CN114277190B

CN114277190B - Specific DNA fragment, primer, kit and detection method for detecting exogenous gene residues in hiPSC

Info

Publication number: CN114277190B
Application number: CN202111668786.0A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Nuwacell Ltd
Current assignee: Anhui Zhongsheng Traceability Biotechnology Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2024-07-23
Anticipated expiration: 2041-12-31
Also published as: CN114277190A

Abstract

The present invention provides a specific DNA fragment, a primer set, a kit and a detection method for detecting exogenous gene residues in hiPSC, and the present invention belongs to the field of stem cell technology. The present invention uses the free vector sequence used to induce the preparation of hiPSC to screen specific DNA sequences, and designs detection primers to respectively design the specific DNA sequences obtained by screening, so as to obtain primer pairs with good amplification effect and high detection sensitivity. The present invention detects the residual exogenous genes in hiPSC based on the qPCR method, does not need to extract genomic DNA, and obtains the genome by directly lysing cells. The whole process can be completed in about 5 to 6 hours, which is suitable for hiPSC product quality control, more economical, more time-saving, and more guaranteed for monitoring the safety, economy and productivity of clinical-grade hiPSC.

Description

Specific DNA fragment, primer, kit and detection method for detecting exogenous gene residues in hiPSC

Technical Field

The invention belongs to the technical field of stem cells, and particularly relates to a specific DNA fragment, a primer group, a kit and a detection method for detecting exogenous gene residues in hiPSC.

Background

Human pluripotent stem cells (humanpluripotent stem cell, hpscs), including human embryonic stem cells (human embryonic stem cell, hESC) and human induced pluripotent stem cells (human induced pluripotent stem cell, hiPSC), are a class of stem cells that can proliferate indefinitely in vitro and have the potential to differentiate into virtually all functional cells of an adult. Based on the two characteristics, the hPSC has wide clinical application prospect in the treatment of difficult and complicated diseases for which no effective therapy exists at present. Currently, with the rapid development of stem cell technology, more and more hPSC-derived functional cell products have been put into clinical trials in various countries.

HiPSC is a pluripotent stem cell obtained by reprogramming by expressing a specific gene or a specific gene product in a differentiated somatic cell, which not only avoids the social ethical problems involved in embryo-derived hESC, but also has rich sources and unrestricted genetic background, and is expected to overcome the immune-typing problems faced by cell transplantation. Based on these advantages of hipscs, in recent years, there have been a variety of hiPSC-derived functional cell products that have entered clinical use. Early iPSC reprogramming uses a retroviral/lentiviral vector to express a foreign gene. The vector can realize long-term stable expression of exogenous genes, so that higher reprogramming efficiency is obtained, but insertional gene mutation is caused, and the function and the tumorigenicity of iPSC are seriously affected by exogenous gene expression. Hipscs used to prepare clinical grade functional cell products should be free of foreign DNA residues, so a reprogramming method that preferentially selects non-integrated reprogramming systems is needed. A common non-integrated hiPSC reprogramming system is often constructed based on an episomal DNA plasmid vector of OriP/EBNA 1. The two critical sequences OriP/EBNA1 of this vector are derived from the double stranded DNA herpes virus (EBV). Approximately 90% of adults worldwide are infected with EBV, which, during latency, is episomally isolated from the cell genome in the form of circular DNA plasmids, oriP and EBNA1 being viral origins ^[1] required for stable maintenance of their episomal unique. The OriP/EBNA1 vector is capable of establishing stable episomes (about 5 to 50 plasmids/cell) in a portion of cells after transfection of the cells, thereby stably expressing the exogenous gene to ensure success of reprogramming. The defect of replication and distribution of the OriP/EBNA1 vector resulted in about 5% of the plasmid lost per cell division, thus facilitating the screening of traceless ipscs ^[2]. In 2009, yu et al obtained a human traceless iPSC ^[3] for the first time using the OriP/EBNA1 vector, and the combination with small molecules improved the reprogramming Cheng Xiaolv ^[4] of this system significantly. compared with other methods, the OriP/EBNA1 vector has a plurality of advantages: the stable free state can be established in various cells, so that the method is not limited by the cell types; the plasmid can be prepared by a conventional method, and viral packaging is not required; only one transfection is needed, and the operation is simple; the cell with free state is established to contain only a small amount of vector, so that the risk of genome integration is reduced; due to the defects of vector replication and distribution and silencing of viral promoters expressing EBNA1 in PSCs, traceless iPSCs can be obtained by conventional subculture without the need for cumbersome single cell cloning screening work. This method is therefore more common in the preparation of clinical grade ipscs.

Although OriP/EBNA1 vectors can be lost after multiple passages through hipscs, there is still the potential for incomplete rejection and the risk of reprogramming plasmids remaining within hipscs, thereby increasing the safety risk of tumorigenicity of the cell product. In international and domestic research, the strict requirements on exogenous foreign matters (exogenous genes) are seen in detail in the guidelines of cell therapy product research and evaluation technology in China. Although the preparation methods of hiPSC are various, the quality standard of the product is strictly controlled according to the control principle of the exogenous gene, so that when the hiPSC cells are induced to be formed by the exogenous gene, a detection method which is suitable for industrial scale production and has high sensitivity is needed to detect the residue of the exogenous gene in the hiPSC cell products so as to ensure that the products are in line with clinical use and downstream application.

In addition to hiPSC obtained by re-editing induction using episomal vector based on OriP/EBNA1, S/MAR system plasmid vector, SV40 system plasmid vector and S/MAR system plasmid vector can be used for construction.

Introduction of S/MAR System plasmid vector: in the nucleus of eukaryotes, the genome is anchored to the nuclear framework network by the nuclear framework attachment region (SAR) or nuclear Matrix Attachment Region (MAR) of DNA (abbreviated S/MAR). S/MAR has certain characteristics and diversity, and is considered to be involved in various nuclear biochemical processes such as DNA replication regulation and transcription regulation. By recombination, transfection of genes or transgenic animals and plants with S/MARs on one or both sides of the gene of interest, it was found that the gene expression after integration can sometimes be enhanced several-fold, even ten-thousand-fold, and/or exhibit a site-independent effect. Some studies have also reported that S/MARs can overcome the problem of transgene silencing during passage. These effects make S/MAR a useful cis-regulatory element in genetic engineering.

The SV40 system episomal vector belongs to an SV40 DNA-plasmid DNA hybrid vector, and the recombinant SV40DNA molecule has to be packaged to have infectivity, so that the inserted exogenous DNA fragment cannot be too large. In order to maximize the SV40 loading, some gene segments must be deleted, so that the recombinant SV40 molecule must co-infect recipient cells with wild-type virus to form an infectious recombinant viral particle. The S/MAR system plasmid vector can efficiently express foreign genes, but has small loading capacity.

The BKV system episomal vector is a novel episomal vector based on human papillomavirus sequences. Human papillomaviruses (BKV) belong to the genus papillomaviruses of the family polyomaviridae, whose genome is double-stranded DNA, and which, after infection of host cells, do not integrate and autonomously replicate independent of the chromosome, up to 500 copies per host cell. Genes encoding viral core and coat proteins are deleted and spliced with E.coli plasmids to form shuttle vectors which do not integrate and are also unable to form mature virion lytic recipient cells. Installing cytochrome P45 dioxan mediated enhancer DRE to control mouse mammary cancer promoter MMTP, and driving the expression of exogenous gene.

