Multiplex fluorescent quantitative PCR detection composition, method and kit for detecting residual induced pluripotent stem cells in differentiated cells

Abstract

The invention discloses a multiplex fluorescent quantitative PCR detection composition, method and kit for detecting induced pluripotent stem cell residues in differentiated cells, the detection method is non-diagnostic or therapeutic, and comprises the following steps: S1, extracting an RNA template of a sample to be detected; s2, removing genome DNA and carrying out reverse transcription on RNA; s3, establishing a fluorescent quantitative PCR reaction system by adopting the detection composition in claim 1; s4, carrying out multiple fluorescent quantitative PCR (Polymerase Chain Reaction) detection; and S5, analyzing the result of the to-be-detected sample. The kit can be used for accurately and quickly detecting iPSC residues in a sample.

Description

Technical Field

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

[0002] Induced pluripotent stem cells (iPSCs) are a type of cell similar to embryonic stem cells that is obtained by reprogramming adult cells through specific transcription factors. They have the ability to self-renew and differentiate into a variety of cell types. Currently, iPSCs have great potential in the fields of regenerative medicine, disease model research, and cell therapy, and are receiving increasing attention.

[0003] Compared with embryonic stem cells, iPSCs are derived from adult cells, avoiding ethical issues. Autologous iPSC-derived products can also effectively reduce immune rejection reactions. With the deepening of iPSC cell research and the increase in iPSC product pipelines, safety assessment of stem cell therapy products has become crucial.

[0004] The preparation process of iPSC-derived cell therapy products is complex, and relevant regulations and guidelines require cell bank management and quality control at all stages of the entire process. Undifferentiated or incompletely differentiated iPSCs may lead to the formation of teratomas or other tumors, so the detection of iPSC residues in terminally differentiated cells is an important part of quality control and helps ensure the safety, effectiveness and consistency of iPSC-derived stem cell products. Summary of the invention

[0005] The purpose of the present invention is to provide a multiplex fluorescence quantitative PCR detection composition, method and kit for detecting residual induced pluripotent stem cells (iPSCs) in differentiated cells. The kit can accurately and quickly detect residual iPSCs in a sample.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a multiplex fluorescent quantitative PCR detection composition for detecting residual induced pluripotent stem cells in differentiated cells, wherein the detection composition comprises primers and probes for the following genes:

[0008] Primers and probe for the internal reference gene GAPDH: the upstream primer is shown in SEQ ID NO: 1, the downstream primer is shown in SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3;

[0009] Primers and probe for the internal reference gene β-actin: the upstream primer is shown in SEQ ID NO: 4, the downstream primer is shown in SEQ ID NO: 5, and the probe is shown in SEQ ID NO: 6;

[0010] Primers and probe for the target gene Lin28A: the upstream primer is shown in SEQ ID NO: 7, the downstream primer is shown in SEQ ID NO: 8, and the probe is shown in SEQ ID NO: 9;

[0011] Primers and probe for the target gene ESRG: the upstream primer is shown in SEQ ID NO: 10, the downstream primer is shown in SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12;

[0012] Primers and probe for the target gene SOX2: the upstream primer is shown in SEQ ID NO: 13, the downstream primer is shown in SEQ ID NO: 14, and the probe is shown in SEQ ID NO: 15;

[0013] Primers and probe for the target gene POU5F1: the upstream primer is shown in SEQ ID NO: 16, the downstream primer is shown in SEQ ID NO: 17, and the probe is shown in SEQ ID NO: 18;

[0014] Primers and probe for the target gene CDH1: the upstream primer is shown as SEQ ID NO: 19, the downstream primer is shown as SEQ ID NO: 20, and the probe is shown as SEQ ID NO: 21.

[0015] In a second aspect, the present invention provides a multiplex fluorescence quantitative PCR detection kit for detecting residual induced pluripotent stem cells in differentiated cells, wherein the detection kit comprises the above-mentioned detection composition.

[0016] In the above technical solution, the detection kit also includes a positive control standard, a negative control standard and a fluorescent quantitative PCR reaction reagent.

[0017] In the above technical solution, the fluorescent quantitative PCR reaction reagent is a fluorescent quantitative PCR reaction premix, including probe method qPCR special reagent Premix Ex Taq, ROX Reference Dye internal reference dye, nuclease-free water, and the above detection composition.

[0018] In a third aspect, the present invention provides a multiplex fluorescence quantitative PCR detection method for detecting residual induced pluripotent stem cells in differentiated cells, wherein the detection method is not for diagnosis or treatment purposes and comprises the following steps:

[0019] S1, extracting RNA template of the sample to be tested;

[0020] S2, removal of genomic DNA and reverse transcription of RNA;

[0021] S3, using the above detection composition to establish a fluorescent quantitative PCR reaction system;

[0022] S4, performing multiplex fluorescence quantitative PCR detection;

[0023] S5. Result analysis of the tested samples.

