CN114277190A

CN114277190A - Specific DNA fragment, primer, kit and detection method for detecting foreign gene residues in hiPSC

Info

Publication number: CN114277190A
Application number: CN202111668786.0A
Authority: CN
Inventors: 不公告发明人
Original assignee: Nuwacell Ltd
Current assignee: Nuwacell Ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-05

Abstract

The invention provides a specific DNA fragment, a primer group, a kit and a detection method for detecting foreign gene residues in a hipSC, and belongs to the technical field of stem cells. According to the invention, the free vector sequences used in the preparation of the hipscs by induction are used for screening specific DNA sequences, and detection primers are respectively designed according to the screened specific DNA sequences, so that a primer pair with good amplification effect and high detection sensitivity is obtained. The method is used for detecting the residual condition of the foreign genes in the hiPSC based on the qPCR method, does not need to extract genome DNA, obtains the genome in a direct cell cracking mode, can complete the whole process within about 5-6 hours, is suitable for the quality control of the hiPSC product, is more economic and time-saving, and is more guaranteed for monitoring the safety, the economy and the productivity of clinical-grade hiPSC.

Description

Specific DNA fragment, primer, kit and detection method for detecting foreign 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 foreign gene residues in a hiPSC.

Background

Human pluripotent stem cells (hpscs), including human embryonic stem cells (hescs) and human induced pluripotent stem cells (hipscs), are a class of stem cells that can proliferate indefinitely in vitro and have the potential to differentiate into almost 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 without effective therapy at present. Currently, with the rapid development of stem cell technology, more and more hPSC-derived functional cell products have been approved in various countries to enter clinical trials.

The hiPSC is a pluripotent stem cell obtained by reprogramming in a way of expressing a specific gene or a specific gene product in differentiated somatic cells and the like, not only avoids the social ethical problem related to embryo source hESC, but also has rich sources and unlimited genetic background, and is expected to overcome the immune match problem faced by cell transplantation. Based on these advantages of hipscs, in recent years, several classes of hiPSC-derived functional cell products have entered clinical use. Early iPSC reprogramming used reverse transcription/lentiviral vectors to express foreign genes. The vector can realize long-term stable expression of exogenous genes, so that higher reprogramming efficiency is obtained, but insertional gene mutation can be caused, and the function and tumorigenicity of iPSC can be seriously influenced by exogenous gene expression. Hipscs used to prepare clinical grade functional cell products should not contain foreign DNA residues, so a reprogramming method that preferentially selects a non-integrative reprogramming system is needed. Commonly used non-integrative hiPSC reprogramming systems are often constructed based on the episomal DNA plasmid vector of OriP/EBNA 1. The two key sequences OriP/EBNA1 of this vector are derived from the double-stranded DNA herpes virus (Epstein-Barr virus, EBV). Around 90% of adults worldwide are infected with EBV, which is free from the cell genome in the form of circular DNA plasmid during the incubation period, and OriP and EBNA1 are virus elements required for stably maintaining the free state^[1]. The OriP/EBNA1 vector can establish a stable free state (about 5-50 plasmids/cell) in partial cells after transfecting the cells, thereby stably expressing exogenous genes to ensure the success of reprogramming. The defect of replication and distribution of the OriP/EBNA1 vector ensures that about 5 percent of cells lose plasmids in each cell division, so that traceless iPSC can be easily screened^[2]. In 2009, Yu et al obtained human traceless iPSC for the first time by using OriP/EBNA1 vector^[3]The combined use of small molecules greatly improves the reprogramming efficiency of the system^[4]. Compared with other methods, the OriP/EBNA1 vector has a plurality of advantagesPoint: can establish stable free state in various cells, thus being not limited by cell types; the plasmid can be prepared by a conventional method without virus packaging; only one transfection is needed, and the operation is simple; the cell in a free state is established and only contains a small amount of vectors, so that the risk of genome integration is reduced; due to the defects of vector replication and distribution and the silencing of the virus promoter for expressing the EBNA1 in the PSC, traceless iPSC can be obtained only by conventional subculture, and complicated single cell clone screening work is not needed. Therefore, the method is more commonly used in the preparation of clinical-grade ipscs.

Although the OriP/EBNA1 vector can be lost after repeated passages of the hiPSC, there is still a risk that incomplete deletion and the reprogramming plasmid remain in the hiPSC, thereby increasing the potential safety hazard of tumorigenicity of the cell product. In international and domestic research, the exogenous foreign matter (exogenous gene) is strictly required in the cell therapy product research and evaluation technical guide principle of China. Although the methods for preparing hipscs are various, the control principle of exogenous genes is strictly followed to control the product quality standard, so that when the hipscs cells are induced and formed by the exogenous genes, a detection method which is suitable for industrial scale production and has high sensitivity is required to detect the residues of the exogenous genes in the hipscs cell products so as to enable the products to meet clinical use and downstream application.

Besides obtaining the hipscs by using the episomal vector weight editing induction based on OriP/EBNA1, the hipscs can be constructed by using an S/MAR system plasmid vector, an SV40 system plasmid vector and an S/MAR system plasmid vector.

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

The SV40 system episomal vector belongs to SV40 DNA-plasmid DNA hybrid vector, and the recombinant SV40DNA molecule must be packaged to have infection capability, so that the inserted exogenous DNA fragment cannot be too large. In order to increase the SV40 loading capacity as much as possible, some gene segments must be deleted, so that the recombinant SV40 molecule must co-infect recipient cells with wild-type virus in order to form infectious recombinant viral particles. The S/MAR system plasmid vector can express foreign genes with high efficiency, but has small loading capacity.

