CN115725749A - Mouse source cell STR detection kit, method and application - Google Patents
Mouse source cell STR detection kit, method and application Download PDFInfo
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Abstract
The invention discloses a detection kit and a detection method for mouse source cell STR, wherein the kit comprises four pairs of fluorescent channel primers and a universal primer aiming at least one mouse source cell STR identification site, the channel primers are nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.8, the universal primer consists of a connecting sequence at the 5 'end and a specific sequence at the 3' end, and the connecting sequence is one of the channel primers. The kit can realize simultaneous detection of a plurality of STR loci, can also realize simultaneous detection of one biogenic locus and 1 mycoplasma locus, and has low cost. The invention can solve the problem that mouse source cell STR can not be typed and cell line identification can not be carried out in the market.
Description
Technical Field
The invention belongs to the field of gene detection in biotechnology, and particularly relates to a mouse source cell STR detection kit, a method and application.
Background
At present, STR, as an important genetic marker system, has been widely used in the fields of tumor biochemical research, forensic individual identification, paternity testing, population genetic analysis, and the like. STRs have a high information content, are widely distributed in genomes, can be detected by PCR amplification, electrophoretic typing, etc., have been widely used in various fields of biology in a short time, and have made remarkable progress.
The cells are special commodities, have reproducibility, are easily copied by merchants and then sold, but the times of cell passage, culture conditions, whether cross contamination exists or not and the like cannot be guaranteed. Mouse cell lines are used next to human cells in the laboratory, and are widely used in human disease research, the recombinant protein industry, and stem cell research. The accuracy of using the mouse source cells is ensured, and the method plays a crucial role in experimental research. Currently, STR genotyping has been applied to cell strain identification as a gold standard by the authorities such as ATCC and DSMZ, and as for STR (Short Tandem Repeat) it consists of Short Tandem repeats with a length of 3-7 base pairs, which are widely present in the human genome and can be used as highly polymorphic markers called DNA fingerprints of cells. And comparing the obtained STR typing result with a cell STR database to deduce the name of the cell line to which the test sample belongs. In recent years, therefore, authoritative journals such as Nature and Science have been widely requested by authors to provide source descriptions and identification results of cells used in order to avoid the problem of distortion of scientific data due to cell errors. The method for STR typing and identification by using multiplex fluorescence PCR technology is the most widely applied method at present, the STR identification sites of mouse cells have 18-3,4-2,6-7,19-2,1-2,7-1,1-1,3-2,8-1,17-2,12-1,5-5, X-1,13-2,2-1,15-3,6-4,11-2 and 18 STR sites, and the 18 STR sites can be used for STR typing and identification of mouse cells. The cross contamination of human source cell, mouse source cell and mycoplasma is easy to occur during cell culture. It is essential to simultaneously identify whether a plurality of sites of mouse-derived cells contain human-derived cell pollution or mycoplasma pollution, but related products for identifying mouse-derived cell STRs and detecting human-derived and mouse-derived pollution at the same time are not provided in the market at present. Meanwhile, the multiple fluorescence PCR technology required to be applied to cell STR identification needs to be used for synthesizing the fluorescent primers with the same number of STR sites, so that the cost of the reagents is greatly increased.
Therefore, the mouse source cell STR identification kit which can simultaneously detect human source, mouse source and mycoplasma pollution has good application value.
Disclosure of Invention
One of the purposes of the invention is to provide a mouse derived cell STR detection kit, which can realize simultaneous detection of a plurality of STR loci and further can simultaneously realize detection of one human derived locus and 1 mycoplasma locus, and has low cost.
The technical scheme for achieving the aim comprises the following steps.
A detection kit for mouse source cell STR comprises four pairs of fluorescent channel primers and a universal primer aiming at least one mouse source cell STR identification site, wherein the channel primers are nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.8, the universal primer is composed of a connecting sequence at the 5 'end and a specific sequence at the 3' end, and the connecting sequence is one of the channel primers.
In some of these embodiments, the mouse-derived cell STR identifies 18 sites, wherein the universal primers for the 18-3,4-2,6-7,19-2, F sites are used for the same channel 1; the universal primers for the 1-2,7-1, 3-2, and 8-1_F sites were used for the same channel 2; the universal primers for the 2-1,15-3,6-4, and 11-2 _Fsites were used for the same channel 3; the universal primers for the 17-2,12-1,5-5, X-1, and 3-1 _Fsites were used for the same channel 4.
