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CN114134239B - Kit for rapidly evaluating quality of mammalian cells by PCR method and detection method thereof - Google Patents

Kit for rapidly evaluating quality of mammalian cells by PCR method and detection method thereof Download PDF

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CN114134239B
CN114134239B CN202111417378.8A CN202111417378A CN114134239B CN 114134239 B CN114134239 B CN 114134239B CN 202111417378 A CN202111417378 A CN 202111417378A CN 114134239 B CN114134239 B CN 114134239B
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CN114134239A (en
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林锦梅
陈少锐
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Guangzhou Yeshan Biotechnology Co ltd
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Abstract

The invention relates to the field of cell identification, in particular to a kit for rapidly evaluating the quality of mammalian cells by a PCR method and a detection method thereof. The kit comprises a nucleotide sequence shown as SEQ ID NO:1 to 12. The invention selects fragments with higher conservation in the types of commonly possibly polluted cells as primers, can detect the cell species and the types, has short detection time, can quickly obtain results, can obtain the accurate growth quality of infected bacteria, mycoplasma and cells or the types and sexes of cross infection of cells, is convenient for subsequent experimental treatment or protection, has wide detection range, can detect multiple types of possibly polluted cells, reduces the possibility of pollution or false positive, and can comprehensively identify cell lines.

Description

Kit for rapidly evaluating quality of mammalian cells by PCR method and detection method thereof
Technical Field
The invention relates to the field of cell identification, in particular to a kit for rapidly evaluating the quality of mammalian cells by a PCR method and a detection method thereof.
Background
Cells cultured in vitro are increasingly being used in scientific research and production. Meanwhile, the use of cross-contaminated cells is becoming serious, and the quality of scientific research and the quality of products are seriously affected. In addition to research, cultured cells are used for disease diagnosis and production of bioactive substances, and the quality of the cells is particularly important. Therefore, the method is widely focused by students, and the quality requirement is put forward. The current approach to the identification of research cell lines is comprehensive.
The core problems existing in the use of in vitro cultured cells are: pathogen, microbial contamination, cross-contamination of cells or misidentification, and genetic information changes due to excessive passage of cells. The reasons for these changes are mainly: cell culture operation technique is not standard, cell sources are not clear, passage numbers are not clear, cell errors are introduced, and the like.
Mycoplasma pollution is a problem which is easy to occur, difficult to avoid and difficult to find in the cell culture process. When cells (particularly passaged cells) are contaminated with mycoplasma, the intracellular DNA, RNA and protein expression changes, while the growth rate of the cells is generally not significantly affected, so that contamination of the cells with mycoplasma is generally not noticeable.
The mycoplasma detection generally employs the following methods:
1. culture method: the method is the most traditional mycoplasma pollution detection method and is also the earliest detection method, the basic experimental steps are that firstly, a cell culture is placed in a mycoplasma broth liquid culture medium, after the culture is carried out for 3 days at 37 ℃, the cell culture is centrifuged, sediment is transferred into a mycoplasma solid agar culture medium, the culture is carried out under the same condition, and if similar omelet-like colonies appear in the culture medium, the cell is polluted by mycoplasma.
Dna fluorescent staining method: the fluorescent dye is combined with the A-T enrichment region of DNA by using a fluorescent reagent Hoechst 33258 and the like to label cells and mycoplasma DNA, and can be dyed because the A-T content in the mycoplasma DNA is majority, and the dye generates yellow green fluorescence under the excitation of ultraviolet light, so that a fluorescent microscope is used for observing whether mycoplasma exists. The normal nucleus region has clear fluorescence, and the cytoplasmic region has no fluorescence. The cells contaminated by mycoplasma can see a plurality of fluorescent small spots with uniform size not only on the cell nucleus but also outside the cell nucleus and on the cell membrane, namely mycoplasma DNA, which proves that the cells are contaminated by mycoplasma.
