CN117535390A - Primer pair, kit and detection method for detecting exogenous gene residues in cell products - Google Patents

Abstract

The invention provides a primer pair, a kit and a detection method for detecting exogenous gene residues in a cell product, and belongs to the technical field of biology. The primer pair qPCR method provided by the invention is used for detecting exogenous gene residues in cell products, the detection limit (sensitivity) can reach 0.0003 plasmid copy number/cell number (the converted plasmid concentration is 0.0876 fg/mu l), and the method has the characteristics of high sensitivity, good specificity and high detection efficiency. Therefore, the invention can detect the exogenous gene residue in the cell product with high sensitivity, and can better ensure the clinical application safety of the cell product.

Description

Primer pair, kit and detection method for detecting exogenous gene residues in cell products

Technical Field

The invention relates to the technical field of biology, in particular to a primer pair, a kit and a detection method for detecting exogenous gene residues in cell products.

Background

Human induced pluripotent stem cells (human induced pluripotent stem cell, hiPSC) are a class of stem cells that can proliferate indefinitely in vitro and differentiate into the potential of multiple cell types in vivo. Among them, hiPSC is a pluripotent stem cell obtained by reprogramming by expressing a specific gene or a specific gene product or compound treatment in a differentiated somatic cell, and has several advantages of avoiding social ethical problems, abundant sources, unrestricted genetic background, and the like. Therefore, hiPSC has wide application prospect in clinical cell therapy.

Current clinical grade hiPSC reprogramming systems are often constructed based on episomal DNA plasmid vectors, whereby the ipscs obtained may risk reprogramming DNA plasmid vector residues. In addition, gene Editing (Gene Editing) techniques are also often used for iPSC. The gene editing is a genetic engineering technology capable of modifying specific target genes in a cell genome, and can realize the directed editing of the specific genes. In vitro, the modification system constructed by adopting the genetic engineering technology can effectively transfer genetic materials and the like into specific target cells, and is used for modifying the genetic materials of the target cells, changing the gene expression mode, regulating the biological characteristics of the cells and the like. Through gene editing, endogenous genes can be modified in fixed points in the iPSC or exogenous genes can be introduced, and the screened monoclonal iPSC can be used for producing functional cell products. However, foreign gene DNA fragments may remain during gene editing.

The foreign gene remaining in hiPSC is mainly derived from episomal vectors or gene editing vectors used in the preparation of hiPSC. These vectors will typically carry a sequence of a resistance gene for an antibiotic. Kanamycin resistance gene (Kanamycin, kana) is a common resistance gene and can be used for detecting exogenous DNA residues in cells transfected with a plasmid containing the Kana resistance gene. Therefore, detection of DNA of Kana gene at a molecular level can be used to specifically detect the residue of a plasmid containing Kana resistance gene, thereby calculating and judging the residual level of foreign DNA. At present, in an effective method for detecting exogenous DNA residues of cell products, a quantitative qPCR detection method is to add a fluorescent group into a PCR reaction system, monitor and analyze fluorescent signals in the reaction process to quantitatively detect nucleic acid, and is a detection means with higher reliability in the safety control of the cell products. The prior art discloses a technical scheme for detecting exogenous plasmid residues by combining a primer pair and a probe for a Kana resistance gene or a truncated fragment thereof by using real-time fluorescence quantitative PCR, but the detection sensitivity of the scheme is 20 fg/. Mu.l, and the sensitivity is required to be further improved so as to ensure the safety of functional cell products.

Foreign genes (reprogramming vector residues, or gene editing vector residues) remaining in hipscs are impurities associated with cell product processes that persist as residual impurities in clinically treated functional cells with potential safety risks, particularly infectious, oncogenic risks. Therefore, it is necessary to establish a high-sensitivity detection method for detecting the residual amount of the foreign gene to ensure the safety of the functional cell product.

Disclosure of Invention

In order to solve the above problems, the present invention provides a primer pair for detecting foreign gene residues in a cell product, the primer pair comprising an upstream primer and a downstream primer for amplifying a specific DNA fragment in the foreign gene, the specific DNA fragment comprising a truncated sequence of a resistance gene and a truncated sequence of an adjacent element thereof, a position at which one of the upstream primer and the downstream primer binds to the specific DNA fragment being on the adjacent element or bridged to the adjacent element and the resistance gene, and a position at which the other primer binds to the specific DNA fragment being on a downstream segment of 650bp or less in length of the resistance gene.