[0024] In the above technical solution, the fluorescent quantitative PCR reaction system is 20 μL, including 18 μL of fluorescent quantitative PCR reaction premix and 2 μL of template;

[0025] The templates are cDNA of the sample to be tested, a positive control standard and a negative control standard;

[0026] The fluorescent quantitative PCR reaction premix comprises a probe method qPCR special reagent Premix Ex Taq, ROX Reference Dye internal reference dye, nuclease-free water, and the detection composition according to claim 1.

[0027] In the above technical solution, the reaction conditions of the fluorescent quantitative PCR reaction system are: pre-denaturation at 95°C for 30s, denaturation at 95°C for 5s, annealing at 60°C for 1min, and 40 cycles.

[0028] The beneficial effects of the present invention are:

[0029] 1. When screening and designing primers, the present invention selects target gene sequences spanning intron segments, which has strong specificity. At the same time, the genome-removing operation is introduced to eliminate the interference of cell genome DNA.

[0030] 2. The detection composition of the present invention, wherein the probe fluorescent reporter group can be selected from FAM, VIC or Cy5, and the fluorescent quencher group can be selected from BHQ1 or BHQ2, can meet the requirements of detecting any stem cell marker gene, and can detect any marker gene combination.

[0031] 3. By selecting target genes that are highly expressed in positive cells and lowly expressed or not expressed in negative cells, a primer-probe combination spanning the intron segment is designed, and finally a primer-probe with good specificity and high sensitivity is screened.

[0032] 4. The detection kit for detecting iPSC residues of the present invention contains positive control standards and negative control standards for detection, and tests the qPCR detection method at the same time. The primers have strong specificity and there is no cross-reaction between the primer probes. By carrying different fluorescent reporter groups on the probes, it can meet the requirements of simultaneously detecting the expression of multiple target genes in one program.

[0033] 5. Through repeated screening and optimization of the reaction system, including the concentrations of primers and probes, the detection method 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.

[0034] 6. By adding the spiked cells, without cell culture, different gradients of positive cells are added to 1 million negative cells, and the final sensitivity can reach 0.001%, that is, 1 positive cell can be detected in 100,000 negative cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Test amplification curves for multiplex qPCR;

[0036] Figure 2 is the amplification curve of GAPDH;

[0037] Figure 3 is the amplification curve of Lin28A;

[0038] Figure 4 This is the ESRG amplification curve. DETAILED DESCRIPTION

[0039] 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.

[0040] 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.

[0041] 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).

[0042] The experimental materials used in the embodiments of the present invention are:

[0043] RNA extraction kit was purchased from Tiangen, catalog number: DP419;

[0044] RNA reverse transcriptase kit was purchased from TaKaRa, catalog number: RR047Q;

[0045] The probe qPCR reagent Premix Ex Taq (Probe qPCR) was purchased from TaKaRa (containing ROX Reference Dye internal reference dye), catalog number: RR390A;

[0046] Nuclease-free water was purchased from Invitrogen, catalog number: 10977-015.

[0047] Example 1 Primer probe design and synthesis

[0048] By consulting references and comparing with the NCBI database, the target gene mRNA sequence information was confirmed, representative sequences spanning intronic regions were selected, and primer probes were designed and tested. The relevant primer probe information is shown in Table 1.

[0049] Table 1 Primer probe sequence information

[0050]

[0051]

[0052] Example 2: Sample setup, RNA extraction and reverse transcription

[0053] In the negative control group, human cells such as 293T and 2BS were selected as negative cells.

[0054] Undifferentiated iPSC cells were selected as positive cells in the positive control group.

[0055] The negative spike group selected 1×10 6 293T cells were cultured and iPSC positive cells were added at different ratios such as 1 / 100, 1 / 1000, 1 / 10000, and 1 / 100000.

[0056] Collect the cell pellet by centrifugation, add 1 ml of lysis buffer RZ, add 200 μl of chloroform, shake for 15 seconds, and leave at room temperature for 3 minutes.

[0057] 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.

[0058] 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.

[0059] Transfer the adsorption column to a new 1.5ml centrifuge tube, add 30-100μl RNase-Free ddH2O, place at room temperature for 2min, centrifuge at 12,000rpm at 4℃ for 2min to obtain the RNA of the sample to be tested, measure the concentration and store at -80℃.

