method can be used to detect the stemness and differentiation potential of induced pluripotent stem cells. The relative expression calculation formula is:

[0115] If the marker gene expression of the control group is set to 1, the relative expression of the marker gene in the experimental group, i.e. the difference multiple, is 2. -ΔΔCt =2 -[(实验组标志基因Ct-实验组内参基因Ct)-(对照组标志基因Ct-对组照内参基因Ct)] .

[0116] Example 6 Method Specificity Test

[0117] According to the preliminary test results in Example 5, iPSC cells from different sources purchased from ATCC were selected for induction experiments, the induction time was increased, and the cells were collected after 21 days of induction for RNA extraction, inversion and qPCR detection, and specificity testing was performed. The test results are shown in Table 10, Figure 7 As shown:

[0118] Table 10 Specificity test results

[0119]

[0120] According to the results in Table 10, cells from different sources were selected. After 21 days of induction, the expression levels of the three germ layer marker genes were also upregulated to varying degrees compared with undifferentiated iPSCs. By comparing with the internal reference gene GAPDH data, the relative expression levels and the fold changes were calculated and it was found that the fold changes of all marker genes were >5.

[0121] Example 7 Method Durability Test

[0122] The end-point cell samples induced in Example 6 were selected, and different experimenters were selected to perform RNA extraction, inversion and qPCR detection to verify the durability of this method. The results are shown in Table 11. Figure 7 As shown:

[0123] Table 11 Durability test results

[0124]

[0125] Combined with the results of Example 5 and Example 6, according to Table 9-Table 11, Figure 5-Figure 7The results show that the expression trends of the three germ layer marker genes are consistent when changing small variables such as cell generation and endpoint detection experimenters. This method has good specificity and durability.

[0126] Based on the above experimental data, the experimental validity judgment standard of this method was determined, that is, the iPS cells were tested to be able to induce the formation of embryoid bodies, and the induced embryoid bodies were tested by qPCR. If the difference in the relative expression of the three germ layer marker genes was ≥2, the cells were considered to have the potential for three germ layer differentiation.

[0127] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A detection kit for detecting the differentiation potential of induced pluripotent stem cells (iPSCs), characterized in that: The detection kit includes a primer combination for detecting the differentiation potential of iPSCs; Inducing iPSCs to form embryoid bodies, wherein the embryoid bodies have three germ layers, namely, ectoderm, mesoderm and endoderm, Ectodermal marker genes include CDH9, COL2A1, DLK1, DMBX1, DRD4, EN1, FABP7, HES5, LHX2, LMX1A, MAP2, MYO3B, NCAM1, NES, NOS2, NR2F1, NR2F2, OLFM3, OTX2, PAMR1, PAPLN, PAX3, PAX6, POU4F1, PRKCA, RAX, SDC2, SOX1, TFAP2A, TRPM8, WNT1, ZBTB16, Mesoderm marker genes include ABCA4, ALOX15, APLNR, BMP10, CDH5, CDX2, COLEC10, CXCR4, DES, ESM1, FCN3, FOXF1, HAND1, HAND2, HEY1, HOPX, HOXB7, IL6ST, ISL1, MEOX1, MIXL1, NCAM1, NKX2-5, ODAM, PDGFRA, PLVAP, RGS4, SNAI2, T, TBX3, TBX6, TM4SF1, Endoderm marker genes include AFP, CABP7, CDH20, CER1, CLDN1, CPLX2, CXCR4, ELAVL3, EOMES, FOXA1, FOXA2, FOXP2, GATA4, GATA6, GSC, HHEX, HMP19, HNF1B, HNF4A, KIT, KLF5, LEFTY1, LEFTY2, NODAL, PHOX2B, POU3F3, PRDM1, RXRG, SOX17, SOX7, SST, 2-3 marker genes are selected for each germ layer, and GAPDH is selected as an internal reference gene. The primer composition includes primers for the marker genes selected for each germ layer and the internal reference gene GAPDH.