Preferably, the resistance gene is one selected from the group consisting of kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, and neomycin resistance gene.

Preferably, the adjacent element is an upstream promoter or a downstream origin of replication.

Preferably, the upstream promoter is one selected from the group consisting of an AmpR promoter, a CMV promoter, an EF1a promoter, an SV40 promoter, a T7 promoter, and a T7lac promoter.

Preferably, the downstream origin of replication is one selected from the group consisting of a pUC origin of replication, a pBR322 origin of replication, an F1 origin of replication, a ColE1 origin of replication and a pMB1 origin of replication.

Preferably, the resistance gene is a kanamycin resistance gene and the upstream promoter is an AmpR promoter.

Preferably, the resistance gene is a kanamycin resistance gene, and the downstream origin of replication is a pUC origin of replication.

Preferably, the primer pair is SEQ ID NO:13 and SEQ ID NO: 14.

Preferably, the primer pair is any one of the following primer pairs:

SEQ ID NO:15 and SEQ ID NO:16, a primer pair shown in FIG. 16;

SEQ ID NO:17 and SEQ ID NO:18, a primer pair shown in FIG; and

SEQ ID NO:19 and SEQ ID NO: 20.

Preferably, the primer pair is SEQ m NO:17 and SEQ ID NO:18, or the primer set forth in SEQ ID NO:19 and SEQ ID NO: 20.

Preferably, the specific DNA fragment is a sequence selected from the group consisting of SEQ ID NOs: 24. SEQ ID NO: 25. SEQ ID NO:26 and SEQ ID NO: 27.

Preferably, the resistance gene is a kanamycin resistance gene, and the downstream segment of the resistance gene having a length of 650bp or less consists of SEQ ID NO: 28.

The invention provides a kit for detecting exogenous gene residues in a cell product, which comprises the primer pair.

The invention provides a method for detecting exogenous gene residues in a cell product, which comprises the following steps:

lysing the cell product to obtain a test article;

lysing the negative cells to obtain a negative control;

mixing the negative reference substance with positive reference substances with different copy numbers to obtain a positive gradient reference substance;

the primer pair or the kit is used for qPCR detection on a negative control substance and a positive gradient standard substance, so that the detection limit is determined according to the detection value;

using the primer pair or the kit, and carrying out qPCR detection on a sample by taking a positive standard substance corresponding to the detection limit in the positive gradient standard substances as a reference to obtain a detection value of the sample;

and comparing the detection value of the test sample with the detection value of the positive standard sample corresponding to the detection limit, and judging the residual limit of the exogenous gene in the cell product.

Preferably, the cell product is a human induced pluripotent stem cell product, or a human induced pluripotent stem cell-derived cell product.

Advantageous effects

The primer pair provided by the invention is a specific binding fragment which takes a specific DNA fragment containing a resistance gene truncated sequence and an adjacent element truncated sequence thereof as a template (instead of taking the resistance gene or the truncated fragment thereof as the template), and the primer pair is used for detecting exogenous gene residues in cell products by a combined qPCR method, so that the primer pair has the characteristics of good specificity, high sensitivity and good amplification efficiency. The primer pair provided by the invention has the detection limit (sensitivity) reaching 0.0003 plasmid copy number/cell number (the converted plasmid concentration is 0.0876 fg/. Mu.l), and compared with the prior art, the primer pair greatly improves the detection sensitivity and can better ensure the clinical application safety of cell products. The detection method using the primer pair of the invention is accurate and reliable, has high sensitivity and high detection efficiency, and after the detection limit of the primer pair is confirmed, the standard curve sample detection is not needed in the detection process, the detection is simple and convenient, and the detection efficiency is obviously improved.

Drawings

The invention will be further described with reference to the drawings and examples. In the drawings:

FIG. 1 is an amplification melting curve of primer pair 1;

FIG. 2 is an amplification melting curve of primer pair 2;

FIG. 3 is an amplification melting curve of primer pair 3;

FIG. 4 is an amplification melting curve of primer pair 4;

FIG. 5 is an amplification melting curve of primer pair 5;

FIG. 6 is an amplification melting curve of primer pair 6;

FIG. 7 is an amplification melting curve of primer pair 7;

FIG. 8 is an amplification melting curve of primer pair 8;

FIG. 9 is an amplification melting curve of primer pair 9;

FIG. 10 is an amplification melting curve of primer pair 10.

