CN117418024A - Detection kit and method for identifying mouse-derived cell line - Google Patents
Detection kit and method for identifying mouse-derived cell line Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and particularly discloses a detection kit and a method for identifying a mouse-derived cell line, wherein the detection kit comprises a primer pair capable of amplifying 9 mouse STR loci simultaneously; the 9 mouse STR sites are: 4-2, 5-5, 12-1, X-1, 18-3, 6-4, 6-7, 9-2, 15-3. According to the invention, 10 pairs of fluorescent primers originally needed are reduced to 2 pairs by the specific primers, so that the detection cost is greatly reduced; the identification method of the mouse-derived cell line established by using the kit can simultaneously amplify the introduced specific genes of the human-derived cell line under the cooperation of specific primer proportion and two rounds of multiplex PCR, thereby achieving the purpose of verifying whether the pollution of the human-derived cell line exists in the mouse-derived cell line and solving the problem of cross pollution of the common species cell line in a laboratory.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mouse short tandem repeat multiplex amplification detection kit and a method for identifying a mouse-derived cell line.
Background
The cell line is used as a common material for research in many fields of life science, biomedicine and the like, has important roles, and is an important source for carrying out in vitro experiments. Common quality problems for cell lines include cell aging, altered cell properties, cell contamination, etc., common cell contamination including bacterial/fungal contamination, mycoplasma infection, cross-cell contamination. Cell banks are subject to varying degrees of cross-contamination according to literature and ATCC authority data: in the German cell bank, 18% of cells of the 252 newly established lines have cross contamination, the cross contamination of human tumor cell lines is up to 29%, and 18.8% of cells in the Iran cell bank have cross contamination. Part of the scientific papers are removed from the manuscript because of the misuse of the cell lines, and huge economic loss and time and labor cost waste are caused. Cell cross contamination is difficult to find by morphological detection methods, and common detection methods include isozymogram detection methods, chromosome karyotyping detection methods, HLA genotyping detection methods, STR genotyping detection methods, SNP detection methods, flow detection methods, immunofluorescent staining detection methods and the like, wherein the STR genotyping detection method is the most widely used method.
STRs are a class of DNA sequences with length polymorphisms found in genomic DNA, consisting of a tandem of 2-6 base repeat units, which can be used as highly polymorphic markers, also known as DNA fingerprints of cells. Cross-contamination detection and identification of 278 human cell lines of 28 study units by STR typing technique was performed by Tautz D et al and Edwards A et al, and it was found that 46% of the cells were cross-contaminated, 40% of which were from China, and 67% of the cell contamination sources were Hela cells and subcellular strains thereof hybridized with other cells (Simple sequences, curr Opin Genet Dev 1994; 4:832-837;DNA typing and genetic mapping with trimeric andtetrameric tandem repeats. Am. J. Hum Genet 49, 746-56). Mouse cell lines are the most common cell lines except human cell lines, from which mouse cell lines can be identified by the presence of a characteristic short tandem repeat in the mouse genome. Patent CN111718999B discloses a multiplex amplification system of a mouse short tandem repeat, which can be used for identifying mouse cells, but the multiplex amplification system requires 19 pairs of primers, and requires classified fluorescent labeling of the primers, which is costly and cannot judge cross contamination of human and mouse cells.
Disclosure of Invention
In order to solve the problems, the invention provides a mouse short tandem repeat multiplex amplification detection kit, which comprises a primer pair capable of amplifying 9 mouse STR sites simultaneously; the 9 mouse STR sites are: 4-2, 5-5, 12-1, X-1, 18-3, 6-4, 6-7, 9-2, 15-3;
the primer pair consists of the following nucleotide sequences:
primer pair SEQ ID NO: 1-2;
amplification of primer pair SEQ ID NO: 3-4;
amplification of primer pair SEQ ID NO: 5-6;
amplifying the primer pair SEQ ID NO of X-1 to 7-8;
primer pair SEQ ID NO: 9-10;
amplification of primer pair SEQ ID NO: 11-12;
amplifying the primer pair SEQ ID NO of 6-7 to 13-14;
amplifying the primer pair SEQ ID NO of 9-2 by 15-16;
and amplifying the primer pair SEQ ID NO 17-18 of 15-3.
Further, the kit also comprises a primer pair capable of amplifying the human gene D5S818 at the same time; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 19-20.