The BKV system episomal vector is a novel episomal vector based on human papilloma virus sequences. Human papilloma virus (BKV) belongs to the genus papillomavirus of the family polyomaviridae, the genome of the papillomavirus belongs to double-stranded DNA, the genome is not integrated after infecting host cells, the genome autonomously replicates independently of chromosomes, and each host cell can achieve up to 500 copies. Genes encoding the viral core and coat proteins are deleted and spliced with E.coli plasmids to form shuttle vectors which do not integrate and also do not form mature viral particle lytic receptor cells. The cytochrome P45dioxin mediated enhancer DRE is installed to control mouse breast cancer promoter MMTP, so that the promoter can drive the expression of exogenous gene.

The conventional PCR method is subjected to gel electrophoresis analysis after PCR, and the method is inaccurate, has too large error and cannot meet the requirement of high-precision detection. qPCR is a common method, but the sensitivity, limit and stability of the current qPCR method for detecting the hiPSC exogenous gene residue are influenced by various factors (such as the dosage of reaction system components, reaction conditions and the like in the qPCR method), and the requirement of high-sensitivity hiPSC exogenous gene residue detection cannot be met. In addition, in the existing detection methods, cell genome DNA is mostly required to be extracted, the operation is troublesome, the detection process is long, the cost is high, and the quantity control of the hiPSC cytoplasm cannot be effectively monitored. Therefore, there is a need for a stable, highly sensitive, more economical, and time-saving detection method that is more safe, economical, and productive for monitoring clinical-grade hipscs. Only if a safe hiPSC cell bank is established, downstream applications can be better supplied, which is also a problem that the cell therapy field always pursues and needs to solve for monitoring cell quality.

Reference documents:

[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 hipscs, which have the characteristic of high sensitivity.

The invention provides a specific DNA fragment for detecting foreign gene residues in hipscs, wherein the specific DNA fragment is derived from an episomal vector used for preparing the hipscs by induction.

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

Preferably, the free vector based on OriP/EBNA1 includes pCEP-EGFP, nEP4-E-EGFP, PMEP4-C-EGFP and PKEP 4-E-EGFP.

Preferably, when the episomal vector is an episomal vector based on OriP/EBNA1, the specific DNA fragment is selected from at least one of the following sequences:

A. intercepting a truncated DNA sequence with the total length of more than 70bp of an SV40 polyA signal region;

B. intercepting a truncated DNA sequence with the full length of more than 70bp of an OriP sequence fragment region;

C. intercepting a truncated DNA sequence with the total length of more than 70bp of an EBNA1 sequence fragment region;

D. and (3) intercepting a truncated DNA sequence with the total length of more than 70bp of the bacterial source DNA fragment region on the plasmid.

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

1) DNA segment with nucleotide sequence as shown in SEQ ID No. 1;

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

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

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

1) DNA segment with nucleotide sequence shown in SEQ ID NO. 2;

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

3) 2 on the basis of SEQ ID NO. 2 through adding, deleting and inserting one or more nucleotide sequences to form a DNA fragment;

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

A. the nucleotide sequence is shown as SEQ ID NO: 3, or a DNA fragment thereof;

B. and SEQ ID NO: 3 complementary DNA fragments;

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

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

1) DNA segment with nucleotide sequence shown in SEQ ID NO. 27;

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

3) 27 on the basis of SEQ ID NO. 27 through adding, deleting and inserting one or more nucleotide sequences to form a DNA fragment;

preferably, the specific DNA fragment comprises one or more of the following DNA fragments: EBNA1 truncated fragments, OriP truncated fragments, SV40 truncated fragments, and bacterially derived DNA fragments on plasmids;

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

the OriP truncated fragment comprises an OriP-1 with a nucleotide sequence shown as SEQ ID NO. 21, an OriP-2 with a nucleotide sequence shown as SEQ ID NO. 22 and an 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 foreign gene residues in a hiPSC, which comprises a primer for specifically detecting the specific DNA fragment;

preferably, the primers comprise one or more of the following pairs of primers: a primer pair for specifically detecting the EBNA1 truncated segment, a primer pair for specifically detecting the OriP truncated segment, a primer pair for specifically detecting the SV40 truncated segment and a primer pair for specifically detecting the bacterial-derived DNA truncated segment on the plasmid;

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

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

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

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

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

the nucleotide sequence of EBNA1-3-F is shown in SEQ ID NO: 8 is shown in the specification;

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

preferably, the primer pair for specifically detecting the OriP truncated fragment 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 OriP-1-R is shown as SEQ ID NO: 11 is shown in the figure;

the nucleotide sequence of OriP-2-F is shown as SEQ ID NO: 12 is shown in the specification;

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

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

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

preferably, the primer pair for specifically detecting the SV40 truncated fragment comprises SV40-F/SV 40-R;

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

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

The invention provides a kit for detecting foreign gene residues in hipscs based on a qPCR technology, which comprises the primers.

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

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

1) screening specific DNA fragments carried by the free carrier as target DNA;

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

3) mixing the hESC and the plasmid standard substance according to different proportions to prepare a positive gradient standard substance by taking the free type vector in the step 1) as the plasmid standard substance;

4) and (3) cracking the cells of the hiPSC to be detected to obtain a sample to be detected, carrying out qPCR detection on the sample to be detected, the positive gradient standard substance and the primer, and judging the residual limit of the hiPSC exogenous gene 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 sample to be detected.