Detailed Description

The primer pair for detecting foreign gene residues in cell products provided by the invention comprises an upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are used for amplifying a specific DNA fragment in the foreign gene, the specific DNA fragment comprises a resistance gene truncated sequence and an adjacent element truncated sequence thereof, the position of one primer in the upstream primer and the downstream primer combined with the specific DNA fragment is on the adjacent element or bridged on the adjacent element and the resistance gene, and the position of the other primer combined with the specific DNA fragment is on a downstream segment with the length of 650bp or less of the resistance gene.

In the present invention, the foreign gene remaining in the cell product is mainly derived from the reprogramming vector or the gene editing vector used in the preparation of hiPSC. The reprogramming or gene editing vector may be a conventional vector such as GenCRISPR ^TM A plasmid or other suitable vector, so long as it can be used to deliver a reprogramming factor or an endonuclease that recognizes and cleaves specific targeting DNA. Such endonucleases include, but are not limited to, zinc finger enzymes, transcription activator-like effector nucleases and CRISPR-Cas nucleases. Vectors for delivery of reprogramming factors or endonucleases include, but are not limited to, plasmids, phages, animal viruses and cosmids. The vector may be an expression vector, including eukaryotic expression vectors and viral expression vectors. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesvirusesToxic and lentiviral.

The primer pair according to the present invention uses a specific DNA fragment in the foreign gene as an amplified fragment. The specific DNA fragment according to the present invention is not a resistance gene or a truncated fragment thereof, but comprises or consists of a resistance gene truncated sequence and its adjacent element truncated sequence, or comprises or consists of a resistance gene partial sequence and its adjacent element partial sequence. In other words, a specific DNA fragment according to the invention spans the resistance gene truncated sequence and its adjacent element truncated sequences (two sequences are contiguous). In the present invention, a truncated sequence or a truncated fragment refers to a partial sequence or fragment taken from the full-length sequence of a gene or element. The primer pair according to the present invention has higher sensitivity than a primer pair having a resistance gene or a truncated fragment thereof as an amplified fragment.

According to the present invention, the location at which the upstream and downstream primers bind to the specific DNA fragment has a significant effect on the amplification efficiency and sensitivity of the primers. In the present invention, one of the upstream primer and the downstream primer is bound to the specific DNA fragment at a position on the adjacent element or bridged to the adjacent element and the resistance gene, and the other primer is bound to the specific DNA fragment at a position on the downstream segment of the resistance gene having a length of 650bp or less. In other words, one of the upstream and downstream primers complementarily pairs with one corresponding DNA strand in the specific DN a fragment at a position on or bridging the adjacent element to the resistance gene, while the other primer complementarily pairs with the other corresponding DNA strand in the specific DNA fragment at a position on the downstream segment of the resistance gene of 650bp or less in length. The primer pair obtained by the method has better specificity, better amplification efficiency and high sensitivity.

In the present invention, the downstream segment of the resistance gene is the segment 3 'to the resistance gene, and the upstream segment of the resistance gene is the segment 5' to the resistance gene. In the present invention, the adjacent element may be an upstream promoter or a downstream origin of replication. When the upstream adjacent element of the resistance gene is a promoter and the downstream adjacent element thereof is an origin of replication, the downstream segment of the resistance gene is a segment thereof near one end of the origin of replication, and the upstream segment of the resistance gene is a segment thereof near one end of the promoter. In the present invention, the length of the downstream segment of the resistance gene is 650bp or less. Examples of such downstream segments of 650bp or less in length may include SEQ ID NO:28, a sequence shown in seq id no:

SEQ ID NO:29, a sequence shown in seq id no:

the method comprises the steps of carrying out a first treatment on the surface of the Or (b)

SEQ ID NO:30, a sequence shown in seq id no:

in the present invention, the upstream promoter may be a eukaryotic promoter or a prokaryotic promoter. Preferably, the upstream promoter is one selected from the group consisting of an AmpR promoter, a CMV promoter, an EF1a promoter, an SV40 promoter, a T7 promoter, and a T71ac promoter. In the present invention, the promoter may be an AmpR promoter, the sequence of which is SEQ ID NO:21:

in the present invention, the downstream origin of replication may be a conventional plasmid origin of replication. Preferably, the downstream origin of replication is one selected from the group consisting of a pUC origin of replication, a pBR322 origin of replication, an F1 origin of replication, a ColE1 origin of replication and a pMB1 origin of replication. The origin of replication may be a pUC origin of replication, i.e. an origin of replication of the UC protein, the sequence of which is SEQ ID NO:23:

in the present invention, the resistance gene may be a common resistance gene selected from the group consisting of kanamycin gene (Kana), ampicillin gene (AmpR), tetracycline gene (TetR), chloramphenicol gene (CmrR) and neomycin resistance gene (NeoR), and preferably the resistance gene is kanamycin gene. The resistance genes of the present invention are common resistance genes used to screen positive clones, and thus, the remaining reprogramming or gene editing vectors in the cell product will typically comprise the specific DNA fragments of the present invention. Therefore, the specific DNA fragment provided by the invention can be widely applied to detection of exogenous DNA residues in cells after reprogramming or gene editing as a detection target, and has a wider application range compared with other detection targets such as an origin of replication, EBNA and the like. Kana has the sequence of SEQ ID NO:22

In the present invention, the primer pair may be any one of the following primer pairs:

the upstream primer SEQ ID NO:13:the downstream primer SEQ ID NO:14: />

The upstream primer SEQ ID NO:15:the downstream primer SEQ ID NO:16: />

The upstream primer SEQ ID NO:17:the downstream primer SEQ ID NO:18: />And

the upstream primer SEQ ID NO:19:the downstream primer SEQ ID NO:20: />

In the present invention, in view of better amplification efficiency and higher detection sensitivity, the sequence of SEQ ID NO:15 and SEQ ID NO:16, the primer pair shown in SEQ ID NO:17 and SEQ ID NO:18, or the primer set forth in SEQ ID NO:19 and SEQ ID NO:20, a primer pair shown in FIG. 20; more preferably the primer pair is SEQ ID NO:17 and SEQ ID NO:18, or the primer set forth in SEQ ID NO:19 and SEQ ID NO: 20. Most preferably the primer pair is SEQ ID NO:19 and SEQ ID NO:20, because the primer pair has better specificity in addition to better amplification efficiency and higher detection sensitivity.

The primer set of the present invention can be synthesized by primer synthesis methods well known in the art, such as chemical synthesis methods.

In the present invention, the specific DNA fragment comprises a truncated sequence of the resistance gene and truncated sequences of adjacent elements thereof. Specific examples of the specific DNA fragment may include the following sequences:

SEQ ID NO：24：

SEQ ID NO：25：

SEQ ID NO：26：

SEQ ID NO：27：

the invention provides a kit for detecting exogenous gene residues in a cell product, which comprises the primer pair. The kit preferably further comprises a qPCR detection reagent, and the source of the qPCR detection reagent is not particularly limited, and any qPCR detection reagent such as qPCR Mix and DNA polymerase known in the art can be used. The kit of the invention also contains a fluorescent dye such as SYBR Green I. The kit preferably further comprises a positive gradient standard. The positive gradient standard may be plasmids (reprogramming plasmids or gene editing plasmids) containing specific DNA fragments of the invention at different copy numbers. The kit preferably further comprises a negative control. The negative control may be a DNA solution obtained after lysing the negative cells. The kit preferably further comprises a blank. The blank may be a cell lysate. The cell lysate is a conventionally used cell lysate.

The detection method of the invention takes the specific DNA fragment containing the resistance gene truncated sequence and the adjacent element truncated sequence as the detection target. The method comprises the following steps: lysing the cell product to obtain a test article; lysing the negative cells to obtain a negative control; mixing the negative reference substance with positive reference substances with different copy numbers to obtain a positive gradient reference substance; the primer pair or the kit is used for qPCR detection on a negative control substance and a positive gradient standard substance, so that the detection limit is determined according to the detection value; the primer pair or the kit of the invention is used, a positive standard corresponding to the detection limit in the positive gradient standard is used as a reference, and qPCR detection is carried out on a sample to be detected to obtain a detection value; and comparing the detection value of the test sample with the detection value of the positive standard sample corresponding to the detection limit, and judging the residual limit of the exogenous gene in the cell product.

The cell products described herein are human induced pluripotent stem cell (hiPSC) products or hiPSC derived cell products. The hiPSC product comprises a genetically modified hiPSC cell product or an unmodified hiPSC cell product. hiPSC derived cell products may Include Cardiomyocytes (iCM), NK cells (iNK), T cells (iT), dopaminergic precursor cells (imDAP), mesenchymal stem cells (iMSC), islet cells, etc., as well as CAR-iNK cells, CAR-iT cells, etc. The cell products of the invention may be prepared using conventional reprogramming or gene editing techniques or other suitable reprogramming or gene editing techniques. hiPSC cells can be obtained, for example, by the method described in CN 108373998. For example, iNK cells may be obtained with reference to the method described in CN202010153358.3, imDAP cells may be obtained with reference to the method described in CN202210329415.8, immsc cells may be obtained with reference to the method described in CN201910884482.4, and so on.