Further, the kit also comprises a fluorescent primer capable of marking the amplified fragment; the kit also comprises a fluorescent primer pair capable of marking amplified fragments; the fluorescent primer pair consists of FAM-F with a nucleotide sequence shown as SEQ ID NO. 21, ROX-F with a nucleotide sequence shown as SEQ ID NO. 22 and STR-R with a nucleotide sequence shown as SEQ ID NO. 23.
Further, the kit also comprises PCR amplification buffer solution, dNTP, taq enzyme and rubusoside.
Still further, the Taq enzyme is Takara R007 enzyme.
The invention also provides a method for identifying a mouse-derived cell line, which comprises the following steps:
(1) Extracting genome total DNA of a cell sample to be detected as template DNA;
(2) Performing PCR amplification on the template DNA by using the mouse short tandem repeat multiplex amplification detection kit to obtain an amplification product;
(3) And (3) carrying out fluorescence detection on the amplified product on a gene sequencer, determining the allele number of the STR locus of DNA in the cells to be detected according to the detected data, and determining whether the cells are mouse-derived cell lines according to the allele number.
Further, the concentration of the template DNA in the step (1) is 10 ng/MuL.
Further, the PCR amplification in the step (2) is performed in two rounds, and the system of the first round of PCR amplification comprises:
the multiplex primer Mix is prepared by mixing the following primer pairs at a concentration of 10 mu M in volume ratio, and then mixing with ddH 2 O is mixed according to the volume ratio of 9:1:
the volumes of the upstream primer and the downstream primer in each primer pair are equal.
The system of the second round of PCR amplification comprises the following components:
the fluorescent primer Mix is prepared by mixing the following primers with the concentration of 100 mu M according to the volume ratio, and then mixing with ddH 2 O is mixed according to the volume ratio of 4:96:
。
further, the first round of PCR amplification is performed by the following steps:
the procedure for the second round of PCR amplification was:
。
the invention has the beneficial effects that:
according to the mouse short tandem repeat multiplex amplification detection kit, 10 pairs of fluorescent primers originally needed are reduced to 2 pairs through the specific primers, so that the detection cost is greatly reduced; the identification method of the mouse-derived cell line established by using the kit can simultaneously amplify the introduced specific genes of the human-derived cell line under the cooperation of specific primer proportion and two rounds of multiplex PCR, thereby achieving the purpose of verifying whether the pollution of the human-derived cell line exists in the mouse-derived cell line and solving the problem of cross pollution of the common species cell lines (human sources and mouse sources) in a laboratory. Proved by experiments: the identification method can determine the types of at least 15 common mouse cell lines, judge whether human cell pollution exists, and has accurate and reliable results.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a graph of the analysis results of a first generation sequencing fragment of a test Raw264.7 cell line;
FIG. 2 is a graph showing the analysis results of a JEKO-1 cell line first generation sequencing fragment;
FIG. 3 is a graph showing the results of a first generation sequencing fragment analysis of the hCMCD 3 cell line;
FIG. 4 is a graph showing the analysis results of a first generation sequencing fragment of the MCF-7 cell line;
FIG. 5 is a graph showing the analysis result of a first generation sequencing fragment of MDA-MB-436 cell line;
FIG. 6 is a graph showing the analysis result of a first generation sequencing fragment of SH-SY5Y cell line;
FIG. 7 is a graph of the results of a first generation sequencing fragment analysis of the test H9C2 cell line;
FIG. 8 is a graph showing the analysis results of a first generation sequencing fragment of IEC-6 cell line;
FIG. 9 is a graph showing the analysis results of a first generation sequencing fragment of IEC-18 cell line.
Detailed Description
Example 1 kits and detection methods of the invention for the identification of mouse-derived cell lines
1. The invention provides a multiple amplification detection kit component for a mouse short tandem repeat sequence
The primer pair comprises a primer pair for amplifying 9 mouse STR loci shown in SEQ ID NO. 1-18, a primer pair for amplifying a humanized gene D5S818 shown in SEQ ID NO. 19-20, and a fluorescent primer pair shown in SEQ ID NO. 21-23; as shown in table 1:
TABLE 1 primers of the invention
Note that: f is a 5' end primer, R is a 3' end primer, and a fluorescent group is added to the 5' of the primer sequence of FAM-F to form a primer with fluorescence: addition of a fluorescent group 5' to the primer sequence of FAM-TTCCCTACACGACGC, ROX-F forms a fluorescent primer: ROX-GTTCAGACGTGTGCT.
