GLP-1 and FGF21 double-factor high-expression adipose-derived stem cell target gene positive rate detection method

Abstract

The invention provides a GLP-1 and FGF21 double-factor high-expression adipose-derived stem cell target gene positive rate detection method, which adopts a micro-droplet digital polymerase chain reaction technology, takes an inserted WPRE gene, LTR gene or LVRV gene as a detection target, uses a primer and a probe of a target gene, and adopts a reference gene as a detection target for detection. Through an excellent primer probe combination, positive genes of single cells in a GLP-1 and FGF21 double-factor high-expression adipose-derived mesenchymal stem cell population are effectively detected, liquid drops are tight, the cell integrity is high, and a target gene positive rate detection result is good.

Description

Technical Field

[0001] The present invention relates to a method for detecting the gene positive rate of gene-modified cells, and in particular to a method for detecting the target gene positive rate of adipose stem cells with high expression of GLP-1 and FGF21 dual factors. Background Art

[0002] In order to solve the current situation of single clinical treatment mode and poor effect of diabetes, the applicant, based on the extensive research results of mesenchymal stem cell treatment of diseases, innovatively introduced GLP-1 and FGF21 into autologous adipose mesenchymal stem cells, such as 201810283803.0 and 202010180799.2, to obtain a cell therapy product for type 2 diabetes.

[0003] In order to explore the evaluation system for the safety, efficacy and stability of cell therapy products for the treatment of type 2 diabetes mellitus modified with autologous adipose-derived mesenchymal stem cells with dual GLP-1 and FGF21, and to ensure the accuracy and reliability of the results obtained from biological sample analysis using the detection method, the inventors of the present application explored the detection method for the positive rate of the target gene in adipose-derived mesenchymal stem cells with high expression of the dual GLP-1 and FGF21 factors. Summary of the invention

[0004] The inventors adopted droplet digital polymerase chain reaction technology, selected appropriate primer probes for the insertion sequence, and selected appropriate primer probes for the internal reference gene, and effectively detected the positive genes of adipose mesenchymal stem cells with high expression of GLP-1 and FGF21 dual factors through excellent primer probe combination, with compact droplets, high cell integrity, and good detection results of the positive rate of cell target genes, thereby completing the present invention.

[0005] The present invention provides a method for detecting the positive rate of target genes of adipose stem cells with high expression of GLP-1 and FGF21 dual factors. The method adopts droplet digital polymerase chain reaction technology, takes the inserted WPRE gene, LTR gene or LVRV gene as the detection target, uses primers and probes of the WPRE gene, LTR gene or LVRV gene, and adopts an internal reference gene as the detection target for detection.

[0006] In the method of the present invention, the primers for the WPRE gene are selected from one or more pairs of the following primers:

[0007] WPRE Primer Pair 1:

[0008] Upstream primer: 5'-CCGTTGTCAGGCAACGTG-3' (WPRE-F)

[0009] Downstream primer: 5′-AGCTGACAGGTGGTGGCAAT-3′ (WPRE-R);

[0010] WPRE Primer Pair 2:

[0011] Upstream primer: 5'-CCTTTCCGGGACTTTCGCTTT-3'

[0012] Downstream primer: 5′-GCAGAATCCAGGTGGCAACA-3′;

[0013] WPRE Primer Pair 3:

[0014] Upstream primer: 5'-ACTGTGTTTGCTGACGCAAC-3'

[0015] Downstream primer: 5′-AAGGAAGGTCCGCTGGATTG-3′;

[0016] WPRE Primer Pair 4:

[0017] Upstream primer: 5'-CAGCTCCTTTCCGGGACTTT-3'

[0018] Downstream primer: 5′-GGACGATGATTTCCCCGACA-3′;

[0019] WPRE Primer Pair 5:

[0020] Upstream primer: 5'-CACCACCTGTCAGCTCCTTT-3'

[0021] Downstream primer: 5′-ACGGAATTGTCAGTGCCCAA-3′;

[0022] WPRE Primer Pair 6:

[0023] Upstream primer: 5'-TTGGGCACTGACAATTCCGT-3'

[0024] Downstream primer: 5′-CCGAAGGGACGTAGCAGAAG-3′;

[0025] WPRE Primer Pair 7:

[0026] Upstream primer: 5'-CGCTTTCCCCCTCCCTATTG-3'

[0027] Downstream primer: 5′-CACCACGGAATTGTCAGTGC-3′;

[0028] WPRE Primer Pair 8:

[0029] Upstream primer: 5'-CGCTATGTGGATACGCTGCT-3'

[0030] Downstream primer: 5′-GTTGCGTCAGCAAACACAGT-3′;

[0031] WPRE Primer Pair 9:

[0032] Upstream primer: 5'-CTTTCCCCCTCCCTATTGCC-3'

[0033] Downstream primer: 5′-CGGAATTGTCAGTGCCCAAC-3′;

[0034] WPRE Primer Pair 10:

[0035] Upstream primer: 5'-CTTCTGCTACGTCCCTTCGG-3'

[0036] Downstream primer: 5′-AAGGGAGATCCGACTCGTCT-3′;

[0037] WPRE Primer Pair 11:

[0038] Upstream primer: 5'-CAATCCAGCGGACCTTCCTT-3'

[0039] Downstream primer: 5′-AGATCCGACTCGTCTGAGGG-3′;

[0040] WPRE Primer Pair 12:

[0041] Upstream primer: 5'-GCACTGACAATTCCGTGGTG-3'

[0042] Downstream primer: 5′-GAAGGTCCGCTGGATTGAGG-3′;

[0043] Preferred WPRE primer pair 1:

[0044] Upstream primer: 5'-CCGTTGTCAGGCAACGTG-3' (WPRE-F)

[0045] Downstream primer: 5'-AGCTGACAGGTGGTGGCAAT-3' (WPRE-R). In a preferred embodiment of the method of the present invention, a WPRE probe selected from the following is used: 5'-FAM-TGCTGACGCAACCCCCACTGGT-BHQ1-3' (WPRE-P); 5'-FAM-ACTCATCGCCGCCTGCCTTGCC-BHQ1-3';

[0046] 5'-FAM-ATTGCCACCACCTGTCAGCTCCTTT-BHQ1-3';

[0047] 5'-FAM-TGCCACGGCGGAACTCATCGCCGCC-BHQ1-3';

[0048] 5'-FAM-CGGAACTCATCGCCGCCTGCCTTGC-BHQ1-3';