PCR (Polymerase Chain Reaction) method for detecting mycoplasma: mycoplasma as a prokaryote whose rRNA genes are arranged with conserved and mutable sequences at intervals can be used as an index for biological classification. The PCR uses 4 dNTPs as substrates, and under the condition of the existence of a primer, complementary strand extension is carried out at the 3' -end of a DNA template, and a trace amount of template nucleic acid can be amplified exponentially through repeated cycles. The PCR detection method is particularly suitable for detection and identification of mycoplasma which are difficult and time-consuming to culture.
4. Fluorescent quantitative PCR method: fluorescent quantitative PCR is to add fluorescent probes or corresponding fluorescent dyes based on conventional PCR to realize real-time quantification. As the PCR reaction proceeds, the PCR reaction products are continuously accumulated, and the fluorescence signal intensity is also increased in equal proportion. The fluorescence signal is collected every time a cycle is passed, so that the change of the product quantity can be monitored through the change of the fluorescence intensity, and a fluorescence amplification curve is obtained. The Ct value in the real-time fluorescent quantitative PCR technique refers to the number of cycles that each reaction tube undergoes when the fluorescent signal within it reaches a set threshold. The Ct value of each template has a linear relationship with the logarithm of the starting copy number of the template, the more the starting copy number, the smaller the Ct value. A standard curve can be made using standards of known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value. Thus, the initial copy number of an unknown sample can be calculated from the standard curve as long as the Ct value of that sample is obtained.
Bacterial contamination is a common contamination in laboratory cell culture. The method is very important to attach importance to the beginning, so that pollution is prevented, otherwise, the prior work is abandoned, time is wasted, manpower and material resources are wasted, and even irreparable losses are caused. Even if antibiotics (generally preventive doses) are added to the cell culture broth, contamination may occur due to careless handling. The most common are gram-positive bacteria, such as bacillus subtilis, and gram-negative bacteria such as escherichia coli and pseudomonas, among which staphylococcus albus is common.
After bacterial contamination of the cultured cells, turbidity of the culture medium occurs, and the pH changes to yellow. There are also culture solutions which are not changed much by visual inspection, and only contamination can be found under a mirror. Therefore, the observation should be carefully performed every day. After pollution, cells are subjected to pathological changes, intracellular particles are increased and thickened, and finally the cells become round, fall off and die, so that test failure and cell strain loss are caused. Generally, the bacterial contamination process is fast, and the bacterial contamination process can be observed by naked eyes after about one or two days. Bacterial detection generally employs the following methods:
1. visual inspection: bacterial and fungal contamination often occurs after open procedures such as passaging, pipetting, sampling, and the like, and proliferation is rapid and if contaminated, it is evident that it is observed within 48 hours.
2. And (3) inoculation observation: contamination can also be found by inoculating with a common broth or with a culture medium without the addition of a dual-antibody drug.
3. And (3) observing under a microscope: a large number of spherical particles float in the culture solution under the high-power microscope of the inverted microscope, namely bacterial pollution is caused; if there is a filamentous, tubular, dendritic or oval material between the cells, fungal contamination is common.
4. Sequencing and verification: and extracting genome from a proper amount of culture solution, sequencing, and analyzing a sequencing result.
Cell cross-infection detection generally employs the following methods:
cell culture techniques are not standard, cell sources are not clear, passage numbers are not clear, introduced cell errors use cross-contaminated or misidentified cells, leading to erroneous research findings, unrepeatable results, catastrophic consequences of clinical cell therapy, which waste a lot of time, effort and money.
Therefore, NIH, ATCC, nature and Science and the like have called for a plurality of times in recent years, and researchers are required to identify cells, for example, 2011, the national institute of standards for cell STR identification has issued a national standard exclusively; the journal of Science, 2014, month 12, 2015, journal of Science, respectively, monograph cell cross-contamination and error identification severity; 2015, 4 month Nature notification: the Nature flag journal will require the authors to identify the cell lines used in the paper from 5 months; a scientist has been reported to cancel Nature paper by misuse of the cell line in 2015, 6.