In the present invention, cell lysates are used to lyse cell products and negative cells. The cell lysate is a conventionally used cell lysate.

In the present invention, when the cell product to be detected is a hiPSC cell product that has not been genetically modified, human embryonic stem cells (hescs) are used as negative cells. Human embryonic stem cells (hescs) are similar to hiPSC cells in cell structure and function. The hescs are human embryo isolated or obtained without undergoing fertilization for less than 14 days of in vivo development, and are commercially obtained embryonic stem cells or stem cells. When the cell product tested was a genetically modified hiPSC cell product, the non-genetically modified hiPSC cell product was used as a negative cell. When the cell product to be tested is an unmodified hiPSC derived cell product, such as iNK cells, the corresponding primary cells, such as PBNK cells, are used as negative cells. When the cell product to be detected is a genetically modified hiPSC derived cell product, e.g., a CAR-iT cell, the corresponding non-genetically modified hiPSC derived cell product, e.g., an iT cell, is used as a negative cell.

In the invention, protein K can be added after cell lysis to degrade protein in cell lysate and degrade or inhibit endogenous DNase, thereby protecting low copy positive standard such as plasmid concentration and ensuring more accurate and higher sensitivity of the method.

In the present invention, as the positive standard, a positive plasmid, for example, a plasmid used in the preparation of hiPSC, may be used.

qPCR assays are performed in the present invention using qPCR methods, which are well known in the art. qPCR methods may include probe methods and dye methods, and in the present invention, qPCR dye methods are preferably used, and detection of amplified products is performed by adding a fluorescent dye such as SYBR Green I dye to the amplification system.

In the invention, the detection limit of the primer pair can be determined by comparing the detection value of the positive gradient standard substance with the detection value of the negative control substance to determine the positive standard substance with obvious difference in statistics, dividing the limit by the 95% confidence interval of the positive standard substance and judging the detection rate of the positive gradient standard substance and the detection rate of the negative control substance.

Examples

The present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the invention.

The reagents and sources of raw materials in the following examples are described below, and it should be noted that the present invention is not limited to the sources of the reagent materials used.

The cell lysate is prepared by the Anhui middling traceable biotechnology limited company according to the conventional formula of the cell lysate; proteinase K (cat# RT 403) was purchased from Tiangen; qPCR Mix is prepared by the Ministry of advanced biotechnology Co., ltd in Anhui according to the formula disclosed in patent application CN 202111668786.0; SYBR Green I is available from Solabio corporation; NC (DNA solution obtained after cleavage of hiPSC without genetic modification) was derived from the Ministry of Biotechnology Inc. in Anhui; the test sample (DNA solution obtained after the cleavage of the genetically modified hiPSC) is derived from the flourishing biotechnology company of Anhui; the plasmid was synthesized by Nanjing Jinsri biotechnology limited; the primer pair was synthesized by Nanjing Jinsri biosciences.

Example 1: designing specific primer pairs in different sequence sections of plasmid

pUC57 vector-based plasmids (full length 8550 bp) containing the Kana resistance tag, including the upstream AmpR Promoter (Promoter) (SEQ ID NO: 21), the Kana resistance gene (SEQ ID NO: 22), and the downstream pUC origin of replication (pUC origin) (SEQ ID NO: 23). Primers were designed using, as templates, a specific DNA fragment consisting of a Kana resistance gene truncated sequence and an adjacent upstream AmpR promoter truncated sequence, a specific DNA fragment consisting of a Kana resistance gene truncated sequence and an adjacent downstream pUC replication origin truncated sequence, and a Kana resistance gene or a truncated fragment thereof, respectively, as shown in table 1:

TABLE 1 primer pair sequence information

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Example 2: specificity and sensitivity verification of primer pairs

And screening primer pairs with good specificity and high sensitivity through a melting curve and CT values.

The pUC57 vector-based plasmid containing the Kana resistance tag was selected, and the plasmid mass of a prescribed copy number was calculated based on its length and molecular weight, and the concentration and copy number of such plasmid stock was calculated and confirmed therefrom. Plasmid stock was diluted according to copy number gradient, the dilution method is shown in table 2:

TABLE 2 plasmid stock gradient dilution protocol

After accurate counting of negative cells (hiPSCs not genetically modified), 2X 10 cells were taken ⁶ After adding 85 μl of cell lysate into each cell, vortex shaking, adding protease K, and lysing the cells at 56 ℃, and directly using the negative cell DNA solution obtained after lysis as a sample for detection. The negative cell DNA solution and the positive plasmid were mixed as shown in Table 3, and samples having different ratios of the number of copies of the positive plasmid to the number of negative cells were obtained as a standard for confirming the detection limit of the primer.