[0049] 5'-FAM-CTTTCCTTGGCTGCTCGCCTGTGTT-BHQ1-3';

[0050] 5'-FAM-CGCCGCCTGCCTTGCCCGCTGCTGG-BHQ1-3';

[0051] 5'-FAM-TGCCACCACCTGTCAGCTCCTTCC-BHQ1-3';

[0052] 5'-FAM-GCCTGCCTTGCCCGCTGCTGGACAG-BHQ1-3';

[0053] 5'-FAM-TCCTTCCCGCGGCCTGCTGCCGGCT-BHQ1-3';

[0054] 5'-FAM-TGCTGCCGGCTCTGCGGCCTCTTCC-BHQ1-3'; or 5'-FAM-CTCGCCTGTGTTGCCACCTGGATTC-BHQ1-3';

[0055] Preferred:

[0056] 5'-FAM-TGCTGACGCAACCCCCACTGGT-BHQ1-3'(WPRE-P). In the method of the present invention, the primer of the LTR gene is selected from the following primers:

[0057] Upstream primer: 5'-TGTGTGCCCGTCTGTTGTGT-3' (LTR WPRE-F)

[0058] Downstream primer: 5'-GAGTCCTGCGTCGAGAGAGC-3' (LTR WPRE-R).

[0059] As a probe for the LTR gene, one is selected from: 5'-FAM-GTGACTCTGGTAACTAGAGATCCCT-BHQ1-3'.

[0060] In a preferred embodiment of the method of the present invention, ribonuclease P protein subunit p30 (RPP30) is used as the internal reference gene, and its primers are selected from one or more pairs of the following primers:

[0061] RPP30 Primer Pair 1:

[0062] Upstream primer: 5-AGTGCATGCTTATCTCTGACAG-3 (RPP30-F)

[0063] Downstream primer: 5-GCAGGGCTATAGACAAGTTCA-3 (RPP30-R);

[0064] RPP30 Primer Pair 2:

[0065] Upstream primer: 5'-ATTCGGCACGAGGTGGG-3'

[0066] Downstream primer: 5′-GAGCCTTCAGGTCAGAACCC-3′;

[0067] RPP30 Primer Pair 3:

[0068] Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3'

[0069] Downstream primer: 5′-CCTTCTGATGGCCGAGGTTT-3′;

[0070] RPP30 Primer Pair 4:

[0071] Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3'

[0072] Downstream primer: 5′-GAGCCTTCAGGTCAGAACCC-3′;

[0073] RPP30 Primer Pair 5:

[0074] Upstream primer: 5'-AATTCGGCACGAGGTGGG-3'

[0075] Downstream primer: 5′-GCAAGCCTAGATTTGCCACG-3′;

[0076] RPP30 Primer Pair 6:

[0077] Upstream primer: 5'-GGCACGAGGTGGGACTTC-3'

[0078] Downstream primer: 5′-GCAAGCCTAGATTTGCCACG-3′;

[0079] RPP30 Primer Pair 7:

[0080] Upstream primer: 5'-AATTCGGCACGAGGTGGG-3'

[0081] Downstream primer: 5′-TGAATAGCCAAGGTGAGCGG-3′;

[0082] RPP30 Primer Pair 8:

[0083] Upstream primer: 5'-GGCACGAGGTGGGACTTC-3'

[0084] Downstream primer: 5′-TGAATAGCCAAGGTGAGCGG-3′;

[0085] RPP30 Primer Pair 9:

[0086] Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3'

[0087] Downstream primer: 5′-GCAAGCCTAGATTTGCCACG-3′;

[0088] RPP30 Primer Pair 10:

[0089] Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3'

[0090] Downstream primer: 5′-TGAATAGCCAAGGTGAGCGG-3′;

[0091] RPP30 Primer Pair 11:

[0092] Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3'

[0093] Downstream primer: 5′-GGCCGAGGTTTCTTCACTGT′-3′.

[0094] Accordingly, it is preferred to use a probe selected from the following:

[0095] 5'-VIC-TTTCCTGTGAAGGCGATTGACCGA-BHQ1-3'(RPP30-P);

[0096] 5'-VIC-TGGACCTGCGAGCGGGTTCTGACC-BHQ1-3';

[0097] 5'-VIC-TGAGAGCAACTTCTTCAAGGGCCC-BHQ1-3';

[0098] 5'-VIC-TAACTGTAACAGAGAAACTACCAT-BHQ1-3';

[0099] 5'-VIC-GAGAAGGTATACAATTTCCAGTGC-BHQ1-3';

[0100] 5'-VIC-CTATAGCCCTGCTATCAAAGACTC-BHQ1-3';

[0101] 5'-VIC-ATATCGTTGACTTTAAGGAAAAGA-BHQ1-3';

[0102] 5'-VIC-TTAATGTGGCGATTGACCGAGGCC-BHQ1-3';

[0103] 5'-VIC-TTGATGCAAATCTGCAAAGGAAAG-BHQ1-3';

[0104] 5'-VIC-CGGCCATCAGAAGGAGATGAAGAT-BHQ1-3';

[0105] 5'-VIC-TTGTCTATAGCCCTGCTATCAAAG-BHQ1-3';

[0106] Preferred:

[0107] 5'-VIC-TTTCCTGTGAAGGCGATTGACCGA-BHQ1-3'(RPP30-P).

[0108] In another embodiment of the method of the present invention, albumin gene (ALB) is used as an internal reference gene, and its primers are selected from one or more pairs of the following primers:

[0109] ALB Primer Pair 1:

[0110] Upstream primer: 5-TGAAACATACGTTCCCAAAGAGTTT-3 (ALB-F)

[0111] Downstream primer: 5-CTCTCCTTCTCAGAAAGTGTGCATAT-3 (ALB-R);

[0112] ALB Primer Pair 2:

[0113] Upstream primer: 5'-TGCTGCAAGGCTGACGATAA-3'

[0114] Downstream primer: 5′-AGCCCTAGCCTAACCAAACA-3′;

[0115] ALB Primer Pair 3:

[0116] Upstream primer: 5'-GTGCACTTGTTGAGCTCGTG-3'

[0117] Downstream primer: 5′-TCCCTAAGCCCTAGCCTAACC-3′;

[0118] ALB Primer Pair 4:

[0119] Upstream primer: 5'-AAACACAAGCCCAAGGCAAC-3'

[0120] Downstream primer: 5′-CCCTAAGCCCTAGCCTAACCA-3′;