1. Isozymogram detection method: as the cells from different species have differences in isozymogram distribution, after separation by gel electrophoresis, the electrophoresis band type and the relative migration distance are detected, and the method can be used for detecting the cross contamination of cells. The isozymogram detection method is suitable for detecting the cross contamination of cells among species. When the isozymic detection method is used for detecting the cross contamination of cells, the detection of glucose-6-phosphate dehydrogenase (G6 PD), lactate Dehydrogenase (LDH), malate Dehydrogenase (MD) and Nuclear Phosphorylase (NP) is very important. If the mobility of the isozymes of the cell sample to be detected is inconsistent with that of the standard reference substance or a plurality of zymogram bands exist, the cell sample can be judged to have inter-species cell cross contamination.
HLA genotyping assay: because of its high polymorphism, HLA is a stable genetic marker that is most representative of individual specificity and accompanies the individual's life, the chance of HLA type identity being very low between unrelated individuals, and thus by detecting the HLA genotype of cells, it is possible to determine whether or not there is cross-contamination of cells. Since HLA genotypes only exist in human bodies, the HLA genotyping detection method is suitable for detecting cross contamination among different humanized cells (in species). The HLA genotyping method is preferably carried out by PCR amplification using sequence specific primers and HLA high resolution typing detection combined with a second generation sequencing technology. If the HLA genotyping result of the cell sample to be detected is obviously different from that of the target cell, the human cell can be judged to have intra-species cell cross contamination.
3. Cell morphology analysis: the cell morphology detection method generally detects characteristics such as cell growth morphology and growth characteristics, and primarily judges whether cell cross contamination occurs. The cell morphology detection method is suitable for detecting cross contamination of cells growing on the wall, and is not suitable for cells growing in suspension. The detection indicators of the cell morphology detection method generally comprise cell morphology (such as circles, fusiform shapes and the like), cell diameter and cell volume. Because of the low detection accuracy, if the cell morphology detection method detects that cross contamination of the cell is likely to occur, it is important to select other detection methods for further verification and validation.
PCR detection method: the PCR detection method is a cell cross contamination detection method established based on the evolutionary conservation difference among species, and has higher sensitivity and specificity. Currently, conserved genes such as cytochrome b (CYTB) and cytochrome c oxidase 1 (cytochrome coxidasesubunit, col) have large differences among different species, and have been used for detection of cell cross-contamination. The PCR detection method is mainly suitable for detecting the cross contamination of cells among species at present.
STR genotyping assay: the STR genotyping detection method is characterized in that STR patterns are obtained by amplifying and detecting a plurality of STR sites simultaneously according to the polymorphism of cells from different individuals at the STR sites, and whether the cells have cross contamination or not is detected, so that the method has the characteristics of high sensitivity, high discrimination capability, standardized automatic typing and the like. The STR genotyping detection method is suitable for detecting cross contamination of cells in the species. When the STR genotyping detection method is used for detecting the cross contamination of the human cells, the detected STR loci preferably comprise: d13S317, TH01, D5S818, D16S53: TPOX, D7S820, CSF 1PO, vWA and sex determination site Amelogenin (AMEIJ).
6. Immunofluorescent staining assay: some cells have specific antigens or receptor markers (such as specific proteins), and immunofluorescent staining detection methods can observe and detect the biological characteristics of the cells under a fluorescent microscope by labeling fluorescent pigments on the antibodies (or antigens) that do not affect the activity of the antigen-antibody, and then binding the antibodies to their corresponding antigens (or antibodies). The immunofluorescence staining detection method is suitable for detecting the special characteristics of cells; antibodies specific to some species cells are also suitable for detection of cross-contamination of cells between some species by immunofluorescent staining. In performing immunofluorescent staining assays, selection of antigen or receptor markers with both specificity and specificity is important for efficient identification of cell-specific properties.