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

5-35% of 10 multiplied amplification buffer solution, 5-45% of 10 multiplied enhancement buffer solution, 0.01-10% of dNTPs and 0.1-6 mM MgSO 2₄0.1-25 percent, (1.25-10) xSYBR green I0.01-2.5 percent, forward primer 0.5-10 percent, reverse primer 0.5-10 percent, Platinum pfx DNA polymerase 0.01-5.5 percent and template DNA 5-50 percent;

the forward primer and the reverse primer are the primers.

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

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

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

preferably, the number of cells is 1.0X 10 when the cells are lysed⁶～4.0×10⁶Mu.l/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 candidate target DNA sequences are screened, primers are respectively designed for DNA sequences of different sites of an EBNA sequence fragment region, an OriP sequence fragment region, an SV40 truncated fragment, a bacterium-derived DNA sequence fragment region on a plasmid, and a pair of primers is designed for an SV40 truncated fragment, so that a detection result shows that the amplification result of a primer pair for amplifying the EBNA1 truncated fragment or the OriP truncated fragment and a primer pair for an SV40 polyA signal region is good, and the sensitivity reaches 0.1% (1000 cells contain 1copy DNA fragments) or 0.01% (10000 cells contain 1copy DNA fragments).

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

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

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

(3) During detection, operations such as extraction of genome DNA in the traditional experiment are not needed, and only the genome is obtained by directly cracking cells and the preparation of qPCR reagents is needed, so that the quality control operation is simplified, the cost is reduced, and the method is more suitable for industrial preparation of clinical-grade hipscs.

(4) In the establishing method of the invention, protease K is added after cell lysis to play a role in degrading or inhibiting DNA enzyme, thereby protecting the concentration of low-copy plasmid, and ensuring that the method is more accurate, reliable and higher in sensitivity.

Drawings

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

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

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

FIG. 4 is a structural diagram of the positive plasmid PKEP 4-E-EGFP.

Detailed Description

In the present invention, the episomal vector preferably comprises one or more of the following systems: an episomal vector based on one or more of OriP/EBNA1, S/MAR, SV40, BKV and BPV-1. In the present invention, the contents of the specific DNA fragment are specifically described by taking an episomal vector based on OriP/EBNA1 as an example.

In the present invention, the OriP/EBNA 1-based episomal vector preferably comprises pCEP-EGFP, nEP4-E-EGFP, PMEP4-C-EGFP and PKEP4-E-EGFP, and the structural diagrams of the plasmids correspond to FIGS. 1 to 4, respectively. The specific DNA fragment is specifically described below by taking the 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. intercepting a truncated DNA sequence of which the total length of SV40 polyA signal region gene is more than 70 bp;

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 OriP sequence fragment regions; the region shown by EBNA1 and the downstream blank region in FIG. 1 are collectively referred to as EBNA1 sequence fragment regions.

Wherein, the SV40 polyA signal region preferably comprises at least one of the following DNA fragments: 1) DNA segment with nucleotide sequence as shown in 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) DNA segment with nucleotide sequence shown in 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 as SEQ ID NO: 3, or a DNA fragment thereof;

B. and SEQ ID NO: 3 complementary DNA fragments;

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

1) DNA segment with nucleotide sequence shown in SEQ ID NO. 27;

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

3) 27 by adding, deleting and inserting one or more nucleotide sequences.

In the embodiment of the invention, the detection method of the specific DNA fragment is specifically illustrated by using an EBNA1 truncated fragment, an OriP truncated fragment, an SV40 truncated fragment and a DNA fragment derived from bacteria on a plasmid. The EBNA1 truncated fragment preferably 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 OriP truncated 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, a candidate DNA fragment EBNA1 truncated fragment is used as an amplification sequence, and then a primer is designed according to the amplification sequence, and the result shows that EBNA1 has higher amplification effect and higher detection sensitivity in the aspect of detecting the exogenous gene residue in the human induced pluripotent stem cells.

In the invention, a partial sequence of the candidate DNA fragment OriP is used as an amplification sequence, and then a primer is designed according to the amplification sequence, and the result shows that the OriP has higher amplification effect and higher detection sensitivity in the aspect of detecting the exogenous gene residue in the human induced pluripotent stem cell. Meanwhile, the DNA fragment of the SV40 polyA signal region is used as a detection target, and the result shows that the amplification effect is ideal, and the detection sensitivity is not much different from that of the OriP and EBNA1 truncated fragments.

In the invention, candidate SV40 truncated fragments are used as amplification sequences, and primers are designed according to the amplification sequences, and the result shows that an 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 foreign gene residues in hipscs, which comprises a primer for specifically detecting the specific DNA fragment of claim 1;

in the present invention, the primer preferably includes one or more of the following pairs of primers: a primer pair for specifically detecting the EBNA1 truncated fragment, a primer pair for specifically detecting the OriP truncated fragment and a primer pair for specifically detecting the SV40 truncated fragment.