In some of these embodiments, the channel primers for channel 1 are SEQ ID NO.1-SEQ ID NO.2,
the primers for the channel 2 are SEQ ID NO.3-SEQ ID NO.4,
the primers for the channel 3 are SEQ ID NO.5-SEQ ID NO.6,
the primer for channel 4 was SEQ ID No.7SEQ ID No.8.
In some of these embodiments, the specific sequence of the universal primer is selected from the group consisting of:
SEQ ID NO.9 and SEQ ID NO.27 for the 18-3 _Fsite,
aiming at the 4-2 \/F site SEQ ID NO.10 and SEQ ID NO.28,
for the 6-7 \/F sites SEQ ID NO.11 and SEQ ID NO.29,
for the 19-2 \/F site SEQ ID NO.12 and SEQ ID NO.30,
for 1-2 \/F sites SEQ ID NO.13 and SEQ ID NO.31,
for the 7-1_F _ site SEQ ID NO.14 and SEQ ID NO.32,
for 1-1 \/F sites SEQ ID NO.15 and SEQ ID NO.33,
for the 3-2 \/F sites SEQ ID NO.16 and SEQ ID NO.34,
to the 8-1 _Fsite SEQ ID NO.17 and SEQ ID NO.35,
aiming at 2-1 \/F sites SEQ ID NO.18 and SEQ ID NO.36,
to the 15-3 v u F site SEQ ID NO.19 and SEQ ID NO.37,
for the 6-4 \/F site SEQ ID NO.20 and SEQ ID NO.38,
for the 11-2 \/F sites SEQ ID NO.21 and SEQ ID NO.39,
for the 17-2 \/F site SEQ ID NO.22 and SEQ ID NO.40,
to the 12-1 _Fsite SEQ ID NO.23 and SEQ ID NO.41,
for the 5-5 \/F sites SEQ ID NO.24 and SEQ ID NO.42,
for the X-1 \/F sites SEQ ID NO.25 and SEQ ID NO.43,
SEQ ID NO.26 and SEQ ID NO.44 for the 13-1 _Fsite.
In some embodiments, the specific sequence of the universal primer in the kit comprises the above 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide sequences.
In some embodiments, the kit further comprises mycoplasma detection primers, and the mycoplasma detection primers are respectively composed of one channel primer at the 5 'end and a specific sequence at the 3' end.
In some preferred embodiments, the specific sequence of the Mycoplasma detection primer is shown in SEQ ID NO.45 and SEQ ID NO. 46.
In some preferred embodiments, the mycoplasma detection primers are used in channel 2.
In some embodiments, the kit further comprises human cell detection primers, wherein the human cell detection primers respectively consist of one channel primer at the 5 'end and a specific sequence at the 3' end.
In some preferred embodiments, the human cell detection primers are shown as SEQ ID NO.47 and SEQ ID NO. 48.
In some preferred embodiments, the human cell detection primers are used in channel 2.
The invention also aims to provide a method for detecting the mouse source cell STR.
A method for detecting mouse cell STR comprises the following steps:
configuring an amplification reaction system: diluting all primers in the kit, mixing the diluted primers, and mixing the diluted primers with a DNA template and a reaction reagent of a sample to be detected;
performing fluorescence PCR amplification;
and (3) carrying out capillary electrophoresis detection on the product amplified by the fluorescent PCR.
In some of these embodiments, the working concentration of each primer is: the fluorescent universal primer is 5 +/-0.5 nmol/mu L, the specific primer aiming at the mouse STR identification site is 1 +/-0.1 nmol/mu L, the mycoplasma detection primer is 1 +/-0.1 nmol/mu L, and the human cell detection primer is 1 +/-0.1 nmol/mu L.
In some of these embodiments, the procedure at the time of amplification is: 95 plus or minus 2 ℃ for 5 minutes; 10 cycles of: 94 + -2 deg.C for 30 seconds, 56 + -2 deg.C for 1 minute, 70 + -2 deg.C for 1 minute, 19-21 cycles: 94 plus or minus 2 ℃ for 30 seconds, 61 plus or minus 2 ℃ for 1 minute, and 70 plus or minus 2 ℃ for 1 minute; final extension: 60. + -. 2 ℃ for 40 minutes.