However, the above-mentioned detection is not complete, and it is necessary to determine which pollution is detected by a method corresponding to the selection of the pollution through the experience of an operator. In addition, various experimental methods have corresponding defects.
Telomerase detection:
telomerase is an enzyme that synthesizes telomeres at the end of a chromosome. Telomeres (telomere) are natural ends of chromosomes in eukaryotic cells, consist of thousands of 6 base repeats (TTAGGG), are essential genetic components of cells, and function to maintain telomeres of sufficient length to ensure accuracy of genetic information during replication to prevent loss of genetic information at the ends of chromosomes. Under normal conditions, each cell division cycle causes the telomeres to become shorter and shorter, and when the telomeres are short to some extent, signals that the cells stop dividing. The number of passages is large, and telomeres can be shortened when the cell growth state is poor. In the process of cell chromosome replication, the self RNA can be used as a template to synthesize a repetitive sequence at the 3' -end of a chromosome, so that the length of telomeres can be maintained. Telomerase activity was inhibited in normal somatic cells, but a vast array of data suggests expression in some malignancies as high as 85%. Since telomerase is expressed specifically in a variety of tumor cells, and is necessary for the continued division of most tumor cells.
Basic principle: by utilizing the characteristic that telomerase can take the template region of its own RNA as a template in vitro, a repetitive sequence of 6 bases is added at the tail end of a proper oligonucleotide chain, the repetitive sequence is amplified by a PCR method, and then a ladder with 6 base differences is displayed by agarose electrophoresis. The main principle of TRAP method established by Kim in 1994 is as follows: firstly, synthesizing a TS of 18nt as an upstream primer, combining telomerase with GTT of the TS terminal and synthesizing AGGGTTAG, then synthesizing a 6-base repeated sequence of ggttag each time by translocation, adding CX as a downstream primer after the inactivation of the telomerase, and amplifying telomerase extension products through multiple denaturation, annealing and extension. The PCR telomerase activity detection kit is based on the same principle to detect the activity of telomerase. Positive results showed ladder-like bands 6bp apart on gel electrophoresis, with the number, oligo, deep or shallow bands indicating the magnitude of telomerase activity. The advantages of high sensitivity, strong specificity and the like of the traditional TRAP method are maintained. Meanwhile, an internal standard substance is additionally arranged, a positive reference substance is provided, the primer design is optimized, the PCR effect is further improved, and the activity of telomerase in cell and tissue extracts can be detected with high sensitivity.
Mycoplasma detection:
1. the culture method is simple and visual, most mycoplasma can generate typical specific colonies, but the mycoplasma grows slowly, the culture generation needs about 3-4 hours, and the mycoplasma can be judged whether to be polluted or not by culturing for 28 days generally, so that the workload is large, and the risk of mycoplasma transmission is further increased. The mycoplasma is harsh to the culture conditions, the normal growth of the mycoplasma can be influenced by the different component quality or batch of the culture medium, and some mycoplasma can not grow through a culture method, so that the detection result by the culture method is inaccurate, and false negative is easy to generate.
The DNA fluorescent staining sensitivity is lower, and the light mycoplasma pollution of the cells is not easy to detect due to the existence of background fluorescence. Cell lysis death produces debris that can also be stained by fluorescent dyes and mistakenly by mycoplasma staining to cause false positives. Compared with the cell culture method, the DNA fluorescent staining method shortens the detection time, but has low sensitivity and accuracy and requires a fluorescent microscope for detection. The fluorescent staining method does not determine which mycoplasma specifically cause cell contamination. Fluorescent staining generally requires culturing of contaminant-free indicator cells as a control, increasing the detection effort.