2. Detection by the kit of the invention
(1) Extracting total genome DNA of a cell sample to be detected, and using ddH 2 Diluting to10 ng/mu L to obtain template DNA;
(2) Carrying out two-round PCR amplification on the template DNA by using a mouse short tandem repeat multiplex amplification detection kit to obtain an amplification product;
the specific method for amplification by using the kit comprises the following steps:
(1) preparation of multiplex primer Mix: uniformly mixing a primer pair for amplifying 9 mouse STR loci with the concentration of 10 mu M and a primer for amplifying a humanized gene D5S818 in 1 pair according to the following volume ratio, wherein the volumes of upstream primers and downstream primers in each primer pair are equal; then is combined with ddH 2 Mixing O according to the volume ratio of 9:1 to obtain:
(2) preparing a first round PCR amplification system: the components are mixed according to the following volume dosages:
(3) first round PCR amplification
Amplifying according to the following PCR reaction procedure to obtain an amplified fragment;
(4) preparing fluorescent primer Mix, mixing the fluorescent primers with the concentration of 100 mu M respectively according to the following volume ratio, and then mixing with ddH 2 Mixing O according to the volume ratio of 4:96 to obtain the catalyst;
(5) preparing a second PCR amplification system: the components are mixed according to the following volume dosages:
(6) second round PCR amplification
Amplifying according to the following PCR reaction program to obtain an amplified product, and storing the amplified product in a dark place for standby:
(3) Performing fluorescence detection on the amplified product on a first-generation sequencer, analyzing the detected data by using Genemapper 6.0 software, determining the allele number of STR locus of DNA in the mouse-derived cells to be detected, comparing the allele number with a cellosaurus database to obtain the mouse-derived cell line type, wherein the cellosaurus database has no corresponding STR locus allele number, and determining the mouse-derived cell line type according to the following STR data table:
when the gene mapper 6.0 software analysis results have the allele number of the human gene D5S818, the human cells are mixed in the mouse cells to be detected.
The beneficial effects of the present invention are further illustrated by the following test examples
Experimental example 1 investigation of mouse-derived cell line identification Using short tandem repeats
1 Experimental materials
45 strains (15 cells, 3 samples of each cell) of mouse-derived cell line from each institute of the university of Sichuan Huaxi hospital; nucleic acid extraction kit: magpure tissue & blood DNA LQ kit (D6315-03, magen); taq enzyme: takara R007; dye fragment standard: geneScan ™ 600 LIZ ™; hi-Di Formamide carboxamide; PCR amplification buffer (10×, pH8.3, abbreviated as 10 Xbuffer), first generation sequencer electrophoresis gel: POP-7 ™ Polymer for 3730XL; PCR instrument: thermo cycles r; a generation sequencer (capillary electrophoresis sequencer): ABI 3730XL; micro-spectrophotometer: thermo Nanodrop one.
2. Primer design
9 STR sites located in the mouse genome and 1 site located in the human genome are selected, primers are designed by using primer software, and the designed primers are compared in NCBI Blast to show good specificity. Primer sequences and ratios are shown in the following table:
TABLE 2 multiplex primer sequences and ratios
During use, the primers with the concentration of 10 mu M are mixed in proportion, and 10 mu L ddH is added into each 90 mu L of primer mixed solution 2 O was used as a multiplex primer Mix after mixing. Two additional sets of fluorescent primers were designed as shown in the following table:
TABLE 3 fluorescent primer sequences and ratios
During use, the primers with the concentration of 100 mu M are mixed proportionally, and 96 mu L ddH is added into each 4 mu L of primer mixed solution 2 O was used as a fluorescent primer MIX after being mixed uniformly.