The existing method for detecting the exogenous gene residue of the hiPSC cell product comprises a conventional PCR method and a real-time fluorescence quantitative PCR method (qPCR), wherein gel electrophoresis analysis is carried out after the conventional PCR method is carried out, and the method is inaccurate, has too large error and cannot meet the requirement of high-accuracy detection. qPCR is a common method, but the sensitivity, limitation and stability of detecting hiPSC exogenous gene residues by the current qPCR method are affected by various factors (such as the use amount of reaction system components, reaction conditions and the like in the qPCR method), and the requirement of detecting the hiPSC exogenous gene residues with high sensitivity cannot be met. In addition, in the existing detection method, the genomic DNA of the cells needs to be extracted, the operation is troublesome, the detection process is long, the cost is high, and the hiPSC cell quality control cannot be effectively monitored. Therefore, a stable, highly sensitive, more economical and time-saving detection method is needed, and the safety, economical efficiency and productivity of monitoring clinical hiPSC are more ensured. Only a safe hiPSC cell bank was established to better supply downstream applications, which is also a constant pursuit and problem to be solved by the cell therapy field for monitoring cell quality.

Reference is made to:

[1]Yates JL,Warren N,Sugden B.Stable replication of plasmids derived from Epstein-Barrvirus in various mammalian cells.Nature,1985,313:812-815.

[2]Nanbo A,Sugden A,Sugden B.The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells.EMBO J,2007,26:4252-4262.

[3]Yu J,Hu K,Smuga-Otto K,et al.Human induced pluripotent stem cells free of vector andtransgene sequences.Science,2009,324:797-801.

[4]Yu J,Chau KF,Vodyanik MA,et al.Efficient feeder-free episomal reprogramming with small molecules.PLoS One,2011,6:e17557.

Disclosure of Invention

In view of the above, the present invention aims to provide a specific DNA fragment, a primer set, a kit and a detection method for detecting foreign gene residues in hiPSC, which have the characteristic of high sensitivity.

The present invention provides a specific DNA fragment for detecting foreign gene residues in hiPSCs, which is derived from episomal vectors used in the induction preparation of hiPSCs.

Preferably, the episomal vector comprises one or more of the following episomal vectors: episomal vectors based on one or more of OriP/EBNA1, S/MAR, SV40, BKV, BPV-1.

Preferably, episomal vectors based on OriP/EBNA1 include pCEP-EGFP, nEP4-E-EGFP, PMEP4-C-EGFP and PKEP-E-EGFP.

Preferably, when the episomal vector is an OriP/EBNA 1-based episomal vector, the specific DNA fragment is selected from at least one of the following sequences:

A. Intercepting truncated DNA sequence with the length of SV40 polyA signal area being more than 70 bp;

B. intercepting truncated DNA sequences with the total length of more than 70bp of the segment region of the OriP sequence;

C. Intercepting truncated DNA sequences with the total length of more than 70bp of the EBNA1 sequence fragment region;

D. Truncated DNA sequences of more than 70bp of the whole length of a bacterial source DNA fragment region on the plasmid are intercepted.

Preferably, the SV40 polyA signal region comprises at least one of the following DNA fragments:

1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1;

2) A DNA fragment complementary to SEQ ID NO. 1;

3) A DNA fragment formed by adding, deleting and inserting one or more nucleotide sequences on the basis of SEQ ID NO. 1;

preferably, the EBNA1 sequence fragment region comprises at least one of the following DNA fragments:

1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 2;

2) A DNA fragment complementary to SEQ ID NO. 2;

3) A DNA fragment formed by adding, deleting and inserting one or more nucleotide sequences on the basis of SEQ ID NO. 2;

preferably, the OriP sequence fragment region comprises at least one of the following DNA fragments:

A. the nucleotide sequence is shown in SEQ ID NO: 3;

B. And SEQ ID NO: 3a complementary DNA fragment;

C. in SEQ ID NO:3 by adding, deleting and inserting one or more nucleotide sequences;

Preferably, the bacterial derived DNA fragment region on the plasmid comprises at least one of the following DNA fragments:

1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 27;

2) A DNA fragment complementary to SEQ ID NO. 27;

3) A DNA fragment formed by adding, deleting and inserting one or more nucleotide sequences based on SEQ ID NO. 27;

Preferably, the specific DNA fragment comprises one or more of the following DNA fragments: EBNA1 truncated fragments, oriP truncated fragments, SV40 truncated fragments and DNA fragments of bacterial origin on plasmids;

Preferably, the truncated EBNA1 segment comprises EBNA1-1 with a nucleotide sequence shown as SEQ ID NO. 18, EBNA1-2 with a nucleotide sequence shown as SEQ ID NO. 19 and/or EBNA1-3 with a nucleotide sequence shown as SEQ ID NO. 20;

The truncated OriP fragment comprises OriP-1 with a nucleotide sequence shown as SEQ ID NO. 21, oriP-2 with a nucleotide sequence shown as SEQ ID NO. 22 and OriP-3 with a nucleotide sequence shown as SEQ ID NO. 23;

the nucleotide sequence of the SV40 truncated fragment is shown as SEQ ID NO. 24.

The invention provides a primer for detecting exogenous gene residues in hiPSC, which comprises a primer for specifically detecting the specific DNA fragment;

Preferably, the primer comprises one or more pairs of primers as follows: a primer pair for specifically detecting the truncated EBNA1 fragment, a primer pair for specifically detecting the truncated OriP fragment, a primer pair for specifically detecting the truncated SV40 fragment and a primer pair for specifically detecting the truncated bacterial-derived DNA fragment on a plasmid;

preferably, the primer pair for specifically detecting the truncated segment of the EBNA1 comprises EBNA1-1-F/EBNA1-1-R, EBNA1-2-F/EBNA1-2-R, EBNA1-3-F/EBNA1-3-R;

the nucleotide sequence of EBNA1-1-F is shown as SEQ ID NO:4 is shown in the figure;

the nucleotide sequence of EBNA1-1-R is shown as SEQ ID NO:5 is shown in the figure;

the nucleotide sequence of EBNA1-2-F is shown as SEQ ID NO:6 is shown in the figure;

the nucleotide sequence of EBNA1-2-R is shown as SEQ ID NO: shown in figure 7;

The nucleotide sequence of EBNA1-3-F is shown as SEQ ID NO: shown as 8;

the nucleotide sequence of EBNA1-3-R is shown as SEQ ID NO: shown as 9;

preferably, the primer pair for specifically detecting the truncated fragment of the OriP comprises OriP-1-F/OriP-1-R, oriP-2-F/OriP-2-R, oriP-3-F/OriP-3-R;

the nucleotide sequence of OriP-1-F is shown as SEQ ID NO:10 is shown in the figure;

the nucleotide sequence of the OriP-1-R is shown as SEQ ID NO: 11;

The nucleotide sequence of OriP-2-F is shown as SEQ ID NO: shown at 12;

the nucleotide sequence of the OriP-2-R is shown as SEQ ID NO: 13;

the nucleotide sequence of OriP-3-F is shown as SEQ ID NO: 14;

the nucleotide sequence of the OriP-3-R is shown as SEQ ID NO: 15;

Preferably, the primer pair for specifically detecting the SV40 truncated fragment comprises SV40-F/SV40-R;

The nucleotide sequence of the SV40-F is shown as SEQ ID NO:16 is shown as 16

The nucleotide sequence of the SV40-R is shown as SEQ ID NO: shown at 17.

The invention provides a kit for detecting exogenous gene residues in hiPSC based on qPCR technology, which comprises the primer.

The invention provides application of the specific DNA fragment, the primer and the kit in detecting exogenous gene residues of clinical-grade hiPSC.

The invention provides a method for detecting exogenous gene residues of clinical-grade hiPSC based on qPCR technology, which comprises the following steps:

1) Screening a specific DNA fragment carried by the episomal vector as target DNA;

2) Designing a primer according to the target DNA in the step 1);

3) Taking the episomal vector in the step 1) as a plasmid standard substance, and mixing the hESC and the plasmid standard substance according to different proportions to prepare a positive gradient standard substance;

4) And (3) performing cell lysis on the hiPSC to be detected to obtain a sample to be detected, performing qPCR detection on the sample to be detected, the positive gradient standard substance and the primer, and judging the residual quantity of the exogenous gene of the hiPSC in the sample to be detected according to the detection lower limit of the PCR of the positive gradient standard substance and the detection value of the hiPSC product in the corresponding sample to be detected.