The detection kit and the detection method have the following advantages:
1. at present, the method for identifying the STR of the mouse derived cells in the market has less systems, fewer detected sites and immature systems, at present, 18 STR sites cannot be identified simultaneously, and whether the STR sites contain human sources and mycoplasma pollution or not can be identified simultaneously, and the method can be used for detecting 18 mouse derived sites, 1 human source site and 1 mycoplasma site simultaneously. 18 mouse source sites can be used for detecting whether mouse source cells to be detected are polluted by the mouse source cells and identifying the mouse source cell line of a sample to be detected, 1 human source site can be used for detecting whether the sample to be detected is polluted by the human source cell line, and 1 mycoplasma detection site can be used for detecting whether the sample to be detected is polluted by mycoplasma. The detected mouse source sites are consistent with mouse source cell sites published by ATCC and EXPASY, and all the sites detected by the invention can be used for data comparison, so that which mouse cell line the mouse source cell belongs to is accurately identified, and STR typing and cell line identification of the mouse source cell are realized. The invention can solve the problem that mouse source cell STR can not be typed and cell line identification can not be carried out in the market.
2. Studies have shown that of mycoplasma contamination in cell culture, more than 95% of mycoplasma contaminating cells are mycoplasma fermentans (m.fermentans), mycoplasma hyorhinis (m.hyorhinis), mycoplasma oralis (m.orale), mycoplasma arginini (m.arginini), mycoplasma salivarius (m.salivariaum), and mycoplasma hominis (m.hominis). The detection primer designed by the invention can be used for identifying mouse source cells and detecting whether mouse source cell pollution, human source cell pollution and mycoplasma pollution exist. The primer for detecting mycoplasma contamination can detect more than 30 kinds of common mycoplasma (including mycoplasma fermentans, mycoplasma hyorhinis, mycoplasma oralis, mycoplasma arginini, mycoplasma salivarius, and mycoplasma hominis). The method can detect the pollution of mouse source, anthropogenic source and mycoplasma by one-time amplification, greatly improves the experimental efficiency, and also avoids the problem of secondary pollution possibly caused by multiple experiments.
3. The cell STR identification system is established on the multiplex fluorescence PCR technology, the main cost of the technology lies in the synthesis of fluorescence primers, and the synthesis cost of the fluorescence primers is higher. The traditional STR system is established, multiple fluorescence PCR technology is used, and the number of STR sites is the number of needed fluorescent primers. During the amplification period, the fluorescent channel primer is combined with the specific DNA fragment (linker) amplified by the detection site primer, so that the amplified specific DNA fragment carries a fluorescent signal, and the fluorescent DNA fragment can be used for the next capillary electrophoresis detection. The invention can greatly save the cost of the fluorescent primer, thereby reducing the cost.
Drawings
FIG. 1 is a TEST1 cell STR typing map.
FIG. 2 is a schematic diagram showing the comparison of STR typing results of TEST1 cells inputted into EXPASY online database at various sites.
FIG. 3 is a diagram of data after alignment of STR typing results of TEST11 cells.
FIG. 4 is a graph of STR typing of 4T1 cells.
FIG. 5 is a schematic diagram showing that each site of STR typing results of 4T1 cells is inputted into an EXPASY online database for comparison.
FIG. 6 is a schematic diagram of data after comparison of STR typing results of 4T1 cells.
FIG. 7 is a graph of STR typing of 3T3 cells.
FIG. 8 is a schematic diagram showing that each site of the STR typing results of the 3T3 cells is inputted into the EXPASY online database for comparison.
FIG. 9 is a schematic diagram of data after comparison of STR typing results of 3T3 cells.
FIG. 10 is a schematic diagram of the site arrangement of the mouse derived cell STR identification kit.
Fig. 11 is a schematic diagram of the result of single-channel operation in embodiment 3.
FIG. 12 is a schematic diagram showing the results of the single-tube four-channel machining in example 3.
Fig. 13 is a schematic diagram of a sample LLC detection result in embodiment 4.
FIG. 14 is a graph showing the result of MUS detection in example 4.
FIG. 15 is a diagram showing the results of detection of mEC-03 in the sample in example 4.
Detailed Description
The following examples of the present invention are experimental methods without specifying specific conditions, generally according to conventional conditions, or according to conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated object, indicating that there may be three relationships, for example, a and/or B, which may indicate: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In order that the invention may be more fully understood, reference will now be made to the following more detailed description. The following examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and essential concept of the present invention should be construed as equivalents thereof, and are intended to be included in the scope of the present invention.
The present invention will be described in detail with reference to specific examples. The following examples will help one skilled in the art to further understand the present invention.