[0121] ALB Primer Pair 5:

[0122] Upstream primer: 5'-TGCACTTGTTGAGCTCGTGA-3'

[0123] Downstream primer: 5′-CCTAAGCCCTAGCCTAACCA-3′;

[0124] ALB Primer Pair 6:

[0125] Upstream primer: 5'-GTGAAACACAAGCCCAAGGC-3'

[0126] Downstream primer: 5′-AGCCCTAGCCTAACCAAACAT-3′;

[0127] ALB Primer Pair 7:

[0128] Upstream primer: 5'-GCCGAGGAGGTACTACAGTT-3'

[0129] Downstream primer: 5′-TCCCACATGTACAAAGCCTCC-3′;

[0130] ALB Primer Pair 8:

[0131] Upstream primer: 5'-GAAACACAAGCCCAAGGCAA-3'

[0132] Downstream primer: 5′-CCCTAGCCTAACCAAACATGC-3′;

[0133] ALB Primer Pair 9:

[0134] Upstream primer: 5'-GGTTAGGCTAGGGCTTAGGG-3'

[0135] Downstream primer: 5′-TGGCACAATAGAGCAGGAGA-3′;

[0136] ALB Primer Pair 10:

[0137] Upstream primer: 5'-GAGAAGTGCTGCAAGGCTGA-3'

[0138] Downstream primer: 5′-CCTAAGCCCTAGCCTAACCAA-3′;

[0139] ALB Primer Pair 11:

[0140] Upstream primer: 5'-GCTGCAAGGCTGACGATAAG-3'

[0141] Downstream primer: 5′-GCCCTAGCCTAACCAAACAT-3′.

[0142] Accordingly, a probe selected from the following is used:

[0143] 5'-HEX-TGCTGAAACATTCACCTTCCATGCAGA-BHQ1-3'(ALB-P);

[0144] 5'-HEX-TAGTTCTCTTCATTTTAATATGTCC-BHQ1-3';

[0145] 5'-HEX-AGCTGTTATGGATGATTTCGCAGC-BHQ1-3';

[0146] 5'-HEX-ATGGATGATTTCGCAGCTTTTGTA-BHQ1-3';

[0147] 5'-HEX-GAGGAGGTACTACAGTTCTCTTCA-BHQ1-3';

[0148] 5'-HEX-TGTAGAGAAGTGCTGCAAGGCTGA-BHQ1-3';

[0149] 5'-HEX-GGACAGGGATCTTATTTTACAAAC-BHQ1-3';

[0150] 5'-HEX-TTTGTAAGGCCTGAAATATTTTGA-BHQ1-3';

[0151] 5'-HEX-TGTACATGTGGGACAGGGATCTTA-BHQ1-3';

[0152] 5'-HEX-TGCTTATGAATATTAATAGGAATA-BHQ1-3';

[0153] 5'-HEX-TGGCCTGACAGTAACTTTTTTTTA-BHQ1-3'.

[0154] In the most preferred embodiment of the method of the present invention, the inserted WPRE gene is used as the detection target, and the primers for the WPRE gene are WPRE primer pair 1:

[0155] Upstream primer: 5'-CCGTTGTCAGGCAACGTG-3' (WPRE-F)

[0156] Downstream primer: 5'-AGCTGACAGGTGGTGGCAAT-3' (WPRE-R)

[0157] The probes are:

[0158] 5'-FAM-TGCTGACGCAACCCCCACTGGT-BHQ1-3'(WPRE-P);

[0159] As an internal reference gene, RNA P protein subunit p30 was used, and the primers thereof were RPP30 primer pair 1:

[0160] Upstream primer: 5'-AGTGCATGCTTATCTCTGACAG-3' (RPP30-F)

[0161] Downstream primer: 5'-GCAGGGCTATAGACAAGTTCA-3' (RPP30-R)

[0162] The probes are:

[0163] 5'-VIC-TTTCCTGTGAAGGCGATTGACCGA-BHQ1-3'(RPP30-P).

[0164] In the present invention, the droplet digital polymerase chain reaction technology (droplet digital PCR, ddPCR) used is a technology that does not rely on a standard curve to perform absolute quantification of the target gene. The ddPCR system performs droplet processing on a large reaction system before ordinary PCR amplification, that is, it is "divided" into tens of thousands of nanoliter droplets using oil-in-water technology. Each droplet does not contain a nucleic acid target molecule to be tested or contains at least one nucleic acid target molecule to be tested, and each droplet is an independent PCR reaction system. After the PCR amplification is completed, each droplet is detected one by one using a droplet analyzer. Depending on whether a fluorescent signal is detected, the droplet with a fluorescent signal is read as "1", and the droplet without a fluorescent signal is read as "0". Since the DNA fragments are randomly and independently separated into droplets, the Poisson algorithm can determine the absolute copy number in the original sample independently of the standard curve, thereby achieving absolute quantification of the number of nucleic acid target molecules in the initial reaction system.

[0165] In particular, the whole cell droplet digital polymerase chain reaction technology (WC-ddPCR) is to dilute the cells and add them directly to a large reaction system. The cell-containing reaction system is dropletized, and the oil-in-water technology is used to divide the cell-containing reaction system into tens of thousands of nanoliter droplets, each of which contains cells or does not contain cells. A small amount of lysis components is added to the reaction system, and the cells wrapped in oil are lysed in the droplets to release the nucleic acids they contain, so that whether the cells contain the target gene can be detected.

[0166] In the present invention, the WC-ddPCR amplification system is a dual fluorescence signal ddPCR system. A fluorescence signal is designed for the cell internal reference gene, and another set of fluorescence signals is designed for the inserted target gene. After the PCR amplification is completed, each droplet is detected one by one using a droplet analyzer. In the experiment, droplets in which dual fluorescence signals are detected are judged as "positive cells", droplets in which single internal reference gene fluorescence signals are detected are judged as "negative cells", and droplets without fluorescence signals are judged as "negative droplets". The experiment can obtain the integrity of the cells through the Linkage value calculated by the software. The experiment can obtain the proportion of positive cells by calculating the ratio of the inserted target gene nucleic acid concentration to the reference gene nucleic acid concentration.

[0167] The above is a possible speculation on the principle of the present invention, and of course the principle of the present invention is not limited to this.

[0168] In a preferred embodiment of the method of the present invention, the number of cells during detection is 2000 to 4000 cells / μl.