TABLE 3 preparation of qPCR cell samples

Detection targets in NTC (cell lysate), NC (DNA solution obtained after hiPSC cleavage without genetic modification), and positive gradient standard (gradient diluted plasmid and NC mixed solution) were detected by qPCR method, respectively. In the present invention, the qPCR reaction system used is shown in table 4, and the qPCR reaction procedure used is shown in table 5:

TABLE 4 qPCR reaction System

Component (A)	Volume of
		qPCR Mix	5.95μl
Primer F (Forward Primer) (20. Mu.M)	0.2μl
		Primer R (reverse Primer) (20. Mu.M)	0.2μl
Platinum Pfx DNA Polymerase (polymerase)	0.1μl
		Templite (Template)	3.55μl
Total volume (Total volume)	10μl

TABLE 5 qPCR reaction procedure

As shown in FIGS. 1 to 9, the amplification melting curve of the 9 primer pairs was single-peak (. Degree. C.) and the width of the peak was small, indicating that the 9 primer pairs all had good amplification specificity.

The qPCR results for each set of primer pairs are shown in tables 6 and 7:

1) The qPCR result of primer pair 1-4 (control primer pair) designed by taking Kana sequence or truncated fragment thereof as a template shows that on different gradients of which the ratio of plasmid copy number to cell number is 0.0001, 0.001, 0.01, 0.1 and 1 respectively, CT values have no gradient relation, the sensitivity of the 4 pairs of primers is lower, and the positions of the 4 pairs of primers designed select different positions in Kana resistance genes, the detection sensitivity of the primers designed at different positions of the Kana sequence is lower, so that the detection sensitivity of the primer pair designed only according to the Kana sequence or truncated fragment thereof is lower and does not meet the requirement of detecting exogenous DNA residues in cells;

2) qPCR results of primer pair 5-6 (control primer pair) and primer pair 7 (primer pair of the invention) designed by taking the Promoter-Kana sequence as a template show that the primer pair 5 has gradient relation on different gradients of which the ratio of plasmid copy number to cell number is 0.0001, 0.001, 0.01, 0.1 and 1 respectively, but the amplification efficiency of the primer pair 5 is lower according to CT value data in the table 7, meanwhile, the CT value of the NC group is larger than the CT value of the NTC group, and the primer pair 5 is easily influenced by special property difference due to external factors by combining CT value data of different plasmid gradient concentrations, so that the requirement of detecting exogenous DNA residues in cells is not met; the primer pair 6 has no gradient relation in different proportions, which indicates that the primer pair has lower sensitivity and does not meet the requirement of detecting exogenous DNA residues in cells; the amplification efficiency of the primer pair 7 is good, and the gradient relation (sensitivity) is good;

3) The qPCR result of the primer pair (primer pair 8-9) designed by taking the Kana-pUC replication origin sequence as a template shows that the two primer pairs have gradient relations on different gradients of positive plasmid copy number and cell number of 0.0001, 0.001, 0.01, 0.1 and 1 respectively, and compared with the primer pair 7, the amplification efficiency of the primer pair 8-9 is better, particularly the amplification efficiency of the primer pair 9 is better, and the detection sensitivity is also better.

In summary, the position of the upstream primer binding is bridged on the AmpR promoter and the Kana gene, and the position of the downstream primer binding is in a gradient relation with CT values on different gradients of 0.0001, 0.001, 0.01, 0.1 and 1 respectively, and particularly the primer pair 9 has better amplification efficiency and better detection sensitivity. In the result data of primer pair 9, the CT value of the blank (NTC: cell lysate) was slightly lower than 40, and the CT value of the negative control (NC: DNA solution obtained after negative cell lysis) was slightly lower than 35, indicating that the amplification was susceptible to other factors, and further, the primer was continued to be optimized at this position.