In the invention, the primer pair for specifically detecting the EBNA1 preferably comprises EBNA1-1-F/EBNA1-1-R, EBNA1-2-F/EBNA1-2-R, EBNA1-3-F/EBNA 1-3-R. The amplification product of EBNA1-1-F/EBNA1-1-R is EBNA 1-1. The amplification product of EBNA1-2-F/EBNA1-2-R is EBNA 1-2. The amplification product of EBNA1-3-F/EBNA1-3-R is EBNA 1-3. The nucleotide sequence of EBNA1-1-F is shown in SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of EBNA1-1-R is shown as SEQ ID NO: 5, respectively. The nucleotide sequence of EBNA1-2-F is shown in SEQ ID NO: 6 is shown in the specification; 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 in SEQ ID NO: 8 is shown in the specification; 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 comprises OriP-1-F/OriP-1-R, OriP-2-F/OriP-2-R, OriP-3-F/OriP-3-R. The amplification product of OriP-1-F/OriP-1-R is OriP-1. The amplification product of OriP-2-F/OriP-2-R is OriP-2. The amplification 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 OriP-1-R is shown as SEQ ID NO: 11 is shown in the figure; the nucleotide sequence of OriP-2-F is shown as SEQ ID NO: 12 is shown in the specification; the nucleotide sequence of OriP-2-R is shown as SEQ ID NO: 13 is shown in the figure; the nucleotide sequence of OriP-3-F is shown as SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of OriP-3-R is shown as SEQ ID NO: shown at 15. The primer pair for specifically detecting the SV40 truncated fragment comprises SV40-F/SV 40-R. The amplification product of SV40-F/SV40-R is an 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 a primer synthesis method well known in the art may be used.

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

In the present invention, the best isOptionally, qPCR detection reagents, such as amplification buffer, enhancement buffer, dNTPs, SYBR green I, MgSO₄And Platinumpfx DNA polymerase and the like.

1) screening specific DNA fragments carried by the free carrier as target DNA;

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

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

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

In order to achieve the purpose of detecting the foreign gene residues of the clinical-grade hipscs, the specific DNA sequence is a specific DNA fragment which is unique to the foreign gene and is not expressed by the clinical-grade hipscs. In the specific DNA fragment provided by the invention, the origin of the contained OriP truncated fragment and EBNA1 truncated fragment is the sequence of the herpes virus, which is an exogenous gene fragment shared by all plasmids used in the reprogramming process, and the sequence is not expressed in the hiPSC.

In the present invention, the concentration of proteinase K is preferably 1 to 50. mu.g/mL, more preferably 1 to 20. mu.g/mL, and most preferably 1. mu.g/mL, when the cells are lysed. The proteinase K is 1-50 mu g/mL for cell lysis, the detection sensitivity reaches 0.1%, and the effect is best when the proteinase K is treated by 1 mu g/mL.

In the present invention, the cell density is preferably 1.0X 10 at the time of cell lysis⁶～4.0×10⁶Mu.l/10. mu.l. The cell density is good in gradient, the data difference between gradients is large, the sensitivity difference is large, the sensitivity can reach 0.01%, the data are stable, and the repeatability is good. Albeit 1.0 × 10⁶～2.0×10⁶Each 10. mu.l and 5.0X 10⁶～6.0×10⁶The detection sensitivity of each 10 mu l can reach 0.1 percent, and the test is unstable for many times. Selective cleavage of 1.0X 10⁶～4.1.0×10⁶Each 10 μ l, preferably 3.0X 10⁶cells～4.0×10⁶Mu.l/10. mu.l.

In the invention, through screening, one or more of EBNA1, an OriP DNA fragment, an SV40 truncated fragment and a plasmid bacteria-derived DNA fragment are used as exogenous gene fragments to evaluate the exogenous gene residue condition in clinical grade hipSC. The qPCR detection primer is EBNA1-F/EBNA1-R and/or OriP-F/OriP-R.

In the present invention, the reaction system for the qPCR detection is preferably as follows: 5-35% of 10 multiplied amplification buffer solution, 5-45% of 10 multiplied enhancement buffer solution, 0.01-10% of dNTPs and 0.1-6 mM MgSO 2₄0.1-25 percent, (1.25-10) xSYBR green I0.01-2.5 percent, forward primer 0.5-10 percent, reverse primer 0.5-10 percent, platinumfx DNA polymerase 0.01-5.5 percent and template DNA 5-50 percent. Wherein SYBR green I、MgSO₄The concentration of the solution is an important factor affecting the detection result. The dilution times of SYBR green I are preferably 10 x, 5 x, 2.5 x and 1.25 x, and all the dilution times can reach 1% of detection sensitivity, wherein 5 x SYBR green I group data are stable, and the repeatability of repeat wells in the group is good. MgSO (MgSO)₄When the concentration of the solution is 10mM or more, the enzymatic reaction is affected and the sensitivity is lowered. 0.1 to 6mM MgSO₄The detection sensitivity is up to 0.1%, preferably 0.1-4 mM MgSO₄Stable experiment, good repeatability, more preferably 1.5mM MgSO₄. The results of the experiment showed 5 XSSYBR green I and 1.5mM MgSO₄For initially selecting the best combination mode.

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

The following examples are provided to detect a foreign gene residue in a hiPSC, and provide a specific DNA fragment, primers, kit and detection method thereof. The detailed description is given without understanding the invention as it is intended to limit its scope.

The term 'DNA sequence' as used 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 hipscs of the present invention are hipscs prepared by the department of biotechnology limited located upstream in the japan of anhui, see CN 201711462604.8; CN 201810137319.7; CN 201810252408.6; CN 201810400878.2; CN 202010313083.5. The method of the present invention is applicable not only to the hipscs obtained by the above preparation method, but also to other methods in the art for obtaining hipscs. The primers mentioned in the examples were synthesized by Nanjing Kinshire BioInc., and plasmids were prepared by this company.

In the following examples, information related to specific process steps can be omitted, and conventional processes or reagents in the art can be used, and the key information related to the process steps is described in the text.