[0169] The detection method provided by the present invention has the following advantages:

[0170] (1) Using the inserted WPRE gene as a detection target, and then using an internal reference gene such as RNA P protein subunit p30 and albumin gene (ALB) as a detection target of the cell gene as the detection target, the detection can be carried out;

[0171] (2) In particular, when RNA P protein subunit p30 is used as an internal reference gene, the detection effect is particularly reliable by selecting a specific WPRE gene probe and a RNA P protein subunit p30 gene primer and probe combination;

[0172] (3) The method of the present invention is applicable to a wide range of detection objects, and even for cells thawed after freezing, the detection method of the present invention has no obvious effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1 The WPRE+ALB combined ddPCR test results in Example 1 are shown;

[0174] Figure 2 The WPRE+RPP30 combined ddPCR test results in Example 1 are shown;

[0175] Figure 3 The results of the combined ddPCR test of LTR-WPRE+β-globin in Example 1 are shown;

[0176] Figure 4 The LVRV-WPRE+β-globin combined ddPCR test results in Example 1 are shown;

[0177] Figure 5 The WPRE+RPP30 combined ddPCR test results in Example 2 are shown;

[0178] Figure 6 The results of the combined ddPCR test of LTR WPRE+β-globin in Example 2 are shown;

[0179] Figure 7 The results of the combined ddPCR test of LVRV-WPRE+β-globin in Example 2 are shown;

[0180] Figure 8 The MSC-FG-4000ddPCRddPCR test results in Example 3 are shown;

[0181] Fig. 9 The ddPCR test results of MSC-FG-2000 in Example 3 are shown;

[0182] Fig.10The ddPCR test results of MSC-FG-1000 in Example 3 are shown; DETAILED DESCRIPTION

[0183] The present invention is described in detail below through specific implementation modes, and the characteristics and advantages of the present invention will become clearer and more specific with these descriptions.

[0184] The reagents used in the examples are as follows:

[0185] name Specification Manufacturer / Article No. <![CDATA[ddPCR TM 96-Well Plates]]> 25 pieces / pack BIORAD / 12001925 <![CDATA[ddPCR TM Droplet Reader Oil]]> 2x1L / bottle BIORAD / 1863004 DG32Automated Droplet Generator Cartridges 30 pieces / pack BIORAD / 1864108 PCR Plate Heat Seal,foil,pierceable 100 sheets / pack BIORAD / 1814040 <![CDATA[Pipet Tips for the AutoDG TM System]]> 20 boxes / pack BIORAD / 1864120 Automated Droplet Generation Oil for Probes 140ml / bottle BIORAD / 1864110 ddPCR Enhancer Reagent A 1ml / tube BIORAD / 12013425 ddPCR Multiplex Supermix 5x0.6ml / box BIORAD / 12005910 DTT 2x1ml / box BIORAD / 12012171 PBS 500ml / bottle Hyclone / SH30256.01 BSA 20g / bottle Beyotime / ST025-20g SamI 2,000units / tube NEB / R0141S HaeIII 3,000units / tube NEB / R0108S UltraPure Distilled Water 500ml / bottle Invitrogen / 10977015 TE Buffer pH 8.0 500ml / bottle Invitrogen / AM9858

[0186] The equipment used in the embodiment is as follows:

[0187] Device Name factory Device Model Single channel pipettes Rainin XLS Micro low speed centrifuge IKA MINI G Vortex mixer Scientific Industries Gere2 MINI-G Refrigerated centrifuge Eppendorf 5910R Medium Centrifuge Eppendorf 5810 Fully automated droplet digital PCR system Bio-Rad QX200 AutoDG Analytical Balance METTLER XPR105 Biological Safety Cabinets ESCO AC2-4S1

[0188] The primers used in the examples are as follows:

[0189]

[0190]

[0191] The positive cells used in the example are adipose-derived mesenchymal stem cells that highly express both GLP-1 and FGF21, which are adipose-derived mesenchymal stem cells that highly express both GLP-1 and FGF21 provided by the applicant's prior patent 201810283803.0, see "Example 1" for details.

[0192] The negative cells used in the examples are unmodified adipose-derived mesenchymal stem cells.

[0193] Example 1 Primer probe screening

[0194] 1.1 Experimental process

[0195] 1.1.1 Preparation of PBS + 0.1% BSA diluent

[0196] (1) Weigh 0.25 g of BSA particles using a weighing balance, pour into a clean 50 ml centrifuge tube, and add 25 ml of PBS solution to obtain PBS + 1% BSA solution.

[0197] (2) Take a clean 15 ml centrifuge tube and add 1 ml of PBS + 1% BSA solution and 9 ml of PBS solution to obtain PBS + 0.1% BSA solution as the cell diluent.

[0198] 1.1.2 Sample solution replacement

[0199] (1) Place 50 ml centrifuge tubes containing 12 ml of sample 1 (positive cells) and sample 2 (negative cells) in a medium-sized centrifuge and centrifuge at 300 g for 3 min. Pour off the supernatant.

[0200] (2) Add 1 ml of cell dilution to a 50 ml centrifuge tube and pipette the cells several times. Transfer the 1 ml cell suspension in the 50 ml centrifuge tube to a 1.5 ml centrifuge tube and label it as FG and MSC. Then place it in a small centrifuge and centrifuge at 300 g for 3 min. Carefully remove the supernatant with a pipette.

[0201] (3) Add 1 ml of diluent to a 1.5 ml centrifuge tube and pipette the cells several times. Place the cells in a small centrifuge and centrifuge at 300 g for 3 min. Use a pipette to carefully remove the supernatant.

[0202] (4) Add 460 μl of diluent to sample FG and 520 μl of diluent to sample MSC. Thus, samples 1 and 2 with a cell fluid concentration of 10,000 cells / μl were obtained, named FG-10000 and MSC-10000. According to the cell concentration, gradient dilution was performed, and the dilution table is as follows:

[0203] Sample No. Dilution formula Number of cells per μl of dilution FG-5000 50μl FG-10000 + 50μl cell dilution buffer 5000 FG-1000 10μl FG-10000 + 90μl cell dilution buffer 1000 MSC-1000 10μl MSC-10000 + 90μl cell dilution buffer 1000

[0204] 1.1.3 Preparation of 1x ddPCR MIX

[0205] (1) Prepare four sets of primer-probe systems in 1.5 ml centrifuge tubes according to the table below. Mix thoroughly and dispense into 96-well plates.