In some embodiments of the invention, the invention relates to a detection kit and a detection method for identifying mouse source cell STR. The kit and the detection method can simultaneously detect 18 mouse source sites, 1 human source site and 1 mycoplasma site, identify mouse source cells and simultaneously detect whether human source cell pollution and mycoplasma pollution exist, and greatly improve the experimental efficiency.
The invention designs that one of four groups of channel primers is added at the 5' end of a conventional primer of each specific site as a linker sequence and is properly and respectively corresponding to four types of fluorescence, so that only four groups of fluorescent primers are needed to amplify 20 sites simultaneously. Meanwhile, the sequences of the four groups of fluorescent channel linkers are generated by randomizing the same sequence, and the Tm values of the sequences are almost the same, so that the heterogeneity in the multiplex PCR amplification is greatly reduced. In addition, the invention can further add a human source site and a mycoplasma site while detecting 18 sites of the mouse, thereby being convenient for detecting the pollution of human source cells which often appear in the cell culture of the mouse and simultaneously detecting whether the cells are polluted by mycoplasma. These tests are currently performed in other products only by performing three tests. Thus greatly saving the time for detection. The invention only needs 4 fluorescence channel primers, and the other primers are common primers, thus greatly saving the reagent cost compared with other multiple fluorescence PCR technologies. The primers designed by the invention are divided into a fluorescent universal primer (with fluorescence), a mouse STR locus specific primer (without fluorescence), a mycoplasma detection primer (without fluorescence) and a human cell detection primer (without fluorescence). The key points of the designed primer of the invention are as follows: 1. the four groups of fluorescent channel primers can be respectively combined with the universal linker sequences at the 5' ends of the 20 pairs of specific primers, so that a product with fluorescent specific sites can be amplified in the multiplex amplification. 2. The fluorescent universal primer is produced by randomizing a primer sequence, has the same GC content and Tm value, and can more uniformly amplify 20 different specific gene sites. 3. The primer of the fluorescence channel is combined with the DNA fragment amplified by the mouse STR site specific primer, the mycoplasma detection primer and the human cell detection primer, so that the amplified DNA fragment is provided with the fluorescent group, and the DNA fragment with the fluorescent group is detected during capillary electrophoresis. 4. Compared with other STR detection systems and kits, the fluorescent channel primers can carry fluorescent signals on DNA fragments amplified by all detected sites under the condition of only 4 fluorescent primers, and can be used for capillary electrophoresis detection. 5. The product can simultaneously detect whether the cell strain is polluted by human cells and mycoplasma.
The kit and the detection method can be used for detecting whether a sample to be detected is a mouse cell or not and whether cross contamination in mouse species exists or not; whether human cell contamination exists; whether mycoplasma contamination is present; and (4) carrying out mouse cell strain identification on the mouse cells with published STR data.
The detection site arrangement of the mouse cell STR identification kit in the following examples of the invention is shown in FIG. 10.
Example 1
(1) The amplification primers were as follows:
fluorescent channel primers:
mouse STR site-specific primers: the underlined shading is the universal linker sequence, combined with the fluorescent universal primer.
Mycoplasma (Mycoplasma) detection primer
Human cell detection primer
Diluting and mixing all primers, and mixing the final concentration of a primer system: the primer of the fluorescence channel is 5 nmol/mu L, the specific primer of the mouse STR locus is 1 nmol/mu L, the primer of mycoplasma detection is 1 nmol/mu L, and the primer of human cell detection is 1 nmol/mu L.
(2) The amplification reaction system is as follows:
| genomic DNA | 2-50ng |
| 2.5X multiplex PCR Reaction Mix | 10.0μL |
| Primers Mix | 5.0μL |
| Hot start Taq enzyme | 1.0μL |
| ddH 2 O | Adding water to 25.0 μ L |
(3) The amplification procedure was as follows:
cell suspension, cell precipitation, extraction of whole genome DNA by using a genome extraction kit (Egyptian), and detection of DNA concentration by using a micro ultraviolet visible spectrophotometer (Saimerfii NanoDrop 2000 c) QC. Performing multiplex amplification (ABI 9700 PCR System) with mouse STR identification kit, performing capillary electrophoresis and fragment separation with ABI3730XL genetic analyzer (ABI of America), and performing amplification reaction by using the DNA chip
The ID software (version number: ID-X1.5) was used for genotyping. Because the performance of each type of PCR instrument is different, the annealing temperature in the circulation parameters can be properly adjusted within +/-2 ℃.