Among these, human embryonic stem cells (hescs), which are isolated or harvested stem cells from human embryos within 14 days of fertilization without in vivo development, are commercially approved embryonic stem cells or stem cells. Human embryonic stem cells (hESCs) in this application were isolated from umbilical cords discarded after obstetric production in Anhui Hospital.

The information of the reagents involved in the experiment is shown in Table 1 below

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

Example 1

Preparation method of positive gradient standard substance

Culturing hESC, when the cell density reaches 80-90%, absorbing and discarding the culture medium, adding DPBS (without calcium and magnesium), absorbing and discarding DPBS, adding Solase digestive juice to digest cells, adding the culture medium to resuspend and suck the cell suspension to a centrifugal tube, centrifuging and collecting the cells, and discarding the supernatant; collecting cells, namely, after the DPBS is resuspended, subpackaging the cells in an EP tube; centrifuging an EP tube, adding a lysine buffer, repeatedly blowing and cracking, transferring into a PCR tube, carrying out vortex oscillation, and standing for 5-15 min at 70-95 ℃; storing at 4 deg.C, treating flocculent precipitate in the final tube, adding Proteinase K (protease K, 10 μ g/ml), vortex oscillating, and standing at 37 deg.C for 30 min; standing at 70 deg.C for 10 min; and (2) storing at 4 ℃ to obtain an hESC lysed cell sample, and performing gradient dilution on the hESC lysed cell sample and the plasmid sample to prepare a positive gradient standard substance, wherein the specific preparation method is described in the following table 2:

and (4) preparing a positive gradient standard substance, and mixing uniformly before use.

Positive gradient standards were prepared by taking corresponding volumes of hESC lysed cell samples and adding the corresponding plasmid gradient standards as in table 2.

TABLE 2 Positive gradient standards

Example 2

Method for screening candidate target DNA sequence

1. Screening candidate DNA sequences of interest: taking the plasmid pCEP4-EGFP as an example, the DNA sequence expression spectrum of the plasmid is searched through the official website of NCBI, 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 candidate specific DNA sequences EBNA1-1, OriP-1 and SV40 truncated fragments in the plasmid by a qPCR method.

Firstly, qPCR method is used, and designed primers are used for respective amplification, 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 a commercial PCRx Enhancer System with addition of SYBR green I, and the other reagents were: the model is as follows: the manufacturer is Thermo: all operations were performed on ice. The reaction system is 10 mul per well, the specific reaction system is prepared as shown in table 3, each sample is provided with 3 multiple wells, and the qPCR conditions are shown in table 5.

TABLE 5 reaction conditions for qPCR

The CT values are recorded.

The Ct values obtained by qPCR according to the qPCR reaction system and qPCR conditions are shown in Table 6.

TABLE 6 Ct values of qPCR assay results

The qPCR method was used in this experiment to select 3 qPCR primers, of which EBNA1-1 was compared with the data: EBNA1-1 detection data are from #1- #6, and according to the plasmid gradient, the data have good gradient, and the data difference between gradients is large, which indicates that the sensitivity difference is large; and the OriP-1 is screened according to the same screening principle, and finally three primers and DNA fragments corresponding to the EBNA-1, OriP-1 and SV40 truncated fragments 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 factor

This example is the same as the method and procedure of examples 1 and 2 above, wherein the hiPSC residual gene sensitivity was measured using dilution factor of 10 × SYBR green i, 5 × SYBR green i, 2.5 × SYBR green i, 1.25 × SYBR green i, 0.625 × SYBR green i, 0.313 × SYBR green i, 0.156 × SYBR green i.Selection 5.5mM MgSO₄Adding SYBR green I with different dilution times to perform qPCR, and screening the SYBR green I concentration with the optimal dilution time. The reaction system and procedure of this example are shown in Table 9.

TABLE 9 reaction System and reaction procedure in SYBR green I dilution factor optimization experiment

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

TABLE 10 DNA sequence qPCR detection results of OriP-1 under different dilution multiple of SYBR green I

According to the qPCR result, 1% sensitivity (1 copy DNA of 100 cells) can be detected by 10, 5, 2.5 and 1.25 SYBR green I concentrations, wherein the 5 SYBR green I data is stable, the repeatability of the repeat wells in the group is good, and the 5 SYBR green I concentration is finally screened. Therefore, 5 XSSYBR green I of 1.25X to 10X can be used in the experiment.

Example 4

MgSO₄Optimization of solution concentration

This example is the same as the method and procedure of examples 1 and 2 above, using 0.1mM MgSO₄、0.5mM MgSO₄、1.5mM MgSO₄、2mM MgSO₄、4Mm MgSO₄、6mM MgSO₄、10mM MgSO₄Solution detection hiPSC residual gene sensitivity. Wherein different concentrations of MgSO were added with 5 × SYBR green I₄Three samples #1, #3 and #6 were selected for qPCR, respectively, and screened for optimal MgSO₄And (4) concentration.

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

TABLE 11 MgSO₄Reaction system and reaction program in solution concentration optimization experiment

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

TABLE 12 MgSO various concentrations₄DNA sequence qPCR detection result of OriP-1 under condition

According to the qPCR result, MgSO more than 10mM₄The concentration is too high, the enzymatic reaction is influenced, and the sensitivity is reduced; 0.1 to 6mM MgSO₄The detection sensitivity is 0.1 percent and 0.1 to 4mM MgSO₄Stable experiment, good repeatability, and preferably 1.5mM MgSO₄。

The experimental results of example 3 and example 4 can be used to screen out the optimal conditions: 5 XSSYBR green I and 1.5mM MgSO₄For initially selecting the best combination mode.