2. The detection kit according to claim 1, characterized in that: Two marker genes were selected for each germ layer: NCAM1 and PAX6 for the ectoderm, CDX2 and MEOX1 for the mesoderm, AFP and SOX17 for the endoderm, and GAPDH as the internal reference gene. The primer composition comprises Primers for the internal reference gene GAPDH: the upstream primer is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 2; Primers for the ectoderm target gene NCAM1: the upstream primer is shown as SEQ ID NO: 2, and the downstream primer is shown as SEQ ID NO: 4; Primers for the ectoderm target gene PAX6: the upstream primer is shown as SEQ ID NO: 5, and the downstream primer is shown as SEQ ID NO: 6; Primers for the mesoderm target gene CDX2: the upstream primer is shown as SEQ ID NO: 7, and the downstream primer is shown as SEQ ID NO: 8; Primers for the mesoderm target gene MEOX1: the upstream primer is shown in SEQ ID NO: 9, and the downstream primer is shown in SEQ ID NO: 10; Primers for endoderm target gene AFP: the upstream primer is shown as SEQ ID NO: 11, and the downstream primer is shown as SEQ ID NO: 12; Primers for the endoderm target gene SOX17: the upstream primer is shown as SEQ ID NO: 13, and the downstream primer is shown as SEQ ID NO:

14.

3. The detection kit according to claim 1, characterized in that: The detection kit is mainly composed of reagent A and reagent B; Reagent A is a fluorescent quantitative PCR reaction premix, comprising a highly specific qPCR reagent TB Green Premix Ex TaqII, ROX Reference Dye internal reference dye, nuclease-free water, and the primer composition of claim 1; Reagent B is a control, containing iPSC cDNA and embryoid body cDNA.

4. A fluorescent quantitative PCR detection method for detecting the differentiation potential of induced pluripotent stem cells, characterized in that: The following steps are involved: S1. Preparation of embryoid bodies using iPSCs; S2, extracting RNA from iPSCs and embryoid bodies; S3, reverse transcribe the RNA of iPSC and embryoid body into cDNA respectively; S4. Establishing a fluorescent quantitative PCR reaction system using the primer combination described in claim 1; S5, performing fluorescence quantitative PCR detection on the cDNA described in S3; S6. Result analysis.

5. The detection method according to claim 4, characterized in that: In S4, the fluorescent quantitative PCR reaction system is 20 μL, including 18 μL of reagent A and 2 μL of reagent B; the reagent A is a fluorescent quantitative PCR reaction premix, including highly specific qPCR reagent TB Green Premix Ex Taq II, ROX Reference Dye internal reference dye, nuclease-free water, and the primer composition of claim 1; the reagent B is a reference substance, including iPSC cDNA and embryoid body cDNA.

6. The detection method according to claim 4, characterized in that: In S4, the reaction conditions of the fluorescent quantitative PCR reaction system are: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 5 s, annealing at 60°C for 34 s, and 40 cycles.

7. The detection method according to claim 4, characterized in that: In S6, iPSC undifferentiated cells were used as a control, and the relative expression levels of genes in each germ layer were calculated using internal reference genes, and the difference multiples were calculated to determine the differentiation potential of iPSC.

8. The detection method according to claim 7, characterized in that: Relative expression calculation formula: If the marker gene expression of the control group is set to 1, the relative expression of the marker gene in the experimental group, i.e. the difference multiple, is 2. -ΔΔCt =2 -[(实验组标志基因Ct-实验组内参基因Ct)-(对照组标志基因Ct-对组照内参基因Ct)] ; If the difference in the relative expression of the three germ layer marker genes was ≥2, the iPSCs in the experimental group were considered to have the potential for three germ layer differentiation.

9. The detection method according to claim 4, characterized in that: In S1, the method for preparing embryoid bodies using iPSCs comprises the following steps: S1. Use digestion solution to digest iPSC into single cells or small cell clusters; S2. After centrifugation, resuspend the cells in induction medium and adjust the cell volume to 1×10 6 / mL and inoculated into 6cm non-tissue culture treated culture dishes; S3: On the third day, use a polypropylene centrifuge tube to collect the embryoid bodies formed by sedimentation and transfer them to a new culture dish; S4. Based on the growth of the embryoid bodies, the cell clusters are transferred to a tissue culture treated cell dish on the 5th to 7th day, and culture is continued for 7-14 days to obtain the embryoid bodies.

10. The detection method according to claim 9, characterized in that: The differentiation induction medium includes DMEM / F12 medium, KnockOut serum replacement and 2-mercaptoethanol.

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