[0060] Use the RNA reverse transcriptase kit to prepare a genomic DNA removal system as shown in Table 2, and the reaction procedure is shown in Table 3:

[0061] Table 2 Genomic DNA removal system

[0062] Components Addition amount 5×gDNAEraserBuffer 4.00μL gDNAEraser 2.00μL Total RNA 2.00 μg <![CDATA[RNaseFreedH2O]]> Refill to 20.00 μL

[0063] Table 3 Genomic DNA removal procedures

[0064] step temperature Duration Stage 1 42℃ 2min Stage2 4℃ ∞

[0065] After the reaction is completed, the RNA reverse transcription system is prepared as shown in Table 4, and the reverse transcription procedure is shown in Table 5:

[0066] Table 4 RNA reverse transcription system preparation

[0067] Components Addition amount Reaction solution for removing genomic DNA 20.00μL PrimeScriptRTEnzymeMixI 2.00μL RTPrimerMix 2.00μL 5×PrimeScriptBuffer2forRealTime 8.00μL RNaseFreedH2O 8.00μL

[0068] Table 5 Reverse transcription procedure

[0069] step temperature Duration Stage 1 37℃ 15min Stage2 85℃ 5s Stage3 4℃ ∞

[0070] Example 4 Multiplex qPCR Detection Method

[0071] Positive cell and negative cell samples were selected to screen the primer probes. The configuration of the qPCR reaction premix is ​​shown in Table 6 below:

[0072] Table 6 qPCR reaction system

[0073] Components Volume (μl) PremixExTaq(ProbeqPCR)(2×Conc.) 10.0 Primer probe combination 1 50×ROX 0.2 Nuclease-free water 6.8 Samples to be tested 2.0

[0074] Use ABI7500PCR instrument to set up qPCR reaction program according to Table 7 and collect signals at 60°C:

[0075] Table 7 qPCR reaction procedure

[0076]

[0077] Example 5 Gene Screening Test

[0078] 293T cells and 2BS cells were selected as negative cells, and iPS cells were selected as positive cells for primer and probe screening. The results are shown in Tables 8 to 10:

[0079] Table 8293T cell gene expression

[0080] Detection of genes Ct value of duplicate well 1 Duplicate well 2Ct value Average Ct value

[0081] GAPDH 13.83 13.77 13.80 β-actin 22.36 21.63 22.00 Lin28A Not detected Not detected N / A ESRG 34.16 34.29 34.22 SOX2 25.00 24.95 24.97 POU5F1 21.74 21.96 21.85 CDH1 29.54 29.22 29.38

[0082] Table 92 BS cell gene expression

[0083] Detection of genes Ct value of duplicate well 1 2Ct value of duplicate wells Average Ct value GAPDH 15.17 14.83 15.00 β-actin 20.72 20.24 20.48 Lin28A Not detected Not detected N / A ESRG 35.54 35.84 35.69 SOX2 29.84 29.48 29.66 POU5F1 24.80 23.58 24.19 CDH1 31.34 30.72 31.03

[0084] Table 10 iPS cell gene expression

[0085] Detection of genes Ct value of duplicate well 1 2Ct value of duplicate wells Average Ct value GAPDH 16.91 16.50 16.70 β-actin 23.50 23.26 23.38 Lin28A 22.80 22.54 22.67 ESRG 20.40 20.71 20.56 SOX2 19.65 19.96 19.81 POU5F1 17.02 16.99 17.01 CDH1 24.53 24.30 24.42

[0086] According to the results in Tables 8 to 10, genes with low or no expression in negative cells and high expression in positive cells were selected as target genes for iPS detection. The greater the difference in gene expression and the lower the expression in negative cells, the smaller the background interference when used to detect iPS residues, and the better the detection sensitivity may be.

[0087] Example 6 Multiplex qPCR Results Test

[0088] According to the results of Example 5, GAPDH (VIC fluorescent group) was selected as the internal reference gene, Lin28A (FAM fluorescent group) and ESRG (Cy5 fluorescent group) were selected as the target genes, iPS positive cell extraction samples were selected, gradient dilution was performed using nuclease-free water, and multiple qPCR tests were performed. The test results are shown in Table 11 and Figure 1-Figure 4 As shown:

[0089] Table 11 Multiplex qPCR test results

[0090]

[0091]

[0092] According to the results in Table 11, the multiplex qPCR method has good performance. The primer probe combinations used are all adapted to the same reaction system and procedure. After gradient dilution of the positive sample stock solution, the results of the three target genes all have good linearity and can be stably detected at dilutions of more than 10,000 times.