Example 5

Optimization of qPCR reaction cycle number

On the basis of the above example, the method and procedure of the example are the same as those of the above example 1 and example 2, wherein 35, 40, 45, 55 and 60 cycles of detection of hiPSC foreign gene residue sensitivity are respectively selected in the qPCR experiment. Wherein 5 XSSYBR green I and 1.5mM MgSO are used₄qPCR was performed separately on the OriP-1 fragment for optimal combinatorial mode, and the optimal number of qPCR cycles was screened.

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

TABLE 13 optimization of the number of reaction cycles of the qPCR reaction the experimental reaction system and the reaction procedure

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

TABLE 14 DNA sequence results of OriP-1 detection by qPCR

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

Example 6

Optimization of lysis for different cell numbers

On the basis of the above-mentioned example, the method and procedure of the above-mentioned examples 1 and 2 are the same, wherein hescs of different cell numbers are lysed in qPCR experiments, respectively, as templates to detect hiPSC foreign gene residual sensitivity. Wherein 5 XSSYBR green I and 1.5mM MgSO are used₄ Samples #1, #2, #3 and #4 were selected for optimal combination for qPCR, respectively, and the optimal number of cell lysates were screened for qPCR detection. Wherein different cell numbers were optimized in either 10ul or 20ul systems.

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

TABLE 15 optimization of experimental reaction systems and procedures for lysis of different cell numbers

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

TABLE 16 results of the assay with different numbers of lysed cells

As a result: from #1 to #6, according to the plasmid gradient, 3.0X 10⁶cells group and 4.0X 10⁶The cell data has good gradient performance, and the data difference between gradients is large, which shows that the sensitivity difference is large, the sensitivity can reach 0.01%, the sensitivity can reach 0.1% in multiple tests, the data is stable, and the repeatability is good. And 1.0X 10⁶cells and 2.0X 10⁶The sensitivity of the detection result of the cells group can reach 0.1% to the maximum; 5.0X 10⁶The detection sensitivity of the cells group can reach 0.1%, but the detection sensitivity of the cells group is unstable in multiple tests, so that the selective lysis is 1.0 multiplied by 10⁶cells～4.0×10⁶cells are all, preferably 3.0X 10⁶cells～4.0×10⁶cells。

Example 7

Different concentrations of proteinase K treated samples

Based on the above embodiment, which is the same as the method and steps of the above embodiment 1 and embodiment 2, adding different concentrations of proteinase K when lysing the cells (hESC or hipSC) to obtain the template has an important effect on the qPCR detection to screen the optimal proteinase K usage concentration and more stably and accurately detect the foreign gene residue detection in the hipSC, wherein 5 × SYBR green I and 1.5mM MgSO 5 are used₄Respectively carrying out qPCR for the optimal combination mode, and screening the protease k with the optimal concentration to carry out qPCR detection. The reaction system of this example is shown in Table 17.

TABLE 17 Experimental reaction systems and procedures for treating samples with different concentrations of proteinase K

The results of the DNA sequence of OriP by qPCR detection are shown in table 18.

TABLE 18 results of measurement of samples treated with proteinase K at different concentrations

qPCR results: from #1 to #6, the experimental data for each column group was analyzed according to plasmid gradient: the detection data of 1-50 mug/ml proteinase K are shown in corresponding columns and can be selected, and the sensitivity can reach 0.1%; 1-20 mu g/mL proteinase K detection data are displayed in corresponding columns and can be selected, the sensitivity can reach 0.01%, when 1ug/mL proteinase K is used for processing a template, the effect is best, qPCR result data are stable, good gradient difference exists, the gradient between standard product groups is obvious, the low copy DNA concentration of the standard product can be effectively protected, the exogenous DNA added by DNA enzyme degradation in cells is prevented, and the low copy exogenous DNA concentration of iPSC is protected.

Example 8

Random intercepting fragment 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 as follows EBNA1-2, and the sequence is as follows:

CTGGTCCAGATGTGTCTCCCTTCTCTCCTAGGCCATTTCCAGGTCCTGTACCTGGCCCCTCGTCAGACATGATTCACACTAAAAGAGATCAATAGACATCTTTATTAGACGACGCTCAGTGAATACAGGGAGTGCAGACTCCTGCCCCCTCCAACAGCCCCCCCACCCTCATCCCCTTCATGGTCGCTGTCAGACAGATCCAGGTCTGAAAATTCCCCATCCTCCGAACCATCCTCGTCCTCATCACCAATTACTCGCAGCCCGGAAAAC(SEQ ID NO:19，)。

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

CTCTATGTCTTGGCCCTGATCCTGAGCCGCCCGGGGCTCCTGGTCTTCCGCCTCCTCGTCCTCGTCCTCTTCCCCGTCCTCGTCCATGGTTATCACCCCCTCTTCTTTGAGGTCCACTGCCGCCGGAGCCTTCTGGTCCAGATGTGTCTCCCTTCTCTCCTAGGCCATTTCCAGGTCCTGTACCTGGCCCCTCGTCAGACATGATTCACACTAAAAGAGATCAATAGACATCTTTATTAGACGACGCTCAGTGAATACAGGGAGTGCAGACTCCTGCCCCCTCCAACAGCCCCCCCACCCTCAT(SEQ ID NO:20)。

detecting the residue of the foreign gene in the hiPSC by using the EBNA1-2 amplification sequence and the EBNA1-3 amplification sequence, and designing a primer aiming at the sequence; primer sequences were designed as in Table 19 below, with the internal reference and primers identical to those of example 2 above.