3. Method of
3.1 DNA extraction
The cell pellet was washed twice with PBS, centrifuged at 3000rpm, and the supernatant was removed to collect the pellet, followed by DNA extraction. 200 mu L of LPBS is added into the cell sediment, the mixture is blown and stirred, 200 mu L of lysate is added, 10 mu L of proteinase k is added, and 20 mu L of magnetic beads are mixed. Heating and vibrating at 1200rpm and 60 ℃ for 10min in a vibrating instrument. 400 [ mu ] L BD (Binding buffer) was added, and the mixture was stirred at room temperature for 5min. The sample tube was placed on a magnetic rack for 5min and the supernatant was removed. Adding 500 mu L of washing liquid, vibrating the centrifuge tube to enable the magnetic beads to be uniformly mixed in the washing liquid, and adsorbing for 5min on the magnetic rack, and repeating for one time. In the same manner, 500ml of 75% ethanol was used for 2 more washes. After the waste liquid is removed, the cover is opened and dried for 5min, 80 mu L EB is used for eluting DNA, after the DNA is adsorbed on a magnetic rack for 5min, the liquid is sucked out and transferred into a clean EP tube, and DNA extraction is completed.
3.2 DNA quality identification and homogenization
The quality of the DNA is identified by using Thermo Nano Drop one, the required concentration is more than 10 ng/MuL, A260/A280 is more than 1.8, and A260/A230 is more than 1.8. The DNA was diluted to 10 ng/. Mu.L according to the measured concentration.
3.3 First round PCR
The target sites were amplified using multiplex primer Mix and PCR systems and programming are shown in tables 4 and 5. A blank (EB) and a negative and positive control (negative control is human cells JEKO-1, hCMCD 3, MCF-7, MDA-MB-436, SH-SY5Y gDNA, positive control is mouse cells Raw264.7, H9C2, IEC-6, IEC-18 gDNA) were added.
TABLE 4 first round PCR reaction System
TABLE 5 first round PCR procedure
3.4 In the second round of PCR, the amplified fragments were labeled with fluorescent primers, which were noted to be protected from light.
TABLE 6 second round PCR reaction System
TABLE 7 second round PCR procedure
After two rounds of PCR, the products were stored in the dark and were ready for on-machine analysis.
3.5 Preparation for getting on machine
Preparing a pre-mixed liquid of the upper machine product: 8.5 mu L Hi-Di Formamide,0.5 mu L GeneScan ™ LIZ ™ and 1 mu L of the second-step PCR product are added into each reaction, and after being uniformly mixed, the mixture is placed in a PCR instrument for incubation at 95 ℃ for 5min, and then the mixture is immediately placed on ice for cooling.
3.6 panel, bin was set in Genemapper 6.0 software. The panel/bin required for a generation sequencer analysis was made according to the amplified fragment length of each site as shown in table 8.
TABLE 8 amplified fragment analysis template Panel
TABLE 9 amplified fragment analysis queue File bin
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3.7 Generation sequencer running program
The run program was the default program Genemapper50_pop7_1, with specific parameters as shown in Table 10
TABLE 10 STR cell identification fragment analysis program run parameters
4. Results
4.1 The results were analyzed using Genemapper 6.0 software, the analysis method was the default method of the system-microsatelite default. The panel/bin was imported, a first-generation sequencer would automatically analyze the allele numbers at each site of the sample cells, and these allele numbers were entered into the database for alignment. The cellosaurus database (https:// web. Expasy. Org/cellosaurus-str-search /) is used herein, as exemplified by raw264.7, with the results shown in fig. 1 and table 11.
Table 11 test Raw264.7 STR site information and database alignment
4.2 The results of the 15 cell lines are shown in the following table, wherein some cell lines have no related STR sequence data in the cellosaurus database, and the following cell alignment data sets are established according to multi-source comparison, cell morphology and the like.
TABLE 12 statistical table of STR data for common mouse-derived cell lines
4.3 As a result of comparison of human JEKO-1, hCMCD 3, MCF-7, MDA-MB-436 and SH-SY5Y cell lines, only human genome site D5S818 was detected, and no other mouse specific sites were detected, and the results are shown in FIGS. 2-6.
4.4 The results of the comparison of rat cell lines H9C2, IEC-6 and IEC-18 showed no detection signal, and the results are shown in FIGS. 7-9.
From the above results, it can be seen that: according to the invention, through specific primers and dosage proportion, under the cooperation of two rounds of multiplex PCR, cell DNA amplification products are detected by a first-generation sequencer and analyzed by Genemapper 6.0 software, the types of at least 15 common mouse cell lines can be determined, whether human-derived cell pollution exists or not can be judged, and the result is accurate and reliable. Meanwhile, the invention reduces 10 pairs of fluorescent primers originally needed to 2 pairs, greatly reduces the detection cost and has practical popularization and application values.