[0206] WPRE and ALB dual system cell preparation table

[0207] Combination 1 Reaction volume per well (μl) Reaction volume for 7 reaction wells (μl) ddPCR Multiplex Supermix 6.25 43.75 SamI 0.50 3.50 HaeIII 0.50 3.50 WPRE primer mix 1.25 8.75 ALB primer mix 1.25 8.75 DTT 0.34 2.38 ddPCR Enhancer Reagent A 0.50 3.50 UtraPure Distilled Water 8.41 58.87 Total volume 20.00 140.00

[0208] WPRE and RPP30 dual system cell preparation table

[0209] Combination 2 Reaction volume per well (μl) Reaction volume for 7 reaction wells (μl) ddPCR Multiplex Supermix 6.25 43.75 SamI 0.50 3.50 HaeIII 0.50 3.50 WPRE primer mix 1.25 8.75 RPP30primer mix 1.25 8.75 DTT 0.34 2.38 ddPCR Enhancer Reagent A 0.50 3.50 UtraPure Distilled Water 8.41 58.87 Total volume 20.00 140.00

[0210] LTR WPRE and β-globin dual system cell preparation table

[0211]

[0212]

[0213] LVRV WPRE and β-globin dual system formulation table

[0214] Combination 4 Reaction volume per well (μl) Reaction volume for 7 reaction wells (μl) ddPCR Multiplex Supermix 6.25 43.75 SamI 0.50 3.50 HaeIII 0.50 3.50 LVRV WPRE primer mix 1.25 8.75 β-globin primer mix 1.25 8.75 DTT 0.34 2.38 ddPCR Enhancer Reagent A 0.50 3.50 UtraPure Distilled Water 8.41 58.87 Total volume 20.00 140.00

[0215] (2) After repeatedly pipetting the diluted cells to resuspend, add them to the 1x ddPCR system, seal with sealing film, vortex slowly at the lowest speed to mix, and centrifuge at 300g for 1 min in a medium-sized centrifuge.

[0216] (3) Generate droplets: Place the mixed 1x ddPCR mix 96-well plate into the ddPCR droplet generator and generate droplets according to the default program.

[0217] 1.1.4PCR

[0218] The 96-well plate after droplet generation was sealed and placed in a PCR instrument for PCR amplification. The PCR amplification program is shown in the following table.

[0219] ddPCR amplification procedure

[0220]

[0221] 1.2 Analysis

[0222] The 96-well plate after PCR was placed in the droplet analyzer, and the software was set up and analyzed according to the standard operating procedures for the use and maintenance of the fully automated droplet digital PCR system (Bio-Rad QX200 AutoDG) (SOP-SZGN-0010-00). After the instrument analysis was completed, the experimental results were analyzed.

[0223] The results are as follows Figure 1-Figure 4 shown.

[0224] 1.3 Results and Discussion

[0225] (1) The mixed droplets in the amplification graph of the primer combination LTR-WPRE+β-globin are scattered, which is not conducive to analysis;

[0226] (2) The internal reference droplets in the amplification graph of the primer combination LVRV-WPRE+β-globin are scattered, which is not conducive to analysis;

[0227] (3) Primer combinations WPRE+ALB and WPRE+RPP30 are relatively clustered. From the dispersion results of the droplet clusters, the separation degree between the mixed droplets of the WPRE+RPP30 combination and the internal reference droplets is better than that of the WPRE+ALB combination.

[0228] Example 2 Primer-probe secondary screening

[0229] 2.1 Experimental process

[0230] 2.1.1 Preparation of PBS + 0.1% BSA diluent : Same as 1.1.1

[0231] 2.1.2 Sample solution replacement

[0232] (1) Place sample 1 (positive cells) in a small centrifuge and centrifuge at 300 g for 3 min. Carefully remove the supernatant with a pipette.

[0233] (2) Add 0.5 ml of cell dilution solution to the 1.5 ml centrifuge tube of sample 1 and pipette the cells several times. Then place the tube in a small centrifuge and centrifuge at 300 g for 3 min. Use a pipette to carefully remove the supernatant.

[0234] (3) Add 292 μl of cell dilution solution to sample 1 to obtain a sample with a cell solution concentration of 4000 cells / μl, named 221102-4000.

[0235] 2.1.3 Preparation of 1x ddPCR MIX

[0236] (1) Prepare three sets of primer-probe systems in centrifuge tubes according to the table below, mix thoroughly, and dispense into 96-well plates.

[0237] LVRV-WPRE+β-globin dual system cell preparation table

[0238]

[0239]

[0240] WPRE and RPP30 dual system cell preparation table

[0241] Combination 2 Reaction volume per well (μl) Reaction volume for 3 reaction wells (μl) ddPCR Multiplex Supermix 6.25 18.75 SamI 0.50 1.50 HaeIII 0.50 1.50 WPRE primer mix 1.25 3.75 RPP30primer mix 1.25 3.75 DTT 0.34 1.02 ddPCR Enhancer Reagent A 0.50 1.50 UtraPure Distilled Water 8.41 28.23 Total volume 20.00 75.00

[0242] LTR WPRE and β-globin dual system cell preparation table

[0243] Combination 3 Reaction volume per well (μl) Reaction volume for 3 reaction wells (μl) ddPCR Multiplex Supermix 6.25 18.75 SamI 0.50 1.50 HaeIII 0.50 1.50 LTR WPRE primer mix 1.25 3.75 β-globin primer mix 1.25 3.75 DTT 0.34 1.02 ddPCR Enhancer Reagent A 0.50 1.50 UtraPure Distilled Water 8.41 28.23 Total volume 20.00 75.00

[0244] (2) After repeatedly pipetting the diluted cells to resuspend, add them to the 1x ddPCR system, seal with sealing film, vortex slowly at the lowest speed to mix, and centrifuge at 300g for 1 min in a medium-sized centrifuge.

[0245] (3) Generate droplets: Place the mixed 1x ddPCR mix 96-well plate into the ddPCR droplet generator and generate droplets according to the default program.

[0246] 2.1.4PCR

[0247] After the droplets are generated, the 96-well plate is sealed and placed in a PCR instrument for PCR amplification. The PCR amplification procedure is shown in the following table:

[0248] ddPCR amplification procedure

[0249]

[0250]

[0251] 2.2 Analysis

[0252] The 96-well plate after PCR was placed in the droplet analyzer, and the software was set up and analyzed according to the standard operating procedures for the use and maintenance of the fully automated droplet digital PCR system (Bio-Rad QX200 AutoDG) (SOP-SZGN-0010-00). After the instrument analysis was completed, the experimental results were analyzed.