TABLE 19 primer sequence Listing

Primer name	Sequence of
		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)

Selection of the preferred concentrations or ranges for the components or conditions based on the above examples 1-7 was conducted qPCThe details of the R detection are shown in Table 20. Wherein 5 XSSYBR green I, 1.5mM MgSO ₄1. mu.g/mL proteinase K-treated template, 3.0X 10⁶～4.0×10⁶The cell lysis density and the number of qPCR45 cycles are the best combination, and samples #1, #2, #3 and #4 are respectively selected for qPCR.

TABLE 20 reaction System and reaction procedure

The results of detection of the hiPSC foreign gene residues by randomly cutting out the DNA sequence fragment of EBNA1 and detecting the DNA sequence fragment of EBNA1 by qPCR are shown in table 21.

Table 21 results of qPCR detection of fragments of DNA sequence of EBNA1

qPCR results: from #1 to #4, the experimental data for each column group was analyzed according to plasmid gradient: the DNA sequence EBNA1-2 or EBNA1-3 has gradient differences in qPCR results under the condition of plasmid gradient, the gradient differences in each column are obvious, the gradient sensitivity can reach 0.1% correspondingly (compared with the results of #1-0, the difference of the results of # 3-0.1% is larger than 2, effective gradient detection can be considered), and the sensitivity of the detection result of some groups can reach 0.01%. The DNA sequence fragment of the EBNA1 sequence fragment region which is randomly truncated can be used for detecting the residue of the hipSC foreign gene.

Example 9

Randomly intercepting fragments in DNA sequence of OriP in plasmid sequence for hiPSC exogenous gene residue detection

Randomly intercepting the DNA sequence of the fragment region of the OriP sequence in the plasmid sequence, wherein the specific sequence is as follows: TATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGCATATACAGTCAGCATATGATACCCAGTAGTAGAGTGGGAGTGCTATCCTTTGCATATGCCGCCACCTCCCAAGGGGGCGTGAATTTTCGCTGCTTGTCCTTTTCCTGCTGGTTGCTCCCATTCTTAGGTGAATTTAAGGAGGCCAGGCTAAAGCCGTCGCATGTCTGATTGCT (SEQ ID NO: 22').

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

CTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGCATATACAGTCAGCATATGATACCCAGTAGTAGAGTGGGAGTGCTATCCTTTGCATATGCCGCCACCTCCCAAGGGGGCGTGAATTTTCGCTGCTTGTCCTTTTCCTGCTGGTTGCTCCCATTCTTAGGTGAATTTAAGGAGGCCAGGCTAAAGCCGTCGCATGTCTGATTGCTCACCAGGTAAATGTCGCTAATGTTTTCCAACGCGAGAAGGTGT(SEQ ID NO:23)。

detecting the residue of the foreign gene in the hiPSC based on the OriP-2 amplification sequence and the OriP-3 amplification sequence, and designing a primer aiming at the sequence; primer sequences were designed as in Table 22 below, with the internal reference primers identical to those of example 2 above.

TABLE 22 primer sequence Listing

The qPCR assay was performed by selecting the components or conditions based on the above examples 1-7, preferably using concentrations or ranges, as detailed in table 23. Wherein 5 XSSYBR green I, 1.5mM MgSO ₄1. mu.g/mL proteinase K-treated template, 3.0X 10⁶～4.0×10⁶The cell lysis density and the number of qPCR45 cycles are the best combination, and samples #1, #2, #3 and #4 are respectively selected for qPCR.

TABLE 23 reaction System and reaction procedure for qPCR detection

The results of detection of hiPSC foreign gene residues by randomly cutting out a fragment of the OriP DNA sequence and qPCR detection of the fragment of the OriP DNA sequence are shown in table 24.

Table 24 fragment qPCR detection results of DNA sequence of OriP

qPCR results: from #1 to #4, the experimental data for each column group was analyzed according to plasmid gradient: the DNA sequence OriP-2 or OriP-3 has gradient difference in qPCR result under plasmid gradient, and the gradient difference in each column is obvious, the gradient sensitivity can reach 0.1% correspondingly (compared with the result of #1-0, the result difference of # 3-0.1% is greater than 2, which can be regarded as effective gradient detection), and the sensitivity of the detection result of some column groups can reach 0.01%. The random cut DNA sequence fragment of the OriP sequence fragment region can be used for detecting the residue of the hipSC exogenous gene.

Example 10

Detection of foreign gene residues in hipscs of different generations

The method selects 3 different generations of hipscs as cell samples to be detected, and detects the residual quantity of the foreign genes in the hipscs by adopting the detection method of screening target DNA sequences (OriP DNA sequence and EBNA1 DNA sequence) in the above embodiment. The selected hipscs are seed cell Bank (seed), Master cell Bank (Master cell Bank/MCB) and working cell Bank (working cell Bank/WCB). The seed cell bank is primary differentiated cells and is P10 generation; the main cell bank is P15 generation cells; the working cell bank was P20 generation cells. And respectively selecting corresponding secondary cells from the cell banks, and detecting the foreign gene residue in different cell banks. The preferred concentrations and ranges of the components were selected based on the above examples for qPCR detection, as detailed in table 25. Wherein 5 XSSYBR green I, 1.5mM MgSO ₄1. mu.g/mL proteinase K-treated template, 3.0X 10⁶～4.0×10⁶Cell lysis Density, qPCR45 cycles numberThe combination is preferred.