TABLE 6 primer screening based on Kana resistance Gene or truncated fragment design

TABLE 7 primer selection based on Promoter-Kana, kana-pUC replication origin design

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Example 3: further optimization of specific primers

Referring to the experimental results of example 2, primer pair design optimization was performed with reference to the position of primer 9, and the upstream primer of primer pair 10 after optimization was SEQ ID NO:19 (5'-CTATGGAACTGCCTCGGTG-3') the downstream primer is SEQ ID NO:20 (5'-CTGACGCTCAGTGGAACG-3'). The gDNA extraction, positive plasmid dilution, sample mixing, reaction system, reaction procedure, and reaction sequence were performed in the manner described in reference example 2, table 3, table 4, and Table 5, respectively.

As shown in FIG. 10, the amplification melting curve of the optimized primer pair is unimodal and the width of the peak is smaller, indicating that the optimized primer pair has good amplification specificity.

The qPCR verification results of the optimized primer pairs are shown in Table 8, gradient correlation is shown on different gradients of which the ratio of positive plasmid copy number to negative cell number is 0.0001, 0.001 and 0.01 respectively, and CT values of NTC and NC are respectively increased to be more than 40 and more than 35. The optimized primer pair 10 has better specificity and high sensitivity, and is further selected for methodological verification including specificity, detection limit and durability verification.

TABLE 8 qPCR method of optimized primer pair for detecting exogenous DNA residue

Example 4: detection limit verification for detecting Kana resistance gene-containing plasmid by qPCR method

NTC (cell lysate), NC (DNA solution obtained after lysis of hiPSC without genetic modification), different positive plasmid standard gradients (0.00001, 0.0001, 0.0003, 0.001copies plasmid/hiPSC) were set as test samples, positive plasmid dilution procedure is shown in table 9, positive plasmid and cell mixing procedure is shown in table 10. Experimental procedures and procedure for gDNA extraction, reaction system, reaction procedure, etc. were as in example 2. The results (deltact) were analyzed by qPCR method with multiple (9) assays combined with statistics.

Table 9 Positive plasmid gradient dilution protocol (methodological validation)

TABLE 10 preparation of qPCR cell samples (methodological validation)

As a result, as shown in table 11, there was no significant difference in Δct values for the positive plasmid copy number/cell number (copies plasma/hiPSC) of 0.00001 and NC groups, and the difference in Δct values for the 0.0001copies plasma/hiPSC and NC groups was significant, so the upper limit of the 95% confidence interval (Δct= 17.158) of the 0.0001 group was selected as the reference basis for the limitation; the detection rate of each repeated experiment was determined to be 0,0.00001copies plasmid/hiPSC, 33.33%, 66.67%, and 100% for 0.0003copies plasma/hiPSC.

Therefore, the detection limit of the primer set 10 was 0.0003copies plasmid/hiPSC (equivalent plasmid concentration of 0.0876 fg/. Mu.l). When the delta CT value in the test sample is higher than 0.0003copies plasma id/hiPSC delta CT value, the exogenous DNA residue in the test sample is lower than 0.0003copies plasma id/hiPSC, the detection limit is lower, and the exogenous DNA residue in the test sample is judged to be negative.

Table 11 proof of detection limit of Kana-resistant plasmid by qPCR method

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Example 5: specificity verification for detecting Kana-resistant plasmid by qPCR method

By specific is meant that certain components (e.g., impurities, degradants, substrates, etc.) may be present, affecting an accurate and reliable determination of the analyte. Detection targets in NTC (cell lysate), NC (DNA solution obtained after hiPSC cleavage without genetic modification), PC (0.0003 copiesplast/hiPSC), and test sample (DNA solution obtained after hiPSC cleavage with genetic modification) were detected by qPCR, respectively, and the reaction system and reaction procedure were the same as in example 2; positive control PC was prepared with reference to table 9.

The results are shown in Table 12, in which NC set ΔCT is 18.04, limit of detection (PC) set ΔCT is 14.78, test sample set ΔCT is 17.77, and ΔCT of the test sample is higher than that of PC set ΔCT, indicating that the test sample is negative, and the primer can only identify PC, so that the qPCR method is qualified for detecting the specificity of exogenous DNA residue.

Table 12 specificity verification of qPCR method for detecting Kana-resistant plasmid

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Example 6: durability verification for detecting Kana-resistant plasmid by qPCR method

Durability, i.e. the detection method is still reliable when there is a small change in the parameters of the method. The experimental procedure for durability verification was the same as in example 2, and the experimental design was the same as in example 5. Specific DNA fragments in NTC (cell lysate), NC (DNA solution obtained after hiPSC cleavage without genetic modification), PC, and test article (DNA solution obtained after hiPSC cleavage with genetic modification) were detected by qPCR method using different batches of qPCR Mix (lot 1, lot 2) and primers (primer 10-lot 1, primer 10-lot 2), respectively, and the reaction system and reaction procedure were the same as in example 2.