[0253] The results are as follows Figure 5-7 shown.

[0254] ddPCR test results

[0255]

[0256] 2.3 Results and Discussion

[0257] (1) From the comparison of the three groups of ddPCR result maps, it can be seen that the droplet clusters in combination 2 are more compact, which is conducive to the sorting of droplets;

[0258] (2) From the test data, it can be seen that combination 2 has the highest cell integrity and the highest cell positive rate.

[0259] Example 3 Determination of the number of cells loaded

[0260] Overview: Select positive cells MSC-FG for dilution, test with WPRE+RPP30 primer combination, and determine the optimal loading amount based on the test results of different cell numbers.

[0261] 3.1 Experimental process

[0262] 3.1.1 Preparation of PBS + 0.1% BSA diluent

[0263] Take a clean 15 ml centrifuge tube, add 1 ml of PBS + 1% BSA solution frozen at -20°C and 9 ml of PBS solution into the tube to obtain PBS + 0.1% BSA solution as a cell diluent.

[0264] 3.1.2 Sample solution replacement

[0265] (1) Pour the positive cells and negative cells into 15 ml centrifuge tubes respectively, centrifuge at 300g for 5 min in a medium-sized centrifuge, and carefully remove the supernatant.

[0266] (2) Add 1 ml of cell diluent to the centrifuge tubes of the positive cell sample and the negative cell sample respectively, pipette the cells several times, transfer the cell suspension to 1.5 ml centrifuge tubes, name them as sample 1 and sample 2 respectively, centrifuge at 300 g for 5 min in a small centrifuge, and carefully remove the supernatant.

[0267] (3) Add 0.5 ml of diluent to each of the centrifuge tubes for sample 1 and sample 2, and pipette the cells several times. Place the cells after pipetting in a small centrifuge, centrifuge at 300 g for 3 min, and carefully remove the supernatant.

[0268] 3.1.3 Sample dilution

[0269] Add 100 μl of diluent to the centrifuge tubes of sample 1 and sample 2 to obtain a cell fluid concentration of 49100 cell / μl for sample 1 and 20000 cell / μl for sample 2. Perform gradient dilution according to the cell concentration. The dilution table is as follows:

[0270] Cell sample dilution table

[0271] Sample No. Dilution formula Number of cells per μl of dilution MSC-FG-4000 8.15μl sample 1 + 91.85μl cell dilution 4000 MSC-FG-2000 30μl MSC-FG-4000 + 30μl cell dilution buffer 2000 MSC-FG-1000 10μl MSC-FG-4000 + 30μl cell dilution buffer 1000 MSC-4000 20μl sample 2 + 80μl cell dilution 4000

[0272] 3.1.4 Preparation of 1x ddPCR MIX

[0273] According to the table below, prepare the ddPCR groups except cells in centrifuge tubes, mix thoroughly and dispense into 96-well plates.

[0274] ddPCR system preparation table

[0275]

[0276]

[0277] Vortex the diluted cells to resuspend them, add them to the 1x ddPCR system, seal them with sealing film, vortex them thoroughly, and centrifuge them instantly.

[0278] 3.1.5 PCT amplification procedure

[0279] ddPCR amplification program table

[0280]

[0281] 3.1.6 Analysis

[0282] The 96-well plate after PCR was placed in the droplet analyzer, and the software was set up and analyzed according to the standard operating procedures for the use and maintenance of the fully automated droplet digital PCR system (Bio-Rad QX200 AutoDG) (SOP-SZGN-0010-00). After the instrument analysis was completed, the experimental results were analyzed.

[0283] The results are as follows Figure 8-10 As shown, specifically:

[0284] ddPCR results table

[0285]

[0286] As can be seen from the figure, the image is best when the cell count is 2000 cell / μl and 4000 cell / μl. The cell integrity results are all greater than 90%, which has little effect on the results.

[0287] Example 4: Confirmation of cell status for detection

[0288] The cell samples were encapsulated in droplets using ddPCR technology, and then PCR amplification was performed. Due to concerns about cell damage during the cell preparation and generation process, which would interfere with the accuracy of the results, the test selected fresh samples and frozen samples after resuscitation, and compared the cell integrity and results to determine whether the frozen cells could be used as sample tests.

[0289] 4.1.1 Preparation of PBS + 0.1% BSA diluent

[0290] Take a clean 15 ml centrifuge tube, add 1 ml of PBS + 1% BSA solution frozen at -20°C and 9 ml of PBS solution into the tube to obtain PBS + 0.1% BSA solution as a cell diluent.

[0291] 4.1.2 Sample solution replacement

[0292] (1) Place the 50 ml centrifuge tube containing the positive cell sample into a medium-sized centrifuge and centrifuge at 300 g for 5 min. Carefully remove the upper layer of liquid with a pipette.

[0293] (2) Add 1 ml of cell dilution solution to each sample centrifuge tube, pipette the cells several times, transfer the cell suspension to a 1.5 ml centrifuge tube and name it sample 1. Centrifuge at 300 g for 5 min in a small centrifuge and carefully remove the supernatant.

[0294] (3) Add 0.5 ml of diluent to each centrifuge tube of sample 1 and pipette the cells several times. Place the cells after pipetting in a small centrifuge and centrifuge at 300 g for 3 min. Carefully remove the supernatant.

[0295] 4.1.3 Sample dilution

[0296] Add 100 μl of diluent to the centrifuge tube of sample 1 to obtain sample 1 with a cell fluid concentration of 75,000 cells / μl, named MSC-75000. Perform gradient dilution according to the cell concentration. The dilution table is as follows.

[0297] Cell sample dilution table

[0298]

[0299] 4.1.4 Preparation of 1x ddPCR MIX

[0300] According to the table below, prepare the ddPCR groups except cells in centrifuge tubes, mix thoroughly and dispense into 96-well plates.

[0301] ddPCR MIX preparation table

[0302]

[0303]

[0304] Vortex the diluted cells to resuspend them, add them to the 1x ddPCR system, seal them with sealing film, vortex them thoroughly, and centrifuge them instantly.

[0305] 4.1.5 PCR amplification procedure

[0306] ddPCR amplification program table

[0307]

[0308] 4.1.6 Analysis

[0309] The 96-well plate after PCR was placed in the droplet analyzer, and the software was set up and analyzed according to the standard operating procedures for the use and maintenance of the fully automated droplet digital PCR system (Bio-Rad QX200 AutoDG) (SOP-SZGN-0010-00). After the instrument analysis was completed, the experimental results were analyzed.