Preferably, the reaction system of qPCR detection is as follows:

5 to 35 percent of 10 multiplied by amplification buffer solution, 5 to 45 percent of 10 multiplied by enhancement buffer solution, 0.01 to 10 percent of dNTPs, 0.1 to 6mM MgSO ₄, 0.1 to 25 percent of (1.25 to 10) multiplied by SYBR green I, 0.01 to 2.5 percent of forward primer, 0.5 to 10 percent of reverse primer, 0.01 to 5.5 percent of Platinum pfx DNA polymerase and 5 to 50 percent of template DNA;

The forward primer and the reverse primer are the primers.

Preferably, the reaction procedure for qPCR detection is as follows:

94 ℃ for 5min; 15sec at 94 ℃, 30sec at 58 ℃, 30sec at 68 ℃ and 35-55 cycles; 95℃10sec,65℃1min,97℃1min.

Preferably, the concentration of proteinase K is 1-50. Mu.g/ml when the cells in step 1) are lysed;

Preferably, the number of cells upon cell lysis is 1.0X10 ⁶～4.0×10⁶/10. Mu.l.

The invention provides a specific DNA fragment for detecting exogenous gene residues in human induced pluripotent stem cells and a corresponding amplification primer thereof, wherein the primers are designed for the EBNA sequence fragment region, the OriP sequence fragment region, the SV40 truncated fragment and the DNA sequences of different sites of a bacterial source DNA sequence fragment region on plasmids respectively through screening candidate target DNA sequences, and a pair of primers is designed for the SV40 truncated fragment.

The invention provides a method for detecting exogenous gene residues of clinical-grade hiPSC based on qPCR technology, which has the following advantages:

(1) The specific DNA sequence of the plasmid vector or the fragment thereof is used as a detection marker gene, so that the residual quantity of the hiPSC exogenous gene can be detected, the specificity is strong, and the quality safety of the hiPSC product is greatly improved.

(2) The detection system and the detection method are more accurate, high in sensitivity, higher in stability, short in detection time and capable of being completed in about 5-6 hours, and the sensitivity can reach 0.1% which is far higher than the current international standard.

(3) When the method is used for detection, operations such as extracting genome DNA in a traditional experiment are not needed, and the genome and qPCR reagent preparation is only needed by a direct cell lysis mode, so that the quality control operation is simplified, the cost is reduced, and the method is more suitable for industrially preparing clinical-grade hiPSC.

(4) In the establishment method, proteinase K is added after cells are lysed to play a role in degrading or inhibiting DNase, so that the concentration of low-copy plasmids is protected, and the method is more accurate, reliable and high in sensitivity.

Drawings

FIG. 1 is a block diagram of a positive plasmid pCEP 4-EGFP;

FIG. 2 is a block diagram of positive plasmid nEP, 4-E-EGFP;

FIG. 3 is a block diagram of the positive plasmid PMEP 4-C-EGFP;

FIG. 4 is a block diagram of positive plasmid PKEP, 4-E-EGFP.

Detailed Description

In the present invention, the episomal vector preferably comprises one or more of the following episomal vectors: episomal vectors based on one or more of OriP/EBNA1, S/MAR, SV40, BKV, BPV-1. In the present invention, the content of specific DNA fragments is specifically described by taking an OriP/EBNA 1-based episomal vector as an example.

In the present invention, the episomal vector based on OriP/EBNA1 preferably comprises pCEP-EGFP, nEP4-E-EGFP, PMEP4-C-EGFP and PKEP-E-EGFP, and the structure of the plasmids corresponds to FIGS. 1 to 4, respectively. The specific DNA fragment is described in detail below with plasmid pCEP4-EGFP as an example. When the episomal vector is pCEP-EGFP, the specific DNA fragment is selected from at least one of the following sequences:

A. truncated DNA sequences with the total length of more than 70bp of SV40 polyA signal region genes are intercepted;

Wherein, the region shown by SV40 in FIG. 1 is an SV40 polyA signal region, and the region shown by OriP and the downstream blank region in FIG. 1 are collectively called an OriP sequence fragment region; the region indicated by EBNA1 and the downstream blank region in fig. 1 are collectively referred to as an EBNA1 sequence segment region.

Wherein the SV40 polyA signal region preferably comprises at least one of the following DNA fragments: 1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 1;

2) A DNA fragment complementary to SEQ ID NO. 1;

The EBNA1 sequence fragment region preferably comprises at least one of the following DNA fragments:

1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 2;

2) A DNA fragment complementary to SEQ ID NO. 2;

the OriP sequence fragment region preferably comprises at least one of the following DNA fragments:

A. the nucleotide sequence is shown in SEQ ID NO: 3;

B. And SEQ ID NO: 3a complementary DNA fragment;

the bacterial derived DNA fragment region on the plasmid preferably comprises at least one of the following DNA fragments:

1) A DNA fragment with a nucleotide sequence shown as SEQ ID NO. 27;

2) A DNA fragment complementary to SEQ ID NO. 27;

3) DNA fragments formed by adding, deleting and inserting one or more nucleotide sequences on the basis of SEQ ID NO. 27.

In the embodiment of the invention, the detection method of the specific DNA fragment is specifically described by using the EBNA1 truncated fragment, the OriP truncated fragment, the SV40 truncated fragment and the bacterial source DNA fragment on the plasmid. The EBNA1 truncated segment comprises EBNA1-1 with a nucleotide sequence shown as SEQ ID NO. 18, EBNA1-2 with a nucleotide sequence shown as SEQ ID NO. 19 and/or EBNA1-3 with a nucleotide sequence shown as SEQ ID NO. 20. The truncated OriP fragment preferably comprises OriP-1 with a nucleotide sequence shown as SEQ ID NO. 21, oriP-2 with a nucleotide sequence shown as SEQ ID NO. 22 and OriP-3 with a nucleotide sequence shown as SEQ ID NO. 23. The nucleotide sequence of the SV40 truncated fragment is shown as SEQ ID NO. 24.

In the invention, the truncated segment of the candidate DNA segment EBNA1 is used as an amplification sequence, and then a primer is designed according to the amplification sequence, so that the result shows that the EBNA1 has higher amplification effect and higher detection sensitivity in the aspect of detecting exogenous gene residues in human induced pluripotent stem cells.

In the invention, the candidate DNA fragment OriP partial sequence is used as an amplification sequence, and then a primer is designed according to the amplification sequence, so that the result shows that the OriP has higher amplification effect and higher detection sensitivity in the aspect of detecting exogenous gene residues in human induced pluripotent stem cells. Meanwhile, the invention also takes the DNA fragment of the SV40 polyA signal region as a detection target point, and the result shows that the amplification effect is more ideal, and the detection sensitivity is not very low compared with the truncated fragments of OriP and EBNA 1.

In the invention, the candidate SV40 truncated fragment is taken as an amplification sequence, and then a primer is designed according to the amplification sequence, and the result shows that the SV40 signal region has ideal amplification effect and detection sensitivity in the aspect of detecting exogenous gene residues in human induced pluripotent stem cells.

The invention provides a primer for detecting exogenous gene residues in hiPSC, which comprises a primer for specifically detecting a specific DNA fragment in claim 1;

In the present invention, the primer preferably includes one or several pairs of primers of: a primer pair for specifically detecting the truncated segment of EBNA1, a primer pair for specifically detecting the truncated segment of OriP and a primer pair for specifically detecting the truncated segment of SV 40.