The capillary electrophoresis detection is as follows:
centrifuging the PCR amplification product at 3000rpm for 5 minutes, mixing 1 mu L of PCR amplification product with 0.5 mu L of LIZ-500 and 9.5 mu L of deionized formamide, performing denaturation at 95 ℃ for 3 minutes, performing ice bath, and performing capillary electrophoresis detection. The capillary electrophoresis detection parameters are default parameters of fragment analysis in an ABI3730XL capillary electrophoresis apparatus.
(4) And (3) data analysis:
provided using ABI
ID (software version: ID-X1.5) fragment analysis software STR genotyping the original data. And after the STR genotyping is obtained, judging whether the detected mouse source cells contain human source, mouse source or mycoplasma pollution according to the peaks of the STR loci. Comparing the STR data of the mouse source cells published by ATCC and EXPASY with the STR typing data of the tested mouse source cells to identify which cell line the tested mouse source cells belong to.
Example 2
1. Sample information:
(1) Sample name: TEST1 cells or 4T1 cells or 3T3 cells
(2) Sample type: cell pellet
(3) Sample species: mouse derived cell
2. The experimental process comprises the following steps:
(1) Genome extraction: the cell pellet was extracted with whole genome DNA using a genome extraction kit (kit manufacturer: K316-S, egyptian technologies, inc., guangzhou), and the DNA concentration was detected by a micro ultraviolet-visible spectrophotometer (model: saimeifei NanoDrop 2000 c) QC.
(2) Amplification by multiplex fluorescence PCR technology: the kit and the detection method (amplification primer ratio, PCR System, PCR amplification program) described in example 1 were used to perform multiplex amplification (instrument model: ABI9700 PCR System).
(3) Capillary electrophoresis: capillary electrophoresis was performed using an ABI3730XL genetic analyzer (ABI, inc., USA).
(4) And (3) data analysis: raw data analysis adoption
The ID software (software version: ID-X1.5) performs STR genotype analysis. Data comparison mouse derived cell STR typing data published using EXPASY.
3. The experimental results are as follows:
(1) The STR typing map of TEST1 cells is shown in FIG. 1.
According to the STR typing map of the TEST sample TEST1, the peak diagram situation of the TEST sample is briefly analyzed as follows:
remarking: if more than 2 alleles occur at a certain STR locus (or loci), this may be due to: 1. the genome of the tested cells is unstable, and STR sites of partial cells are changed (such as tumor cells cultured in vitro for a long time); 2. chromosomal trisomy or mosaicism due to abnormal segregation during meiosis or mitosis; 3. the cells to be tested are contaminated with cells of other genetic backgrounds (more than 3 STR loci present more than 2 alleles)
STR typing results
And (3) comparing the results:
and (3) inputting each site of the STR typing result into an EXPASY online database for comparison, wherein the data is shown in figure 2.
No signal of mycoplasma and human origin site is detected, and the detected sample is not polluted by mycoplasma and human origin cells. The matching degree of 18 STR loci of TEST sample TEST1 and RAW 264.7 mouse source cell line reaches 100%, the TEST sample belongs to RAW 264.7 mouse source cell line, and the data comparison is shown in FIG. 3.
(2) The STR typing map of 4T1 cells is shown in FIG. 4.
And (3) inputting each site of the STR typing result into an EXPASY online database for comparison, wherein the data is shown in figure 5. Detecting a signal of a mycoplasma site, wherein a signal of a human source site is not detected, and the detected sample is polluted by mycoplasma; the sample to be tested is not contaminated by human cells. The matching degree of 18 STR loci of the 4T1 sample to the 4T1 mouse source cell line reaches 100%, the sample belongs to the 4T1 mouse source cell line, and the data comparison is shown in FIG. 6.
(3) The STR typing map of 3T3 cells is shown in FIG. 7.
And (3) inputting each site of the STR typing result into an EXPASY online database for comparison, wherein the data is shown in figure 8. No signal of mycoplasma and human origin site is detected, and the detected sample is not polluted by mycoplasma and human origin cells. The matching degree of the 18 STR loci of the detected sample 3T3 and the NIH3T3 mouse cell line reaches 100%, the detected sample belongs to the NIH3T3 mouse cell line, and the data comparison is shown in figure 9.
Example 3
TEST1 cell samples from example 2 were tested.
The configuration was made according to the composition of the channel-specific primers in Table 3.1 below.
TABLE 3.1 preparation of primers specific to each channel
Note: the content in parentheses is the site name
Configuring a PCR reaction system according to a single channel and a single tube four-channel:
TABLE 3.2 Single-channel PCR reaction System
TABLE 3.3 Single-tube four-channel PCR reaction System
PCR reaction procedure:
at 95 ℃ for 3min; (94 ℃,30s, 61 ℃,2 min) x 32cycles (cycle); 30min at 60 ℃; and preserving at 12 ℃.