3. Because of the large copy number of the PCR product, extremely trace contamination can cause false positives. The most likely form of contamination of the PCR products is aerosol contamination, which can occur when the reaction tube is shaken relatively vigorously during operation, particularly when the lid of the reaction tube is opened for the electrophoretic deposition process. The presence of large copies of an aerosol particle, and thus false positives due to contamination by aerosols, is a particularly important problem, and PCR can only detect one indicator at a time.
4. The real-time fluorescent quantitative PCR requires a fluorescent quantitative PCR instrument with high price, and has high equipment maintenance cost, complex machine setting operation, and difficult comprehensive popularization by professional staff.
The STR profile analysis method is only suitable for the identification of the intra-species cells of a few species cells of human and mouse sources and the detection of cross contamination.
Bacterial detection:
1. visual inspection: the method is not objective and accurate enough and is easy to misjudge.
2. And (3) inoculation observation: the requirements on culture conditions and operation are high, and the sterile operation is required to be strictly maintained. Therefore, other strains are easy to infect in the operation process, and the result is misjudged.
3. And (3) observing by a microscope: observation under a microscope may deviate from the judgment of the result. This method is not sufficiently stringent as it is not possible to accurately determine whether an infected cell is bacterial or not, and not for other reasons.
4. Sequencing and verification: the laboratory equipment and the technical requirements are high, professional equipment is needed, and the operation is complex.
Cell cross infection detection:
1. isozymogram detection method: subjective judgment may affect the judgment of the result, and no objective data exists. The sensitivity is low.
HLA genotyping assay: the typing identification requirement is high.
3. Cell morphology analysis: suspension cells cannot be detected, and the accuracy is low.
PCR detection method: the cost of experimental equipment is high, and the experiment is more complicated.
STR genotyping assay: the method is only suitable for detecting the cross infection of the human cells at present.
6. Immunofluorescent staining assay: nonspecific staining affects detection sensitivity.
Cell growth quality:
the mechanism of cell senescence has not been thoroughly elucidated to date. Since most immortalized and tumor cells highly express telomerase activity, it is currently believed that changes in telomere length may be a molecular biological clock of cell division and senescence.
Therefore, the detection method has the defects that the detection time is too long, the mycoplasma infection, bacteria, cell types and cell algebra cannot be accurately and qualitatively detected at the same time, the common detection is single, the single detection range is narrow, the cell types cannot be detected, and the observed result cannot be judged only subjectively, and objective data are needed to support the experimental result.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a kit for rapidly evaluating the quality of mammalian cells by a PCR method and a detection method thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a kit for rapidly evaluating the quality of mammalian cells by a PCR method is provided, wherein the kit comprises a nucleotide sequence shown in SEQ ID NO:1 to 12.
As a preferred embodiment of the kit of the present invention, taq Pro Multiplex DNA polymerase is also included in the kit.
As a preferred embodiment of the kit of the present invention, the kit further comprises a 2X Multiplex buffer.
As a preferred embodiment of the kit of the present invention, the 2×multiplex buffer includes: enzyme reaction buffer, 2mM dNTPs and 25mM MgSO 4 。
The invention also provides a detection method for rapidly evaluating the quality of the mammalian cells by a PCR method, wherein the detection method adopts a nucleotide sequence shown in SEQ ID NO:1 to 12, and performing PCR detection.
As a preferred embodiment of the detection method of the present invention, the PCR reaction may employ a standard procedure under the following conditions: pre-denaturation is carried out at 95 ℃ for 30 sec-5 min; denaturation at 95℃for 30sec; annealing at 60 ℃ for 90sec; extending at 72 ℃ for 60sec; thoroughly extending at 72 ℃ for 10min; cycle number: 30-34.
As a preferred embodiment of the detection method of the present invention, the PCR reaction may employ a rapid procedure under the following conditions: pre-denaturation at 95℃for 30sec; denaturation at 95℃for 15sec; annealing at 60 ℃ for 30sec; extending at 72 ℃ for 30sec; thoroughly extending at 72 ℃ for 5min; cycle number: 25-30.