In the present invention, the primer pair for specifically detecting EBNA1 preferably includes EBNA1-1-F/EBNA1-1-R, EBNA1-2-F/EBNA1-2-R, EBNA1-3-F/EBNA1-3-R. The EBNA1-1-F/EBNA1-1-R amplification product is EBNA1-1. The EBNA1-2-F/EBNA1-2-R amplification product is EBNA1-2. The EBNA1-3-F/EBNA1-3-R amplification product is EBNA1-3. The nucleotide sequence of EBNA1-1-F is shown as SEQ ID NO:4 is shown in the figure; the nucleotide sequence of EBNA1-1-R is shown as SEQ ID NO: shown at 5. The nucleotide sequence of EBNA1-2-F is shown as SEQ ID NO:6 is shown in the figure; the nucleotide sequence of EBNA1-2-R is shown as SEQ ID NO: shown at 7. The nucleotide sequence of EBNA1-3-F is shown as SEQ ID NO: shown as 8; the nucleotide sequence of EBNA1-3-R is shown as SEQ ID NO: shown at 9.

In the present invention, the primer pair for specifically detecting OriP preferably includes OriP-1-F/OriP-1-R, oriP-2-F/OriP-2-R, oriP-3-F/OriP-3-R. The amplified product of OriP-1-F/OriP-1-R is OriP-1. The amplified product of OriP-2-F/OriP-2-R is OriP-2. The amplified product of OriP-3-F/OriP-3-R is OriP-3. The nucleotide sequence of OriP-1-F is shown as SEQ ID NO:10 is shown in the figure; the nucleotide sequence of the OriP-1-R is shown as SEQ ID NO: 11; the nucleotide sequence of OriP-2-F is shown as SEQ ID NO: shown at 12; the nucleotide sequence of the OriP-2-R is shown as SEQ ID NO: 13; the nucleotide sequence of OriP-3-F is shown as SEQ ID NO: 14; the nucleotide sequence of the OriP-3-R is shown as SEQ ID NO: 15. Primer pairs for specific detection of SV40 truncated fragments include SV40-F/SV40-R. The amplified product of SV40-F/SV40-R was the SV40 fragment (SEQ ID NO: 24). The nucleotide sequence of the SV40-F is shown as SEQ ID NO:16, the nucleotide sequence of the SV40-R is shown as SEQ ID NO: shown at 17.

The source of the primer is not particularly limited in the present invention, and primer synthesis methods well known in the art may be employed.

The invention provides a kit for detecting exogenous gene residues in induced pluripotent stem cells based on qPCR (quantitative polymerase chain reaction) technical personnel, which comprises the primer group.

In the present invention, reagents for qPCR detection, such as amplification buffer, enhancement buffer, dNTPs, SYBR Green I, mgSO ₄, platinumpfx DNA polymerase, and the like, are preferably further included.

2) Designing a primer according to the target DNA in the step 1);

The kind of clinical-grade hiPSC is not particularly limited in the present invention, and clinical-grade hiPSC known in the art may be used. In the present invention, the method is applicable to hipscs described in CN201711462604.8, CN201810137319.7, CN201810252408.6, CN201810400878.2, CN202010313083.5 patents. The method disclosed by the invention is not only suitable for detecting the hiPSC obtained by the preparation method, but also suitable for detecting the hiPSC obtained by other methods in the field. The hiPSC experiments of different generations show that the seed cell library (seed), the main cell library (MASTER CELL bank/MCB) and the working cell library (working cell Bank/WCB) can all be used for achieving the purpose of exogenous gene detection by adopting the method.

In the present invention, mixing with hescs in different proportions is preferably mixing with plasmid copy number as a percentage of cell number. The plasmid copy number and cell number percentages are preferably 0.01%, 0.1%, 1%, 10% and 100%.

In order to achieve the objective of detecting foreign gene residues of clinical-grade hipscs, specific DNA sequences should be specific for foreign genes, whereas specific DNA fragments not expressed by clinical-grade hipscs. In the specific DNA fragment provided by the invention, the exogenous gene fragment shared by all plasmids used in the reprogramming process contains OriP truncated fragment and EBNA1 truncated fragment which are derived from herpesvirus sequences and are not expressed in hiPSC.

In the present invention, the concentration of proteinase K at the time of cell lysis is preferably 1 to 50. Mu.g/mL, more preferably 1 to 20. Mu.g/mL, most preferably 1. Mu.g/mL. The proteinase K was 1-50. Mu.g/mL for cell lysis and the detection sensitivity reached 0.1% with the best results when treated with proteinase K at 1. Mu.g/mL.

In the present invention, the cell density at the time of cell lysis is preferably 1.0X10 ⁶～4.0×10⁶/10. Mu.l. The cell density has good gradient, and the data difference between gradients is large, which indicates that the sensitivity difference is large, the sensitivity can reach 0.01%, the data is stable, and the repeatability is good. Although the detection sensitivity of 1.0X10 ⁶～2.0×10⁶/10. Mu.l and 5.0X10 ⁶～6.0×10⁶/10. Mu.l could reach 0.1%, the multiple tests were unstable. The cleavage can be selected to be 1.0X10 ⁶～4.1.0×10⁶/10. Mu.l, preferably 3.0X10 ⁶cells～4.0×10⁶/10. Mu.l.

In the present invention, one or more of the DNA fragments of EBNA1, oriP, SV40 truncated fragment and bacterial source DNA fragment on plasmid were selected as exogenous gene fragments to evaluate exogenous gene residues in clinical-grade hiPSC. The qPCR detection primers are EBNA1-F/EBNA1-R and/or OriP-F/OriP-R.

In the present invention, the reaction system for qPCR detection is preferably as follows: 5 to 35 percent of 10 multiplied by amplification buffer solution, 5 to 45 percent of 10 multiplied by enhancement buffer solution, 0.01 to 10 percent of dNTPs, 0.1 to 6mM MgSO ₄, 0.1 to 25 percent of (1.25 to 10) multiplied by SYBR green I, 0.01 to 2.5 percent of forward primer, 0.5 to 10 percent of reverse primer, 0.01 to 5.5 percent of Platinumpfx DNA polymerase and 5 to 50 percent of template DNA. The SYBR green I and MgSO ₄ solution concentrations are important factors influencing the detection result. The dilution factors of SYBR green I are preferably 10×,5×, 2.5×and1.25×, and all can reach 1% detection sensitivity, wherein the 5×SYBR green I group data are stable, and the repeatability of the repeated holes in the group is good. When the concentration of MgSO ₄ solution is 10mM or more, the enzymatic reaction is affected and the sensitivity is lowered. The detection sensitivity of 0.1-6 mM MgSO ₄ to 0.1%, preferably 0.1-4 mM MgSO ₄, is stable in experiment, and good in repeatability, and more preferably 1.5mM MgSO ₄. Experimental results indicate that 5 XSYBR Green I and 1.5mM MgSO ₄ are the best combination for the initial selection.

In the present invention, the reaction procedure for qPCR detection is preferably as follows: 94 ℃ for 5min; 15sec at 94 ℃, 30sec at 58 ℃, 30sec at 68 ℃ and 35-55 cycles; 95℃10sec,65℃1min,97℃1min. The number of cycles in the qPCR reaction procedure is also a key factor affecting sensitivity. The 45-cycle group data has good gradient, and the data difference between gradients is large, which indicates that the sensitivity difference is large, and the sensitivity can reach 0.01%; the sensitivity of the detection result of the 35-cycle and 40-cycle groups can reach 0.1% at the highest, and can reach the highest when the sensitivity is 0.01%, but the difference is not obvious; the detection sensitivity of 55 cycles and 60 cycles can only reach 1%, so that the 45 cycles have the highest sensitivity of 0.01%, and each gradient difference is relatively uniform, and the detection result is stable.

The following examples are presented in connection with specific DNA fragments, primers, kits and methods for detecting foreign gene residues in hiPSC. They are described in detail, but they are not to be construed as limiting the scope of the invention.