See fig. 11 and 12 for results.
As can be seen from the results of FIG. 11, the gene loci in each channel have been substantially successfully amplified; however, the efficiency of amplification is not a particular balance, with small fragment sites being more efficient and large fragment sites being less efficient.
It can be seen from the results in fig. 12 that, when the fluorescence bands (the signal of ROX has a channel crossing) that are added in each channel due to the channel crossing are eliminated and single-tube four-channel amplification is performed, most of the gene loci can be effectively amplified, the detection result is better than that of single-tube amplification, but some signal values are lower than that of single-tube amplification, and the amplification system and the amplification program need to be optimized; the detection results were inferior to those of the amplification system and the amplification procedure shown in example 1.
EXAMPLE 4 sample testing
4.1. Detecting sample LLC
Sample name: LLC
Sample type: cell pellet
The test was carried out as described in example 1, and the STR typing results are shown in FIG. 13.
As can be seen from fig. 13, more than two allelic peaks were found at 4 of the 18 mouse STR loci examined. According to the following steps: if more than 2 alleles occur at a certain STR locus (or loci), this may be due to: 1. the genome of the tested cells is unstable, and STR sites of partial cells are changed (such as tumor cells cultured in vitro for a long time); 2. chromosomal trisomies or mosaicism due to abnormal segregation of meiotic or mitotic processes; 3. the tested cells are polluted by other cells with genetic background (more than 2 alleles appear at more than 3 STR loci), and the tested mouse cell strains are polluted by mouse cell lines. And detecting the existence of a peak with a higher signal at the mycoplasma site, wherein the mycoplasma pollution exists in the detected sample.
4.2. Detecting MUS of a sample
Sample name: MUS
Type of sample: cell pellet
The test was performed as described in example 1, and the results of STR typing are shown in FIG. 14.
As can be seen from fig. 14, more than two allelic peaks were found at 11 out of 18 mouse STR loci examined. According to the following steps: if more than 2 alleles occur at a certain STR locus (or loci), this may be due to: 1. the genome of the tested cells is unstable, and the STR locus of part of the cells is changed (such as tumor cells cultured in vitro for a long time); 2. chromosomal trisomies or mosaicism due to abnormal segregation of meiotic or mitotic processes; 3. the tested cells are polluted by cells with other genetic backgrounds (more than 2 alleles appear at more than 3 STR sites), and the tested mouse cell strains are polluted by mouse cell lines. And detecting the peak of the mycoplasma site signal, wherein the detected sample has mycoplasma pollution. And detecting the peak of the human source site signal, wherein the human source cell line pollution exists in the detected sample.
4.2. Detecting the mEC-03 of the sample
Sample name: mEC-03
Type of sample: cell pellet
The test was carried out as described in example 1, and the STR typing results are shown in FIG. 15.
As can be seen from fig. 15, more than two allelic peaks were found at 6 of the 18 mouse STR loci examined. According to the following steps: if more than 2 alleles occur at a certain STR locus (or loci), this may be due to: 1. the genome of the tested cells is unstable, and STR sites of partial cells are changed (such as tumor cells cultured in vitro for a long time); 2. chromosomal trisomies or mosaicism due to abnormal segregation of meiotic or mitotic processes; 3. the tested cells are polluted by cells with other genetic backgrounds (more than 2 alleles appear at more than 3 STR sites), and the tested mouse cell strains are polluted by mouse cell lines. No peak of mycoplasma site signal is detected, and no mycoplasma contamination exists in the detected sample. No peak of the human source site signal is detected, and the detected sample has no human source cell line pollution.
The specific implementation of the invention is described above, and the invention can accurately identify the cell line to which the mouse source cell belongs by using the above-mentioned primers and performing multiplex amplification on 18 mouse STR loci under the condition that only 4 fluorescent primers are needed. The invention can detect whether the cell line of the mouse source cell is polluted by the human source cell and mycoplasma or not while identifying the cell line of the mouse source cell.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed but not understood to limit the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A detection kit for mouse source cell STR is characterized by comprising four pairs of fluorescent channel primers and universal primers aiming at least one mouse source cell STR identification site, wherein the channel primers are respectively shown as nucleotide sequences in SEQ ID No.1-SEQ ID No.8, the universal primers consist of a connecting sequence at a 5 'end and a specific sequence at a 3' end, and the connecting sequence is one of the channel primers.