TABLE 25 detection results of foreign gene residues in hipscs from different generations

The results of DNA sequence detection by qPCR for OriP-1 and EBNA1-1 are shown in Table 26.

TABLE 26 Ct values of the results of detection of DNA sequences of OriP-1 and EBNA1-1

As can be seen from the results, the test method of the present application was used for the test by the q-PCR method from #1 to #6, and the experimental data of each column group were analyzed according to the plasmid gradient: the data of the standard substance is stable and has good gradient difference, the detection data of Seed, MCB and WCB samples can reach the sensitivity of 0.01-0.1%, the detection of the residual quantity of exogenous genes in the hipSC can be realized by OriP or EBNA1 sequences, the limit is stable and reaches 0.1%, and the sensitivity of MCB and WCB groups can reach 0.01%, and the method is stable and simple.

Example 11

Respectively reprogramming free plasmids nEP4-E-EGFP, PMEP4-C-EGFP and PKEP4-E-EGFP to prepare hipSC, and detecting residue of foreign genes of the hipSC

The residues of foreign genes in the hipscs prepared by reprogramming the episomal plasmid vectors nEP4-E-EGFP, PMEP4-C-EGFP and PKEP4-E-EGFP were detected using the amplification sequences of OriP-1 and primers thereof in examples 2-3 above.

The preferred concentrations and ranges of the components are selected based on the above examples, and qPCR detection is performed, as shown in Table 26. Wherein 5 XSSYBR green I, 1.5mM MgSO ₄1. mu.g/mL proteinase K-treated template, 3.0X 10⁶～4.0×10⁶Cell lysis density, qPCR45 cycle number were the best combinations.

TABLE 26 qPCR reaction System

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

Table 27 DNA sequence results Ct values of OriP-1

As can be seen from the results, the detection method of the present application, which is carried out by the q-PCR method, was used for the analysis of experimental data of each group from #1 to #6 according to the plasmid gradient: the data of the standard substance is stable and has good gradient difference, the detection data of hiPSC samples prepared by reprogramming the free plasmids nEP4-E-EGFP, PMEP4-C-EGFP and PKEP4-E-EGFP respectively can reach the sensitivity of 0.01-0.1 percent, the method, the sequence, the primer and the like can realize the detection of the residual quantity of exogenous genes in the hiPSC, the limit is stable and reaches 0.1 percent, and the higher limit can reach 0.01 percent, and the method is stable and simple.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shang Sheng Yuan Biotechnology Co., Ltd in Anhui

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tgcaatatga tactggggtt attaagatgt gtcccaggca gggaccaaga caggtgaacc 1080

atgttgttac actctatttg taacaagggg aaagagagtg gacgccgaca gcagcggact 1140

ccactggttg tctctaacac ccccgaaaat taaacggggc tccacgccaa tggggcccat 1200

aaacaaagac aagtggccac tctttttttt gaaattgtgg agtgggggca cgcgtcagcc 1260

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 specific DNA fragment for detecting foreign gene residues in hipSCS, wherein the specific DNA fragment is derived from an episomal vector used in preparation of hipSCS.

2. The specific DNA fragment as claimed in claim 1, wherein the episomal vector comprises one or more of the following episomal vectors: episomal vectors based on one or several of OriP/EBNA1, S/MAR, SV40, BKV, BPV-1.

3. The specific DNA fragment of claim 2, wherein 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:

D. intercepting a truncated DNA sequence with the total length of more than 70bp of a bacterial source DNA fragment region on the plasmid;

1) DNA segment with nucleotide sequence as shown in SEQ ID No. 1;

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

1) DNA segment with nucleotide sequence shown in SEQ ID NO. 2;

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

A. the nucleotide sequence is shown as SEQ ID NO: 3, or a DNA fragment thereof;

B. and SEQ ID NO: 3 complementary DNA fragments;

1) DNA segment with nucleotide sequence shown in SEQ ID NO. 27;

2) a DNA fragment complementary to 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 bacterially derived DNA truncated fragments on plasmids;

4. A primer for detecting foreign gene residues in hipSC, comprising a primer for specifically detecting the specific DNA fragment of claim 1;

preferably, the primers comprise one or more of the following pairs of primers: a primer pair for specifically detecting the EBNA1 truncated segment, a primer pair for specifically detecting the OriP truncated segment, a primer pair for specifically detecting the SV40 truncated segment and a primer pair for specifically detecting the bacterium-derived DNA segment on the plasmid;

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

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

5. A kit for detecting foreign gene residues in hipscs based on qPCR technology, comprising the primer of claim 4.

6. Use of the specific DNA fragment of any one of claims 1 to 3, the primer of claim 4, and the kit of claim 5 for detecting foreign gene residues in clinical-grade hipscs.

7. A method for detecting exogenous gene residues based on a qPCR technology clinical-grade hipSC is characterized by comprising the following steps:

1) screening specific DNA fragments carried by the free carrier as target DNA;

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

8. The detection method according to claim 7, wherein the reaction system of qPCR detection is as follows:

the forward primer and the reverse primer are the primers of claim 4.

9. The assay of claim 7, wherein the reaction sequence of the qPCR assay is as follows:

10. The detection method according to any one of claims 7 to 9, wherein the concentration of proteinase K is 1 to 50 μ g/ml when the cells are lysed in step 1);