The term 'DNA sequence' as described herein refers to a DNA sequence encoding a protein, such as, but not limited to, a DNA sequence encoding a protein present in the genome of a cell.

In the following examples, the hiPSC of the present invention is hiPSC manufactured by the company of biosciences, inc. Of the most traceable biotechnology in the security, see CN201711462604.8; CN201810137319.7; CN201810252408.6; CN201810400878.2; CN202010313083.5. The method of the invention is not only applicable to the hiPSC obtained by the preparation method, but also applicable to other methods in the field for obtaining the hiPSC. The primers involved in the examples were synthesized by Nanjing gold Style biosystems, and plasmids were prepared from this company.

In the following examples, information concerning specific procedures and the like may be omitted, and conventional procedures or reagents in the art may be employed, and key information concerning the procedures is described herein.

Wherein the application relates to human embryonic stem cells (hESCs), which are isolated or obtained from human embryos that have not undergone fertilization for less than 14 days of in vivo development, and which are commercially obtained embryonic stem cells or stem cells. The human embryonic stem cells (hESCs) are obtained by separating and culturing umbilical cords discarded after obstetric production in Anhui hospitals as sources.

The experimental reagent information involved in the experiment is shown in Table 1 below

TABLE 1 description of sources of drugs and reagents involved in examples of the present invention

Example 1

Preparation method of positive gradient standard substance

Culturing hESC, sucking and discarding the culture medium when the cell density reaches 80% -90%, adding DPBS (without calcium and magnesium), sucking and discarding DPBS, adding Solase digestive juice to digest cells, adding the culture medium to resuspension and suck out cell suspension into a centrifuge tube, centrifuging and collecting cells, and discarding the supernatant; collecting cell DPBS, re-suspending, and sub-packaging in an EP tube; centrifuging an EP tube, adding a Lysis buffer, repeatedly blowing and cracking, transferring to a PCR tube, carrying out vortex oscillation, and standing for 5-15 min at 70-95 ℃; preserving at 4deg.C, treating flocculent precipitate in the tube, adding Proteinase K (10 μg/ml) into the tube, vortexing, standing at 37deg.C for 30min; standing at 70deg.C for 10min; preserving at 4 ℃ to obtain a hESC lysed cell sample, and then carrying out gradient dilution on the hESC lysed cell sample and a plasmid sample to prepare a positive gradient standard, wherein the specific preparation method is as follows in table 2:

positive gradient standard preparation, and mixing before use.

A positive gradient standard was prepared by taking a corresponding volume of hESC lysed cell sample, adding the corresponding plasmid gradient standard as in table 2.

TABLE 2 Positive gradient Standard

Example 2

Screening method of candidate target DNA sequence

1. Screening candidate DNA sequences of interest: taking plasmid pCEP4-EGFP as an example, the DNA sequence expression profile of the plasmid is searched through NCBI official website, and a series of DNA sequences which are highly expressed in the plasmid and are not expressed by hiPSC cells are screened. And detecting the expression level of the truncated fragments of the candidate specific DNA sequences EBNA1-1, oriP-1 and SV40 in the plasmids by a qPCR method.

Firstly, the qPCR method is utilized, the primers are respectively amplified, and the reaction system is shown in Table 3.

TABLE 3 qPCR reaction system

The primers used are shown in Table 4.

Table 4 design primer sequence Listing

QPCR was performed using commercial PCRx ENHANCER SYSTEM plus SYBR Green I, other reagents: the model is as follows: the manufacturer is Thermo: all operations were performed on ice. The reaction system was 10. Mu.l per well, the specific reaction system was formulated as shown in Table 3,3 multiplex wells were set per sample, and qPCR conditions are shown in Table 5.

Table 5 qPCR reaction conditions

CT values were recorded.

The Ct values of the results of qPCR detection according to the qPCR reaction system and qPCR conditions are shown in table 6.

Table 6 qPCR test results Ct value

This experiment utilized qPCR method, 3 qPCR primers were selected, wherein EBNA1-1 and data comparison analysis: the EBNA1-1 detection data is from #1 to #6, the data has good gradient according to the plasmid gradient, and the data difference between gradients is large, which indicates that the sensitivity difference is large; the same screening principle of OriP-1 is adopted, and finally three primers and DNA fragments corresponding to the truncated fragments of EBNA-1, oriP-1 and SV40 are screened according to the experimental result, so that the amplification result is better, and the sensitivity reaches 0.1% (1000 cells 1copy DNA) or 0.01% (10000 cells 1copy DNA).

Example 3

Optimization of SYBR Green I dilution

This example is the same as the methods and procedures of examples 1 and 2 above, in which the hiPSC residual gene sensitivity was measured using a dilution factor of 10×SYBR greenⅠ、5×SYBR greenⅠ、2.5×SYBR greenⅠ、1.25×SYBR greenⅠ、0.625×SYBR greenⅠ、0.313×SYBR greenⅠ、0.156×SYBR greenⅠ. Different dilution SYBR green I was added to 5.5mM MgSO ₄ for qPCR, and the SYBR green I concentration at the optimal dilution was selected. The reaction system and the reaction procedure of this example are shown in Table 9.

Table 9 SYBR green I reaction System and reaction procedure in dilution fold optimization experiment

The results of the detection by qPCR are shown in Table 10.

TABLE 10 results of qPCR detection of the DNA sequence of OriP-1 at various dilution factors of 10 SYBR green I

According to qPCR results, 1% sensitivity (100 cells 1copy DNA) was detected at 10×,5×, 2.5×, and 1.25×SYBR green I concentrations, with 5×SYBR green I group data being more stable, repeated wells within the group being more reproducible, and 5×SYBR green I concentrations being screened. Therefore, in the experiment, 1.25 to 10X of 5 XSYBR Green I can be selected.

Example 4

Optimization of MgSO ₄ solution concentration

This example is the same as the methods and procedures of examples 1 and 2 above, in which 0.1mM MgSO₄、0.5mM MgSO₄、1.5mM MgSO₄、2mM MgSO₄、4Mm MgSO₄、6mM MgSO₄、10mM MgSO₄ solutions were used to detect hiPSC residual gene sensitivity. Three samples #1, #3 and #6 were selected for qPCR with 5 XSYBR green I added to different concentrations of MgSO ₄, respectively, and the optimal MgSO ₄ concentrations were selected.

The reaction system of this example is shown in Table 11.

Table 11 optimization experiment of MgSO ₄ solution concentration reaction System and reaction procedure

The results of the detection by qPCR are shown in Table 8.

TABLE 12 results of qPCR detection of the DNA sequence of OriP-1 under different concentrations of MgSO ₄

According to qPCR results, the concentration of MgSO ₄ above 10mM is too high, which affects enzymatic reaction and reduces sensitivity; the detection sensitivity is 0.1% by 0.1-6 mM MgSO ₄, the experiment of 0.1-4 mM MgSO ₄ is stable, the repeatability is good, and 1.5mM MgSO ₄ is preferable.

The best conditions can be selected from the experimental results of example 3 and example 4: 5 XSYBR Green I and 1.5mM MgSO ₄ were the best combination for the initial selection.

Example 5

Optimization of qPCR reaction cycle number

Based on the above examples, the same methods and steps as those of the above examples 1 and 2 were carried out, wherein 35, 40, 45, 55, 60 cycles were used for qPCR experiments to detect the residual sensitivity of the hiPSC exogenous gene. Wherein qPCR was performed on OriP-1 fragments with 5 XSYBR green I and 1.5mM MgSO ₄ as optimal combinations, respectively, and the optimal qPCR cycle numbers were selected.

The reaction system of this example is shown in Table 13.

Table 13 qPCR optimization of reaction cycle number experiment reaction System and reaction procedure

The results of the detection of the DNA sequence of OriP-1 by qPCR are shown in Table 14.