2. The test kit for detecting the mouse derived cell STR as claimed in claim 1, wherein the number of the mouse derived cell STR identification sites is 18,
wherein the universal primers for the 18-3,4-2,6-7,19-2, F sites are used for the same channel 1;
the universal primers for the 1-2,7-1,1-1,3-2, and 8-1 _Fsites were used for the same channel 2;
the universal primers for the 2-1,15-3,6-4, and 11-2 _Fsites were used for the same channel 3;
the universal primers for the 17-2,12-1,5-5, X-1, and 3-1 _Fsites were used for the same channel 4.
3. The kit for detecting STR of mouse derived cells according to claim 2, wherein the kit comprises,
the channel primer aiming at the channel 1 is SEQ ID NO.1-SEQ ID NO.2,
the primers for the channel 2 are SEQ ID NO.3-SEQ ID NO.4,
the channel primers for the channel 3 are SEQ ID NO.5-SEQ ID NO.6,
the primer for channel 4 was SEQ ID No.7SEQ ID No.8.
4. The kit for detecting STR of mouse derived cells as claimed in claim 3, wherein the specific sequence of said universal primer is selected from the following:
SEQ ID NO.9 and SEQ ID NO.27 for the 18-3 _Fsite,
aiming at the 4-2 \/F site SEQ ID NO.10 and SEQ ID NO.28,
for the 6-7 \/F sites SEQ ID NO.11 and SEQ ID NO.29,
to the 19-2 \/F site SEQ ID NO.12 and SEQ ID NO.30,
aiming at 1-2 \/F sites SEQ ID NO.13 and SEQ ID NO.31,
for the 7-1_F _ site SEQ ID NO.14 and SEQ ID NO.32,
for the 1-1 \/F site SEQ ID NO.15 and SEQ ID NO.33,
for the 3-2 \/F sites SEQ ID NO.16 and SEQ ID NO.34,
for the 8-1 \/F sites SEQ ID NO.17 and SEQ ID NO.35,
aiming at 2-1 \/F sites SEQ ID NO.18 and SEQ ID NO.36,
to the 15-3 v u F site SEQ ID NO.19 and SEQ ID NO.37,
for the 6-4 \/F site SEQ ID NO.20 and SEQ ID NO.38,
for the 11-2 \/F sites SEQ ID NO.21 and SEQ ID NO.39,
aiming at the 17-2 \/F site SEQ ID NO.22 and SEQ ID NO.40,
to the 12-1 _Fsite SEQ ID NO.23 and SEQ ID NO.41,
to the 5-5 v/u F site SEQ ID NO.24 and SEQ ID NO.42,
for the X-1 \/F sites SEQ ID NO.25 and SEQ ID NO.43,
SEQ ID NO.26 and SEQ ID NO.44 for the 13-1 _Fsite.
5. The kit for detecting the mouse derived cell STR as recited in any one of claims 1-4, characterized in that, the kit further comprises mycoplasma detection primers, and the mycoplasma detection primers are respectively composed of a 5 'end channel primer and a 3' end specific sequence; preferably, the specific sequences of the mycoplasma detection primers are shown in SEQ ID No.45 and SEQ ID No.46, and more preferably, the mycoplasma detection primers are used in channel 1.
6. The kit for detecting the mouse derived cell STR as claimed in any one of claims 1 to 4, wherein the kit further comprises human derived cell detection primers, and the human derived cell detection primers respectively comprise a channel primer at the 5 'end and a specific sequence at the 3' end; preferably, the specific sequences of the human cell detection primers are shown as SEQ ID NO.47 and SEQ ID NO.48, and more preferably, the human cell detection primers are used for the channel 2.
7. The detection method of the mouse cell STR is characterized by comprising the following steps:
configuring an amplification reaction system: diluting all primers in the detection kit according to any one of claims 1 to 7, mixing, and mixing with a DNA template and a reaction reagent of a sample to be detected;
performing fluorescent PCR amplification;
and (3) carrying out capillary electrophoresis detection on the product amplified by the fluorescent PCR.
8. The method for detecting mouse-derived cell STR according to claim 7, wherein the method further comprises,
the 18-3_F, 4-2_F, 6-7_F, 19-2_F locus detection and mycoplasma source detection aiming at the mouse source are set as the same channel 1;
the same channel 2 was set for the mouse-derived 1-2,7-1,1-1,3-2, and 8-1 v u F site detection, as well as the human-derived cell detection;
the same channel 3 was set for 2-1,15-3,6-4, and 11-2 v u F site detection of mouse origin;
the same channel 4 was set for the 17-2,12-1,5-5, X-1, and 3-1, positions of mouse origin.