[0093] Example 7 Detection Limit Test

[0094] The detection limit refers to the lowest amount of the analyte that can be detected in the sample. Based on the test results of Example 6, in this method, positive cells spiked with different gradient cell amounts under the same negative cell amount are used to study the lowest detection limit of the method. The specific experimental design groups are shown in Table 12:

[0095] Table 12 Detection limit group settings

[0096] Group Name Sensitivity Group Information Negative control N / A <![CDATA[1×10 6 293T cells]]> Positive Control N / A <![CDATA[1×10 6 iPSCs]]> Detection limit 1 10％ <![CDATA[1×10 6 1×10 5 iPSCs]]> Detection limit 2 1％ <![CDATA[1×10 6 1×10 4 iPSCs]]> Detection limit 3 0.1％ <![CDATA[1×10 6 1×10 3 iPSCs]]> Detection limit 4 0.01％ <![CDATA[1×10 6 100 iPSCs spiked into 293T cells]]> Detection limit 5 0.001％ <![CDATA[1×10 6 10 iPSCs spiked into 293T cells]]>

[0097] The test results are shown in Table 13:

[0098] Table 13 Detection limit results

[0099]

[0100]

[0101] According to the results in Table 13, by spiking different gradients of iPS positive cells in 293T negative cells, at a sensitivity of 0.001%, Lin28A and ESRG were both stable, and the Ct value was <35. At the same time, these two target genes had no specific amplification curve or were not detected in the negative cell samples, and could be used as target genes for iPSC residual detection.

[0102] Based on the above experimental data, the experimental validity judgment standard of this method was determined, that is, the Ct value of the reference gene detection channel of all groups should be between 10 and 25; the Ct value of the target gene detection channel of the positive control group was <35, and the test result was positive; the target gene detection channel detection result of the negative control group was not detected or the Ct value was ≥35, and there was no obvious amplification curve.

[0103] 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 multiplex fluorescence quantitative PCR detection composition for detecting residual induced pluripotent stem cells in differentiated cells, characterized in that: The detection composition includes primers and probes for the following genes: Primers and probe for the internal reference gene GAPDH: the upstream primer is shown in SEQ ID NO: 1, the downstream primer is shown in SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3; Primers and probe for the internal reference gene β-actin: the upstream primer is shown in SEQ ID NO: 4, the downstream primer is shown in SEQ ID NO: 5, and the probe is shown in SEQ ID NO: 6; Primers and probe for the target gene Lin28A: the upstream primer is shown in SEQ ID NO: 7, the downstream primer is shown in SEQ ID NO: 8, and the probe is shown in SEQ ID NO: 9; Primers and probe for the target gene ESRG: the upstream primer is shown in SEQ ID NO: 10, the downstream primer is shown in SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12; Primers and probe for the target gene SOX2: the upstream primer is shown in SEQ ID NO: 13, the downstream primer is shown in SEQ ID NO: 14, and the probe is shown in SEQ ID NO: 15; Primers and probe for the target gene POU5F1: the upstream primer is shown in SEQ ID NO: 16, the downstream primer is shown in SEQ ID NO: 17, and the probe is shown in SEQ ID NO: 18; Primers and probe for the target gene CDH1: the upstream primer is shown as SEQ ID NO: 19, the downstream primer is shown as SEQ ID NO: 20, and the probe is shown as SEQ ID NO:

21.

2. A multiplex fluorescence quantitative PCR detection kit for detecting residual induced pluripotent stem cells in differentiated cells, characterized in that: The detection kit comprises the detection composition according to claim 1.

3. The detection kit according to claim 2, characterized in that: The detection kit also includes a positive control standard, a negative control standard and a fluorescent quantitative PCR reaction reagent.

4. The detection kit according to claim 3, characterized in that: The fluorescent quantitative PCR reaction reagent is a fluorescent quantitative PCR reaction premix, which includes a probe method qPCR special reagent Premix Ex Taq, ROX Reference Dye internal reference dye, nuclease-free water, and the detection composition according to claim 1.

5. A multiplex fluorescence quantitative PCR detection method for detecting residual induced pluripotent stem cells in differentiated cells, characterized in that: The detection method is not for diagnosis or treatment purposes and comprises the following steps: S1, extracting RNA template of the sample to be tested; S2, removal of genomic DNA and reverse transcription of RNA; S3. Using the detection composition of claim 1 to establish a fluorescent quantitative PCR reaction system; S4, performing multiplex fluorescence quantitative PCR detection; S5. Result analysis of the tested samples.

6. The detection method according to claim 5, characterized in that: The fluorescent quantitative PCR reaction system is 20 μL, including 18 μL of fluorescent quantitative PCR reaction premix and 2 μL of template; The templates are cDNA of the sample to be tested, a positive control standard and a negative control standard; The fluorescent quantitative PCR reaction premix comprises a probe method qPCR special reagent Premix Ex Taq, ROX Reference Dye internal reference dye, nuclease-free water, and the detection composition according to claim 1.

7. The detection method according to claim 5, characterized in that: The reaction conditions of the fluorescent quantitative PCR reaction system are: pre-denaturation at 95° C. for 30 s, denaturation at 95° C. for 5 s, annealing at 60° C. for 1 min, and 40 cycles.

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