Table 14 qPCR results of detecting the DNA sequence of OriP-1

According to qPCR results: from #1 to #6, the 45-cycle group data shows good gradient according to plasmid gradients, and the data difference between gradients is large, which indicates that the sensitivity difference is large, and the sensitivity can reach 0.01%; the sensitivity of the detection result of the 35-cycle and 40-cycle groups can reach 0.1% at the highest, and can reach the highest when the sensitivity is 0.01%, but the difference is not obvious; the detection sensitivity of 55 cycles and 60cycles can only reach 1%, so that the 45 cycles have the highest sensitivity of 0.01%, and each gradient difference is relatively uniform, and the detection result is stable. Thus, 35-55 cycles are all optional, preferably 35-45 cycles, more preferably 45 cycles.

Example 6

Optimization of the number of lysed different cells

Based on the above examples, this example is the same as the methods and procedures of examples 1 and 2 above, in which hescs of different cell numbers were lysed in qPCR experiments, respectively, as templates to detect hiPSC exogenous gene residual sensitivity. Wherein samples #1, #2, #3, #4 were selected for qPCR respectively using 5 XSYBR green I and 1.5mM MgSO ₄ as optimal combinations, and the optimal cell lysis numbers were selected for qPCR detection. Wherein the optimization of the different cell numbers is performed in a 10ul system or a 20ul system.

The reaction system of this example is shown in Table 15.

TABLE 15 optimization of the experimental reaction system and reaction program for lysing different cell numbers

The results of the DNA sequence of OriP-1 detected by qPCR when cells were lysed in different amounts are shown in Table 16.

TABLE 16 detection results of lysis of different numbers of cells

Results: from #1- #6, the 3.0X10 ⁶ cell group and the 4.0X10 ⁶ cell data show good gradient, and the data difference between gradients is large, which indicates that the sensitivity difference is large, the sensitivity can reach 0.01%, the multiple tests can reach 0.1%, the data is stable, and the repeatability is good. The sensitivity of the detection results of the 1.0X10 ⁶ cells and the 2.0X10 ⁶ cells can reach 0.1% at the highest; the detection sensitivity of the 5.0X10 ⁶ cell group can reach 0.1%, but the multiple tests are unstable, so that 1.0X10 ⁶cells～4.0×10⁶ cell, preferably 3.0X10 ⁶cells～4.0×10⁶ cell can be selected for cleavage.

Example 7

Proteinase K treated samples of different concentrations

Based on the above examples, this example is the same as the methods and steps of examples 1 and 2 described above, and when the cells (hESC or hiPSC) are lysed to obtain templates, the addition of proteinase K at different concentrations has an important influence on qPCR detection to screen the optimal proteinase K use concentration, and more stable and accurate detection of foreign gene residues in hiPSC, wherein qPCR is performed with 5×sybr green I and 1.5mM MgSO ₄ as optimal combinations, respectively, and qPCR detection is performed with the optimal proteinase K concentration. The reaction system of this example is shown in Table 17.

Table 17 Experimental reaction System and reaction procedure for treating samples with proteinase K at different concentrations

The results of the DNA sequence of OriP were determined by qPCR and are shown in Table 18.

TABLE 18 detection results of proteinase K treated samples at different concentrations

QPCR results: from #1- #6, experimental data analysis for each column group based on plasmid gradient: 1-50 mug/ml proteinase K detection data are displayed in the corresponding columns, are all selectable, and the sensitivity can reach 0.1%; the 1-20 mug/mL proteinase K detection data are displayed in the corresponding columns, the proteinase K detection data are optional, the sensitivity can reach 0.01%, when the proteinase K with the concentration of 1ug/mL is used for treating the template, the effect is best, the qPCR result data are stable, and good gradient difference exists, the gradient among standard product groups is obvious, the low copy DNA concentration in the standard product can be effectively protected, the DNA enzyme in cells is prevented from degrading the added exogenous DNA, and the low copy exogenous DNA concentration in the iPSC is protected.

Example 8

Random interception of DNA sequence of EBNA1 in plasmid sequence for hiPSC exogenous gene residue detection

Randomly intercepting the DNA sequence of the EBNA1 sequence fragment region in the plasmid sequence, wherein the sequence is EBNA1-2 as follows:

CTGGTCCAGATGTGTCTCCCTTCTCTCCTAGGCCATTTCCAGGTCCTGTACCTGGCCCCTCGTCAGACATGATTCACACTAAAAGAGATCAATAGACATCTTTATTAGACGACGCTCAGTGAATACAGGGAGTGCAGACTCCTGCCCCCTCCAACAGCCCCCCCACCCTCATCCCCTTCATGGTCGCTGTCAGACAGATCCAGGTCTGAAAATTCCCCATCCTCCGAACCATCCTCGTCCTCATCACCAATTACTCGCAGCCCGGAAAAC(SEQ ID NO:19,).

Meanwhile, randomly intercepting the DNA sequence of the EBNA1 sequence fragment region in the plasmid sequence, wherein the sequence is EBNA1-3 as follows:

CTCTATGTCTTGGCCCTGATCCTGAGCCGCCCGGGGCTCCTGGTCTTCCGCCTCCTCGTCCTCGTCCTCTTCCCCGTCCTCGTCCATGGTTATCACCCCCTCTTCTTTGAGGTCCACTGCCGCCGGAGCCTTCTGGTCCAGATGTGTCTCCCTTCTCTCCTAGGCCATTTCCAGGTCCTGTACCTGGCCCCTCGTCAGACATGATTCACACTAAAAGAGATCAATAGACATCTTTATTAGACGACGCTCAGTGAATACAGGGAGTGCAGACTCCTGCCCCCTCCAACAGCCCCCCCACCCTCAT(SEQ ID NO:20).

Detecting exogenous gene residues in hiPSC by using the EBNA1-2 amplification sequence and the EBNA1-3 amplification sequence, and designing primers aiming at the sequences; the primer sequences were designed as shown in Table 19 below, and the internal reference and primers were as described in example 2 above.

Table 19 primer sequence listing

Primer name	Sequence(s)
		EBNA1-2-F	CTGGTCCAGATGTGTCTCC(SEQ ID NO:6)
EBNA1-2-R	GTTTTCCGGGCTGCGAGTAAT(SEQ ID NO:7)
		EBNA1-3-F	CTCTATGTCTTGGCCCTGAT(SEQ ID NO:8)
EBNA1-3-R	ATGAGGGTGGGGGGGCTGTTG(SEQ ID NO:9)

The selection of the components or conditions on the basis of examples 1-7 above preferably uses concentrations or ranges for qPCR detection, see in particular table 20. Wherein, samples #1, #2, #3 and #4 were selected for qPCR respectively by treating the template with 5 XSYBR green I, 1.5mM MgSO ₄, 1. Mu.g/mL proteinase K, 3.0X10 ⁶～4.0×10⁶ cell lysis density and qPCR 45 cycle number as the optimal combination.

TABLE 20 reaction system and reaction procedure

Fragments of the DNA sequence of EBNA1 were randomly excised for hiPSC exogenous gene residue detection, and qPCR detection, and the results of the detection of the fragments of the DNA sequence of EBNA1 are shown in Table 21.

TABLE 21 results of fragment qPCR detection of DNA sequence of EBNA1

QPCR results: from #1- #4, experimental data analysis for each column group based on plasmid gradient: under the condition of plasmid gradient, the DNA sequence EBNA1-2 or EBNA1-3 has gradient difference, the gradient difference of each column is obvious, the gradient sensitivity corresponds to 0.1 percent (compared with the result of #1-0, the result difference of #3-0.1 percent is more than 2, and the detection result sensitivity of some groups can reach 0.01 percent). Fragments of DNA sequences demonstrating randomly truncated EBNA1 sequence fragment regions were used for hiPSC exogenous gene residue detection.