[0310] 4.1.7 Results

[0311] Experimental results table

[0312]

[0313] There was no difference in cell integrity and positive rate when the samples were revived after freezing. The use of frozen samples had no obvious effect on sample testing.

[0314] The present invention is described in detail above in conjunction with specific implementations, exemplary examples and drawings, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting the positive rate of target genes in adipose stem cells with high expression of both GLP-1 and FGF21. The method adopts droplet digital polymerase chain reaction technology, takes the inserted WPRE gene, LTR gene or LVRV gene as the detection target, uses primers and probes of the WPRE gene, LTR gene or LVRV gene, and uses an internal reference gene as the detection target for detection.

2. The method of claim 1, wherein: The primers for the WPRE gene are selected from one or more pairs of the following primers: WPRE Primer Pair 1: Upstream primer: 5'-CCGTTGTCAGGCAACGTG-3' (WPRE-F) Downstream primer: 5′-AGCTGACAGGTGGTGGCAAT-3′ (WPRE-R); WPRE Primer Pair 2: Upstream primer: 5'-CCTTTCCGGGACTTTCGCTTT-3' Downstream primer: 5′-GCAGAATCCAGGTGGCAACA-3′; WPRE Primer Pair 3: Upstream primer: 5'-ACTGTGTTTGCTGACGCAAC-3' Downstream primer: 5′-AAGGAAGGTCCGCTGGATTG-3′; WPRE Primer Pair 4: Upstream primer: 5'-CAGCTCCTTTCCGGGACTTT-3' Downstream primer: 5′-GGACGATGATTTCCCCGACA-3′; WPRE Primer Pair 5: Upstream primer: 5'-CACCACCTGTCAGCTCCTTT-3' Downstream primer: 5′-ACGGAATTGTCAGTGCCCAA-3′; WPRE Primer Pair 6: Upstream primer: 5'-TTGGGCACTGACAATTCCGT-3' Downstream primer: 5′-CCGAAGGGACGTAGCAGAAG-3′; WPRE Primer Pair 7: Upstream primer: 5'-CGCTTTCCCCCTCCCTATTG-3' Downstream primer: 5′-CACCACGGAATTGTCAGTGC-3′; WPRE Primer Pair 8: Upstream primer: 5'-CGCTATGTGGATACGCTGCT-3' Downstream primer: 5′-GTTGCGTCAGCAAACACAGT-3′; WPRE Primer Pair 9: Upstream primer: 5'-CTTTCCCCCTCCCTATTGCC-3' Downstream primer: 5′-CGGAATTGTCAGTGCCCAAC-3′; WPRE Primer Pair 10: Upstream primer: 5'-CTTCTGCTACGTCCCTTCGG-3' Downstream primer: 5′-AAGGGAGATCCGACTCGTCT-3′; WPRE Primer Pair 11: Upstream primer: 5'-CAATCCAGCGGACCTTCCTT-3' Downstream primer: 5'-AGATCCGACTCGTCTGAGGG-3'; or WPRE Primer Pair 12: Upstream primer: 5'-GCACTGACAATTCCGTGGTG-3' Downstream primer: 5′-GAAGGTCCGCTGGATTGAGG-3′; The primers for the LTR gene are the following primers: Upstream primer: 5'-TGTGTGCCCGTCTGTTGTGT-3' (LTR WPRE-F) Downstream primer: 5'GAGTCCTGCGTCGAGAGAGC-3' (LTR WPRE-R).

3. The method of claim 2, wherein: The WPRE probe uses a probe selected from the following: 5'-FAM-TGCTGACGCAACCCCCACTGGT-BHQ1-3'(WPRE-P); 5'-FAM-ACTCATCGCCGCCTGCCTTGCC-BHQ1-3'; 5'-FAM-ATTGCCACCACCTGTCAGCTCCTTT-BHQ1-3'; 5'-FAM-TGCCACGGCGGAACTCATCGCCGCC-BHQ1-3'; 5'-FAM-CGGAACTCATCGCCGCCTGCCTTGC-BHQ1-3'; 5'-FAM-CTTTCCTTGGCTGCTCGCCTGTGTT-BHQ1-3'; 5'-FAM-CGCCGCCTGCCTTGCCCGCTGCTGG-BHQ1-3'; 5'-FAM-TGCCACCACCTGTCAGCTCCTTCC-BHQ1-3'; 5'-FAM-GCCTGCCTTGCCCGCTGCTGGACAG-BHQ1-3'; 5'-FAM-TCCTTCCCGCGGCCTGCTGCCGGCT-BHQ1-3'; 5'-FAM-TGCTGCCGGCTCTGCGGCCTCTTCC-BHQ1-3'; or 5'-FAM-CTCGCCTGTGTTGCCACCTGGATTC-BHQ1-3'; the LTR probe used was a probe selected from the following: 5'-FAM-GTGACTCTGGTAACTAGAGATCCCT-BHQ1-3'.

4. The method of claim 1, wherein: As the internal reference gene, ribonuclease P protein subunit p30 (RPP30) is used, and its primers are selected from one or more pairs of the following primers: RPP30 Primer Pair 1: Upstream primer: 5'-AGTGCATGCTTATCTCTGACAG-3' (RPP30-F) Downstream primer: 5′-GCAGGGCTATAGACAAGTTCA-3′ (RPP30-R); RPP30 Primer Pair 2: Upstream primer: 5'-ATTCGGCACGAGGTGGG-3' Downstream primer: 5′-GAGCCTTCAGGTCAGAACCC-3′; RPP30 Primer Pair 3: Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3' Downstream primer: 5′-CCTTCTGATGGCCGAGGTTT-3′; RPP30 Primer Pair 4: Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3' Downstream primer: 5′-GAGCCTTCAGGTCAGAACCC-3′; RPP30 Primer Pair 5: Upstream primer: 5'-AATTCGGCACGAGGTGGG-3' Downstream primer: 5′-GCAAGCCTAGATTTGCCACG-3′; RPP30 Primer Pair 6: Upstream primer: 5'-GGCACGAGGTGGGACTTC-3' Downstream primer: 5′-GCAAGCCTAGATTTGCCACG-3′; RPP30 Primer Pair 7: Upstream primer: 5'-AATTCGGCACGAGGTGGG-3' Downstream primer: 5′-TGAATAGCCAAGGTGAGCGG-3′; RPP30 Primer Pair 8: Upstream primer: 5'-GGCACGAGGTGGGACTTC-3' Downstream primer: 5′-TGAATAGCCAAGGTGAGCGG-3′; RPP30 Primer Pair 9: Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3' Downstream primer: 5′-GCAAGCCTAGATTTGCCACG-3′; RPP30 Primer Pair 10: Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3' Downstream primer: 5′-TGAATAGCCAAGGTGAGCGG-3′; RPP30 Primer Pair 11: Upstream primer: 5'-GAATTCGGCACGAGGTGGG-3' Downstream primer: 5′-GGCCGAGGTTTCTTCACTGT′-3′.