The results are shown in tables 13 to 16, and in four times of durability experiments using qPCR Mix and primers of different batches, the delta CT of the test sample is higher than the detection limit (PC) delta CT, and the test shows that the two are significantly different, so that the durability of the detection method provided by the invention is qualified.

Table 13 qPCR method for detecting Kana-resistant plasmid durability verification 1

Table 14 qPCR method durability verification 2 for detecting Kana-resistant plasmid

Table 15 qPCR method durability verification 3 for detecting Kana-resistant plasmid

Table 16 qPCR method durability verification 4 for detecting Kana-resistant plasmid

The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications could be made by those skilled in the art without departing from the principles of the invention, which modifications would also be considered to be within the scope of the invention.

Claims (15)

1. A primer pair for detecting foreign gene residues in a cellular product, the primer pair comprising an upstream primer and a downstream primer for amplifying a specific DNA fragment in the foreign gene, the specific DNA fragment comprising a truncated sequence of a resistance gene and a truncated sequence of an adjacent element thereof, one of the upstream primer and the downstream primer binding to the specific DNA fragment at a position on the adjacent element or bridging the adjacent element to the resistance gene, and the other primer binding to the specific DNA fragment at a position on a downstream segment of 650bp or less in length of the resistance gene.

2. The primer pair of claim 1, wherein the resistance gene is one selected from the group consisting of a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, and a neomycin resistance gene.

3. The primer pair of claim 1 or 2, wherein the adjacent element is an upstream promoter or a downstream origin of replication.

4. The primer pair of claim 3, wherein the upstream promoter is one selected from the group consisting of an AmpR promoter, a CMV promoter, an EF1a promoter, an SV40 promoter, a T7 promoter, and a T71ac promoter.

5. The primer pair of claim 3, wherein the downstream origin of replication is one selected from the group consisting of a pUC origin of replication, a pBR322 origin of replication, an F1 origin of replication, a ColE1 origin of replication and a pMB1 origin of replication.

6. The primer pair of claim 4, wherein the resistance gene is a kanamycin resistance gene and the upstream promoter is an AmpR promoter.

7. The primer pair of claim 5, wherein the resistance gene is a kanamycin resistance gene and the downstream origin of replication is a pUC origin of replication.

8. The primer pair of claim 6, wherein the primer pair is SEQ ID NO:13 and SEQ ID NO: 14.

9. The primer pair of claim 7, wherein the primer pair is any one of the following primer pairs:

SEQ ID NO:15 and SEQ ID NO:16, a primer pair shown in FIG. 16;

SEQ ID NO:17 and SEQ ID NO:18, a primer pair shown in FIG; and

SEQ ID NO:19 and SEQ ID NO: 20.

10. The primer pair of claim 9, wherein the primer pair is SEQ ID NO:17 and SEQ ID NO:18, or the primer set forth in SEQ ID NO:19 and SEQ ID NO: 20.

11. The primer pair of claim 3, wherein the specific DNA fragment is a sequence selected from the group consisting of SEQ ID NOs: 24. SEQ ID NO: 25. SEQ ID NO:26 and SEQ ID NO: 27.

12. The primer pair of claim 2, wherein the resistance gene is a kanamycin resistance gene, and a downstream segment of 650bp or less in length of the resistance gene consists of SEQ ID NO: 28.

13. A kit for detecting foreign gene residues in a cellular product, comprising a primer pair according to any one of claims 1-12.

14. A method for detecting foreign gene residues in a cellular product, comprising the steps of:

lysing the cell product to obtain a test article;

lysing the negative cells to obtain a negative control;

mixing the negative reference substance with positive reference substances with different copy numbers to obtain a positive gradient reference substance;

qPCR detection of negative control and positive gradient standard using the primer pair of any one of claims 1 to 12 or the kit of claim 13, thereby determining a detection limit according to the detection value;

using the primer pair or the kit, and carrying out qPCR detection on a sample by taking a positive standard substance corresponding to the detection limit in the positive gradient standard substances as a reference to obtain a detection value of the sample;

and comparing the detection value of the test sample with the detection value of the positive standard sample corresponding to the detection limit, and judging the residual limit of the exogenous gene in the cell product.

15. The method of claim 14, wherein the cell product is a human induced pluripotent stem cell product or a human induced pluripotent stem cell derived cell product.