Example 9

Random interception of fragments in DNA sequence of OriP in plasmid sequence for hiPSC exogenous gene residue detection

Randomly intercepting DNA sequence of OriP sequence fragment region in plasmid sequence, such as OriP-2, specific sequence is as follows ：TATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGCATATACAGTCAGCATATGATACCCAGTAGTAGAGTGGGAGTGCTATCCTTTGCATATGCCGCCACCTCCCAAGGGGGCGTGAATTTTCGCTGCTTGTCCTTTTCCTGCTGGTTGCTCCCATTCTTAGGTGAATTTAAGGAGGCCAGGCTAAAGCCGTCGCATGTCTGATTGCT(SEQ IDNO:22,).

The DNA sequence of the fragment region of the OriP sequence in the randomly intercepted plasmid sequence is shown as the following OriP-3, and the specific sequence is as follows:

CTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGCATATACAGTCAGCATATGATACCCAGTAGTAGAGTGGGAGTGCTATCCTTTGCATATGCCGCCACCTCCCAAGGGGGCGTGAATTTTCGCTGCTTGTCCTTTTCCTGCTGGTTGCTCCCATTCTTAGGTGAATTTAAGGAGGCCAGGCTAAAGCCGTCGCATGTCTGATTGCTCACCAGGTAAATGTCGCTAATGTTTTCCAACGCGAGAAGGTGT(SEQ ID NO:23).

Detecting exogenous gene residues in hiPSC based on the OriP-2 amplified sequence and the OriP-3 amplified sequence, and designing primers aiming at the sequences; primer sequences were designed as shown in Table 22 below, with internal references and primers as described in example 2 above.

Table 22 primer sequence listing

The selection of the components or conditions on the basis of examples 1-7 above preferably uses concentrations or ranges for qPCR detection, see in particular table 23. Wherein, samples #1, #2, #3 and #4 were selected for qPCR respectively by treating the template with 5 XSYBR green I, 1.5mM MgSO ₄, 1. Mu.g/mL proteinase K, 3.0X10 ⁶～4.0×10⁶ cell lysis density and qPCR 45 cycle number as the optimal combination.

Table 23 qPCR shows the reaction system and reaction procedure

Fragments of the DNA sequence of OriP were randomly intercepted for hiPSC exogenous gene residue detection, and qPCR detection, and the results of fragment detection of the DNA sequence of OriP are shown in table 24.

Table 24 shows the results of fragment qPCR detection of OriP DNA sequences

QPCR results: from #1- #4, experimental data analysis for each column group based on plasmid gradient: under the condition of plasmid gradient, the DNA sequence OriP-2 or OriP-3 has gradient difference in qPCR result, and the gradient difference of each column is obvious, the gradient sensitivity corresponds to 0.1% (compared with the result of #1-0, the result difference of #3-0.1% is more than 2, and can be regarded as effective gradient detection), and the detection result sensitivity of some column groups can reach 0.01%. Fragments of DNA sequences demonstrating randomly truncated fragment regions of the OriP sequence were used for hiPSC exogenous gene residue detection.

Example 10

Exogenous gene residue detection in hiPSCs of different generations

3 Different generations of hiPSCs are selected as cell samples to be tested, and the detection method for screening target DNA sequences (OriP DNA sequences and EBNA1 DNA sequences) in the embodiment is adopted to detect the residual quantity of the exogenous genes in the hiPSCs. Wherein the hiPSC is selected from seed cell library (seed), master cell library (MASTER CELL bank/MCB) and working cell library (working cell Bank/WCB). The seed cell bank is primary differentiated cells and is P10 generations; the main cell bank is P15 generation cells; the working cell bank is P20 generation cells. And respectively selecting corresponding generation secondary cells from the cell libraries, and detecting the residual condition of the exogenous genes in different cell libraries. The concentrations and ranges of the components are preferably selected on the basis of the above examples for qPCR assays, see in particular table 25. The template was treated with 5×SYBR green I, 1.5mM MgSO ₄, 1 μg/mL proteinase K, 3.0X10 ⁶～4.0×10⁶ cell lysis density, qPCR 45 cycles as the optimal combination.

TABLE 25 detection results of foreign Gene residuals in hiPSCs of different generations

The results of the DNA sequences of OriP-1 and EBNA1-1 as detected by qPCR are shown in Table 26.

Table 26 Ct value of the results of detection of DNA sequences of OriP-1 and EBNA1-1

As can be seen from the results, the experimental data of each column group were analyzed from #1- #6 according to the plasmid gradient by q-PCR method using the detection method of the present application: the standard data are stable, good gradient difference exists, seed, MCB, WCB sample detection data can reach the sensitivity of 0.01% -0.1%, the OriP or EBNA1 sequence can realize the detection of the residual quantity of the exogenous genes in the hiPSC, the limit is stable and reaches 0.1%, and the sensitivity of the MCB and WCB groups can reach 0.01%, so that the method is stable and simple.

Example 11

Episomal plasmid nEP, 4-E-EGFP, PMEP4-C-EGFP and PKEP-E-EGFP were separately reprogrammed to prepare hiPSC, and detection of exogenous gene residues of hiPSC

Exogenous gene residues in hiPSC preparation by reprogramming episomal plasmid vectors nEP-E-EGFP, PMEP4-C-EGFP and PKEP-E-EGFP were detected using the amplified sequences of OriP-1 and primers thereof in examples 2-3 described above.

The concentrations and ranges of the components are preferably selected on the basis of the above examples to perform qPCR detection, and the reaction system is shown in Table 26. The template was treated with 5×SYBR green I, 1.5mM MgSO ₄, 1 μg/mL proteinase K, 3.0X10 ⁶～4.0×10⁶ cell lysis density, qPCR45 cycles as the optimal combination.

Table 26 qPCR reaction System

The results of the DNA sequence of OriP-1 as determined by qPCR are shown in Table 27.

DNA sequence results Ct values of Table 27 OriP-1

As can be seen from the results, each group of experimental data was analyzed from #1- #6 according to the plasmid gradient by q-PCR method using the detection method of the present application: the detection data of the hiPSC sample prepared by reprogramming the episomal plasmids nEP, PMEP4, C, and PKEP can reach the sensitivity of 0.01-0.1%, and the design method, sequence, primer, etc. can realize the detection of the residual quantity of the exogenous gene in the hiPSC, and the limit can reach 0.1%, and the limit can reach 0.01% higher.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Anhui, shengyuan Biotechnology Co., ltd

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cccacacgcc gccctgcggt tttggactgt aaaataaggg tgtaataact tggctgattg 1320