5. The method of claim 4, wherein: An RPP30 probe selected from the following was used: 5'-VIC-TTTCCTGTGAAGGCGATTGACCGA-BHQ1-3'(RPP30-P); 5'-VIC-TGGACCTGCGAGCGGGTTCTGACC-BHQ1-3'; 5'-VIC-TGAGAGCAACTTCTTCAAGGGCCC-BHQ1-3'; 5'-VIC-TAACTGTAACAGAGAAACTACCAT-BHQ1-3'; 5'-VIC-GAGAAGGTATACAATTTCCAGTGC-BHQ1-3'; 5'-VIC-CTATAGCCCTGCTATCAAAGACTC-BHQ1-3'; 5'-VIC-ATATCGTTGACTTTAAGGAAAAGA-BHQ1-3'; 5'-VIC-TTAATGTGGCGATTGACCGAGGCC-BHQ1-3'; 5'-VIC-TTGATGCAAATCTGCAAAGGAAAG-BHQ1-3'; 5'-VIC-CGGCCATCAGAAGGAGATGAAGAT-BHQ1-3'; 5'-VIC-TTGTCTATAGCCCTGCTATCAAAG-BHQ1-3'.

6. The method of claim 1, wherein: The albumin gene (ALB) is used as an internal reference gene, and its primers are selected from one or more pairs of the following primers: ALB Primer Pair 1: Upstream primer: 5'-TGAAACATACGTTCCCAAAGAGTTT-3' (ALB-F) Downstream primer: 5′-CTCTCCTTCTCAGAAAGTGTGCATAT-3′ (ALB-R); ALB Primer Pair 2: Upstream primer: 5'-TGCTGCAAGGCTGACGATAA-3' Downstream primer: 5′-AGCCCTAGCCTAACCAAACA-3′; ALB Primer Pair 3: Upstream primer: 5'-GTGCACTTGTTGAGCTCGTG-3' Downstream primer: 5′-TCCCTAAGCCCTAGCCTAACC-3′; ALB Primer Pair 4: Upstream primer: 5'-AAACACAAGCCCAAGGCAAC-3' Downstream primer: 5′-CCCTAAGCCCTAGCCTAACCA-3′; ALB Primer Pair 5: Upstream primer: 5'-TGCACTTGTTGAGCTCGTGA-3' Downstream primer: 5′-CCTAAGCCCTAGCCTAACCA-3′; ALB Primer Pair 6: Upstream primer: 5'-GTGAAACACAAGCCCAAGGC-3' Downstream primer: 5′-AGCCCTAGCCTAACCAAACAT-3′; ALB Primer Pair 7: Upstream primer: 5'-GCCGAGGAGGTACTACAGTT-3' Downstream primer: 5′-TCCCACATGTACAAAGCCTCC-3′; ALB Primer Pair 8: Upstream primer: 5'-GAAACACAAGCCCAAGGCAA-3' Downstream primer: 5′-CCCTAGCCTAACCAAACATGC-3′; ALB Primer Pair 9: Upstream primer: 5'-GGTTAGGCTAGGGCTTAGGG-3' Downstream primer: 5′-TGGCACAATAGAGCAGGAGA-3′; ALB Primer Pair 10: Upstream primer: 5'-GAGAAGTGCTGCAAGGCTGA-3' Downstream primer: 5′-CCTAAGCCCTAGCCTAACCAA-3′; ALB Primer Pair 11: Upstream primer: 5'-GCTGCAAGGCTGACGATAAG-3' Downstream primer: 5′-GCCCTAGCCTAACCAAACAT-3′.

7. The method of claim 6, wherein: An ALB probe selected from the following was used: 5'-HEX-TGCTGAAACATTCACCTTCCATGCAGA-BHQ1-3'(ALB-P); 5'-HEX-TAGTTCTCTTCATTTTAATATGTCC-BHQ1-3'; 5'-HEX-AGCTGTTATGGATGATTTCGCAGC-BHQ1-3'; 5'-HEX-ATGGATGATTTCGCAGCTTTTGTA-BHQ1-3'; 5'-HEX-GAGGAGGTACTACAGTTCTCTTCA-BHQ1-3'; 5'-HEX-TGTAGAGAAGTGCTGCAAGGCTGA-BHQ1-3'; 5'-HEX-GGACAGGGATCTTATTTTACAAAC-BHQ1-3'; 5'-HEX-TTTGTAAGGCCTGAAATATTTTGA-BHQ1-3'; 5'-HEX-TGTACATGTGGGACAGGGATCTTA-BHQ1-3'; 5'-HEX-TGCTTATGAATATTAATAGGAATA-BHQ1-3'; 5'-HEX-TGGCCTGACAGTAACTTTTTTTTA-BHQ1-3'.

8. The method of claim 1, wherein: The WPRE gene was used as the detection target, and the primer for the WPRE gene was WPRE primer pair 1: Upstream primer: 5'-CCGTTGTCAGGCAACGTG-3' (WPRE-F) Downstream primer: 5'-AGCTGACAGGTGGTGGCAAT-3' (WPRE-R) The probes are: 5'FAM-TGCTGACGCAACCCCCACTGGT-BHQ1-3'(WPRE-P); As an internal reference gene, RNA P protein subunit p30 was used, and the primers thereof were RPP30 primer pair 1: Upstream primer: 5'-AGTGCATGCTTATCTCTGACAG-3' (RPP30-F) Downstream primer: 5'-GCAGGGCTATAGACAAGTTCA-3' (RPP30-R) The probes are: 5'-VIC-TTTCCTGTGAAGGCGATTGACCGA-BHQ1-3'(RPP30-P).

9. The method of claim 8, wherein: The cell count during detection was 2000-4000 cell / μl.

10. The method of claim 8, wherein: The cells tested were frozen and then revived.

Images