9. The method for detecting STR of mouse-derived cells as claimed in claim 7 or 8, wherein the working concentration of each primer is as follows: the fluorescent universal primer is 5 +/-0.5 nmol/. Mu.L, the specific primer aiming at the mouse STR identification site is 1 +/-0.1 nmol/. Mu.L, the mycoplasma detection primer is 1 +/-0.1 nmol/. Mu.L, and the human cell detection primer is 1 +/-0.1 nmol/. Mu.L.
10. The method for detecting STR of mouse derived cells according to claim 9, wherein the procedure for amplification is as follows: 95 plus or minus 2 ℃ for 5 minutes; 10 cycles of: 94 + -2 deg.C for 30 seconds, 56 + -2 deg.C for 1 minute, 70 + -2 deg.C for 1 minute, 19-21 cycles: 94 plus or minus 2 ℃ for 30 seconds, 61 plus or minus 2 ℃ for 1 minute, and 70 plus or minus 2 ℃ for 1 minute; final extension: 60. + -. 2 ℃ for 40 minutes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117418024A (en) * | 2023-12-19 | 2024-01-19 | 四川大学华西医院 | Detection kit and method for identifying mouse-derived cell line |
| CN117535388A (en) * | 2023-12-28 | 2024-02-09 | 广州赛库生物技术有限公司 | Amplification primer, multiplex amplification system and kit for detecting rat short tandem repeat sequence |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618662A (en) * | 2012-04-26 | 2012-08-01 | 重庆出入境检验检疫局检验检疫技术中心 | GenomeLab eXpress Profiling (GeXP) multiplex quick detection primers and detection method for 6 food-borne pathogens |
| US20170101677A1 (en) * | 2013-07-03 | 2017-04-13 | United States Of America, As Represented By The Secretary Of Commerce | Mouse cell line authentication |
| CN106834545A (en) * | 2017-03-13 | 2017-06-13 | 苏州市立医院 | Detection of high risk human papillomavirus kit and detection method |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
| CN114085913A (en) * | 2021-07-02 | 2022-02-25 | 苏州鉴达生物科技有限公司 | STR locus fluorescence labeling multiplex amplification kit for mouse cell strain identification and application thereof |
| CN114196633A (en) * | 2021-11-29 | 2022-03-18 | 湖南亚大丰晖新材料有限公司 | Kit for removing and detecting mycoplasma and application thereof |
| CN114921562A (en) * | 2022-04-27 | 2022-08-19 | 南方海洋科学与工程广东省实验室(广州) | SSR multiple PCR primer for Oxytropis myriophylla and application thereof |
-
2022
- 2022-11-17 CN CN202211460741.9A patent/CN115725749A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618662A (en) * | 2012-04-26 | 2012-08-01 | 重庆出入境检验检疫局检验检疫技术中心 | GenomeLab eXpress Profiling (GeXP) multiplex quick detection primers and detection method for 6 food-borne pathogens |
| US20170101677A1 (en) * | 2013-07-03 | 2017-04-13 | United States Of America, As Represented By The Secretary Of Commerce | Mouse cell line authentication |
| CN106834545A (en) * | 2017-03-13 | 2017-06-13 | 苏州市立医院 | Detection of high risk human papillomavirus kit and detection method |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
| CN114085913A (en) * | 2021-07-02 | 2022-02-25 | 苏州鉴达生物科技有限公司 | STR locus fluorescence labeling multiplex amplification kit for mouse cell strain identification and application thereof |
| CN114196633A (en) * | 2021-11-29 | 2022-03-18 | 湖南亚大丰晖新材料有限公司 | Kit for removing and detecting mycoplasma and application thereof |
| CN114921562A (en) * | 2022-04-27 | 2022-08-19 | 南方海洋科学与工程广东省实验室(广州) | SSR multiple PCR primer for Oxytropis myriophylla and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 张平平等: "多重生物检测技术研究进", 《军事医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117418024A (en) * | 2023-12-19 | 2024-01-19 | 四川大学华西医院 | Detection kit and method for identifying mouse-derived cell line |
| CN117535388A (en) * | 2023-12-28 | 2024-02-09 | 广州赛库生物技术有限公司 | Amplification primer, multiplex amplification system and kit for detecting rat short tandem repeat sequence |
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