taaccccgct aaccactgcg gtcaaaccac ttgcccacaa aaccactaat ggcaccccgg 1380

ggaatacctg cataagtagg tgggcgggcc aagatagggg cgcgattgct gcgatctgga 1440

ggacaaatta cacacacttg cgcctgagcg ccaagcacag ggttgttggt cctcatattc 1500

acgaggtcgc tgagagcacg gtgggctaat gttgccatgg gtagcatata ctacccaaat 1560

atctggatag catatgctat cctaatctat atctgggtag cataggctat cctaatctat 1620

atctgggtag catatgctat cctaatctat atctgggtag tatatgctat cctaatttat 1680

atctgggtag cataggctat cctaatctat atctgggtag catatgctat cctaatctat 1740

atctgggtag tatatgctat cctaatctgt atccgggtag catatgctat cctaatagag 1800

attagggtag tatatgctat cctaatttat atctgggtag catatactac ccaaatatct 1860

ggatagcata tgctatccta atctatatct gggtagcata tgctatccta atctatatct 1920

gggtagcata ggctatccta atctatatct gggtagcata tgctatccta atctatatct 1980

gggtagtata tgctatccta atttatatct gggtagcata ggctatccta atctatatct 2040

gggtagcata tgctatccta atctatatct gggtagtata tgctatccta atctgtatcc 2100

gggtagcata tgctatcctc atgcatatac agtcagcata tgatacccag tagtagagtg 2160

ggagtgctat cctttgcata tgccgccacc tcccaagggg gcgtgaattt tcgctgcttg 2220

tccttttcct gctggttgct cccattctta ggtgaattta aggaggccag gctaaagccg 2280

tcgcatgtct gattgctcac caggtaaatg tcgctaatgt tttccaacgc gagaaggtgt 2340

tgagcgcgga gctgagtgac gtgacaacat gggtatgccc aattgcccca tgttgggagg 2400

acgaaaatgg tgacaagaca gatggccaga aatacaccaa cagcacgcat gatgtctact 2460

ggggatttat tctttagtgc gggggaatac acggctttta atacgattga gggcgtctcc 2520

taacaagtta ca 2532

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

ggacaccatc tctatgtctt 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

tattcactga gcgtcgtcta 20

<210> 6

<211> 19

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

ctggtccaga tgtgtctcc 19

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

gttttccggg ctgcgagtaa t 21

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

ctctatgtct tggccctgat 20

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

atgagggtgg gggggctgtt g 21

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

gtagtagagt gggagtgcta 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

ctgtcttgtc accattttcg 20

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

tatgctatcc taatttatat c 21

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

agcaatcaga catgcgacgg c 21

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

ctgggtagta tatgctatc 19

<210> 15

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

acaccttctc gcgttggaaa ac 22

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

atggctgatt atgatccggc tg 22

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

tacagacaag ctgtgaccgt ct 22

<210> 18

<211> 266

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

ggacaccatc tctatgtctt ggccctgatc ctgagccgcc cggggctcct ggtcttccgc 60

ctcctcgtcc tcgtcctctt ccccgtcctc gtccatggtt atcaccccct cttctttgag 120

gtccactgcc gccggagcct tctggtccag atgtgtctcc cttctctcct aggccatttc 180

caggtcctgt acctggcccc tcgtcagaca tgattcacac taaaagagat caatagacat 240

ctttattaga cgacgctcag tgaata 266

<210> 19

<211> 270

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

ctggtccaga tgtgtctccc ttctctccta ggccatttcc aggtcctgta cctggcccct 60

cgtcagacat gattcacact aaaagagatc aatagacatc tttattagac gacgctcagt 120

gaatacaggg agtgcagact cctgccccct ccaacagccc ccccaccctc atccccttca 180

tggtcgctgt cagacagatc caggtctgaa aattccccat cctccgaacc atcctcgtcc 240

tcatcaccaa ttactcgcag cccggaaaac 270

<210> 20

<211> 304

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 20

ctctatgtct tggccctgat cctgagccgc ccggggctcc tggtcttccg cctcctcgtc 60

ctcgtcctct tccccgtcct cgtccatggt tatcaccccc tcttctttga ggtccactgc 120

cgccggagcc ttctggtcca gatgtgtctc ccttctctcc taggccattt ccaggtcctg 180

tacctggccc ctcgtcagac atgattcaca ctaaaagaga tcaatagaca tctttattag 240

acgacgctca gtgaatacag ggagtgcaga ctcctgcccc ctccaacagc ccccccaccc 300

tcat 304

<210> 21

<211> 272

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 21

gtagtagagt gggagtgcta tcctttgcat atgccgccac ctcccaaggg ggcgtgaatt 60

ttcgctgctt gtccttttcc tgctggttgc tcccattctt aggtgaattt aaggaggcca 120

ggctaaagcc gtcgcatgtc tgattgctca ccaggtaaat gtcgctaatg ttttccaacg 180

cgagaaggtg ttgagcgcgg agctgagtga cgtgacaaca tgggtatgcc caattgcccc 240

atgttgggag gacgaaaatg gtgacaagac ag 272

<210> 22

<211> 309

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 22

tatgctatcc taatttatat ctgggtagca taggctatcc taatctatat ctgggtagca 60

tatgctatcc taatctatat ctgggtagta tatgctatcc taatctgtat ccgggtagca 120

tatgctatcc tcatgcatat acagtcagca tatgataccc agtagtagag tgggagtgct 180

atcctttgca tatgccgcca cctcccaagg gggcgtgaat tttcgctgct tgtccttttc 240

ctgctggttg ctcccattct taggtgaatt taaggaggcc aggctaaagc cgtcgcatgt 300

ctgattgct 309

<210> 23

<211> 272

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 23

ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc tcatgcatat 60

acagtcagca tatgataccc agtagtagag tgggagtgct atcctttgca tatgccgcca 120

cctcccaagg gggcgtgaat tttcgctgct tgtccttttc ctgctggttg ctcccattct 180

taggtgaatt taaggaggcc aggctaaagc cgtcgcatgt ctgattgctc accaggtaaa 240

tgtcgctaat gttttccaac gcgagaaggt gt 272

<210> 24

<211> 97

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 24

atggctgatt atgatccggc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac 60

acatgcagct cccggagacg gtcacagctt gtctgta 97

<210> 25

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 25

cacctgcact tattcttggc ag 22

<210> 26

<211> 22

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 26

tattgggctc agaccaggag tc 22

<210> 27

<211> 2018

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 27

gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat 60

ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 120

gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 180

tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 240

ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 300

ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 360

gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 420

gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 480

acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 540

actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 600

ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 660

cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 720

gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 780

caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 840

aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 900

ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 960

tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1020

ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 1080

ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 1140

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1200

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1260

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1320

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 1380

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1440

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 1500

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 1560

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 1620

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 1680

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 1740

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 1800

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 1860

gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttgaagctgt ccctgatggt 1920

cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag 1980

cgagaagaat cataatgggg aaggccatcc agcctcgc 2018

Claims

1. A primer for detecting foreign gene residue in hiPSC is characterized by being a primer for specifically detecting a specific DNA fragment derived from an OriP/EBNA1 episome vector used in preparation of the hiPSC, wherein the specific DNA fragment is OriP-1 with a nucleotide sequence shown as SEQ ID NO. 21;

The primer is an OriP-1-F, oriP-1-R primer pair;

The nucleotide sequence of the OriP-1-F is shown as SEQ ID NO:10 is shown in the figure;

the nucleotide sequence of the OriP-1-R is shown as SEQ ID NO: 11.

2. A kit for detecting an exogenous gene residue in hiPSC based on qPCR technology, comprising the primer of claim 1.

3. Use of the primer of claim 1 or the kit of claim 2 for detecting exogenous gene residues of clinical-grade hipscs.

4. The method for detecting the exogenous gene residue of the clinical-grade hiPSC based on the qPCR technology is characterized by comprising the following steps of:

1) Screening a specific DNA fragment carried by an OriP/EBNA1 episome vector as target DNA, wherein the specific DNA fragment is OriP-1 with a nucleotide sequence shown as SEQ ID NO. 21;

2) Designing the primer according to claim 1 according to the target DNA in the step 1);

5. The method according to claim 4, wherein the reaction system of qPCR detection is as follows:

10X amplification buffer solution 5-35%, 10X enhancement buffer solution 5-45%, dNTPs 0.01-10%, 0.1 mM-6 mM MgSO ₄ 0.1-25%, 1.25X-10X SYBR green I0.01-2.5%, forward primer 0.5-10%, reverse primer 0.5-10%, platinum pfx DNA polymerase 0.01-5.5%, template DNA 5-50%;

The components are in volume percent.

6. The method according to claim 4, wherein the qPCR assay is performed according to the following reaction procedure:

7. The method according to any one of claims 4 to 6, wherein the concentration of proteinase K is 1 to 50. Mu.g/ml when the cells are lysed in step 4).

8. The method according to any one of claims 4 to 6, wherein the number of cells is 1.0X10 ⁶~4.0×10⁶/10. Mu.l when the cells are lysed.