Claims (9)
1. A multiple amplification detection kit for a mouse short tandem repeat sequence is characterized in that: the kit comprises a primer pair capable of amplifying 9 mouse STR loci simultaneously; the 9 mouse STR sites are: 4-2, 5-5, 12-1, X-1, 18-3, 6-4, 6-7, 9-2, 15-3;
the primer pair consists of the following nucleotide sequences:
primer pair SEQ ID NO: 1-2;
amplification of primer pair SEQ ID NO: 3-4;
amplification of primer pair SEQ ID NO: 5-6;
amplifying the primer pair SEQ ID NO of X-1 to 7-8;
primer pair SEQ ID NO: 9-10;
amplification of primer pair SEQ ID NO: 11-12;
amplifying the primer pair SEQ ID NO of 6-7 to 13-14;
amplifying the primer pair SEQ ID NO of 9-2 by 15-16;
and amplifying the primer pair SEQ ID NO 17-18 of 15-3.
2. The mouse short tandem repeat multiplex amplification detection kit according to claim 1, wherein: the kit also comprises a primer pair capable of amplifying the humanized gene D5S818 at the same time; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 19-20.
3. The mouse short tandem repeat multiplex amplification detection kit according to claim 1, wherein: the kit also comprises a fluorescent primer pair capable of marking amplified fragments; the fluorescent primer pair consists of FAM-F with a nucleotide sequence shown as SEQ ID NO. 21, ROX-F with a nucleotide sequence shown as SEQ ID NO. 22 and STR-R with a nucleotide sequence shown as SEQ ID NO. 23.
4. The mouse short tandem repeat multiplex amplification detection kit according to claim 1, wherein: the kit also comprises PCR amplification buffer solution, dNTP, taq enzyme and rubusoside.
5. The kit for multiplex amplification of mouse short tandem repeats according to claim 4, wherein: the Taq enzyme is Takara R007 enzyme.
6. A method of identifying a cell line of mouse origin, comprising: the method comprises the following steps:
(1) Extracting genome total DNA of a cell sample to be detected as template DNA;
(2) Performing PCR amplification on template DNA by using the mouse short tandem repeat multiplex amplification detection kit according to any one of claims 1-5 to obtain an amplification product;
(3) And (3) carrying out fluorescence detection on the amplified product on a gene sequencer, determining the allele number of the STR locus of DNA in the cells to be detected according to the detected data, and determining whether the cells are mouse-derived cell lines according to the allele number.
7. The method according to claim 6, wherein: and (3) the concentration of the template DNA in the step (1) is 10 ng/mu L.
8. The method according to claim 6, wherein: the PCR amplification in the step (2) is carried out in two rounds, and the system for the first round of PCR amplification comprises the following components:
the multiplex primer Mix is prepared by mixing the following primer pairs at a concentration of 10 mu M in volume ratio, and then mixing with ddH 2 O is mixed according to the volume ratio of 9:1:
the volumes of the upstream primer and the downstream primer of each primer pair are equal;
the system of the second round of PCR amplification comprises the following components:
the fluorescent primer Mix is prepared by mixing the following primers with the concentration of 100 mu M according to the volume ratio, and then mixing with ddH 2 O is mixed according to the volume ratio of 4:96:
。
9. the method according to claim 8, wherein: the first PCR amplification procedure was:
the procedure for the second round of PCR amplification was:
。
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| US20140066322A1 (en) * | 2013-07-03 | 2014-03-06 | United States Of America As Represented By The Secretary Of Commerce, Nist | Mouse cell line authentication |
| US20170101677A1 (en) * | 2013-07-03 | 2017-04-13 | United States Of America, As Represented By The Secretary Of Commerce | Mouse cell line authentication |
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| CN115725749A (en) * | 2022-11-17 | 2023-03-03 | 广州艾基生物技术有限公司 | Mouse source cell STR detection kit, method and application |
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| US20140066322A1 (en) * | 2013-07-03 | 2014-03-06 | United States Of America As Represented By The Secretary Of Commerce, Nist | Mouse cell line authentication |
| US20170101677A1 (en) * | 2013-07-03 | 2017-04-13 | United States Of America, As Represented By The Secretary Of Commerce | Mouse cell line authentication |
| CN106701988A (en) * | 2017-02-10 | 2017-05-24 | 上海荻硕贝肯医学检验所有限公司 | Primers, kit and method for detecting short tandem repetitive sequence |
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