The invention has the beneficial effects that:
1. the detection time is short, and the result can be obtained rapidly;
2. the method can obtain the accurate cross infection type or cell growth condition (telomere length) and sex of the infected bacteria, mycoplasma or cells, and is convenient for subsequent experimental treatment or protection;
3. the designed kit is simple and convenient for experimental detection;
4. the detection range is wide, and a plurality of types of possibly polluted cells can be detected;
5. reducing the likelihood of contamination or false positives;
6. fragments with higher conservation in the types which can pollute cells generally are selected as primers;
7. cell species and types may be detected (optionally with the addition of sex identification primers).
Drawings
FIG. 1 is a graph of the results of mycoplasma detection; m: DNA2000 Marker;1: PK-15 cell positive control; 2: a Hela cell positive control; 3: BMSCs;4-5: THP-1 cells; 6: complete culture medium for culturing THP-1 cells; 7: hyclone 1640 medium; 8: hyclone Australian foetus calf serum; 9: blank control.
FIG. 2 is a 1.5% agarose electrophoresis chart of bacterial detection results; lane M: DL2000 DNA markers; lane 1: sample amplification products; lane 2: positive control amplification product; lane 3: negative control amplification product; lane M: DL2000 DNA Marker.
FIG. 3 is an identification chart of sample species; m: and (5) Marker. The sizes from top to bottom are 700, 600, 500, 400, 300, 200 and 100bp respectively. 1484: no band was detected, which was not one of the nine species we tested (homosapiens 391bp,Cricetulus griseus315bp,Macaca mulatta287bp,Cercopithecus aethiops222bp,Rattus norvegicus196bp,Canis familiaris172bp,Mus musculus150bp,Bos Taurus102bp,IC 70bp). 1485: the sample is temporarily a human.
FIG. 4 is a graph showing the results of cell telomere length detection.
FIG. 5 is a diagram of primer positions.
FIG. 6 is a sexing electrophoretogram.
Detailed Description
In order to more clearly describe the technical solution of the present invention, the following description is further given by way of specific examples, but not by way of limitation, only some examples of the present invention.
Example 1
The present example provides a method for rapidly evaluating the quality of mammalian cells by PCR, wherein the primers selected in the invention amplify the following products:
the primer sequences used were:
1. detection method
And (5) sucking the culture supernatant of the THP-1 cells, and adding the culture supernatant into a PCR reaction system. The enzyme used was Taq Pro Multiplex DNA polymerase from Toyo-spun. The specific content of each component in the PCR reaction system is as follows: 2×multiplex buffer; the PCR conditions are shown in the following table:
standard procedure:
quick procedure:
note that:
a. in many cases, a default annealing temperature is used. If the amplification effect is poor, the optimal annealing temperature can be searched through an annealing temperature gradient experiment; rapid procedures lose some of the amplification yield, and standard procedures are recommended for initial amplification.
b. The pre-denaturation time can be adjusted according to different template types. The extracted nucleic acid was pre-denatured for 30sec; when whole blood, blood card, etc. are directly expanded, the pre-denaturation time can be properly prolonged to 5min.
c. When the low copy template, the long fragment or the amplified fragment is amplified, the annealing time can be prolonged to 3min properly so as to improve the amplification efficiency.
d. The extension time is based on the longest segment. However, too long an extension time leads to increased non-specific amplification, and the amplification specificity can be improved by shortening the extension time.
e. When a trace sample is amplified, the amount of amplified product can be increased by increasing the number of cycles. However, too many cycles can result in increased non-specific amplification, and the amplification specificity can be increased by reducing the number of amplification cycles.
2. Detection result
(1) The mycoplasma detection results are shown in FIG. 1, the primer sequences adopted by the invention can identify 12 mycoplasma simultaneously, and the amplification size is 261-576 (the reference is listed below, and the specific sequencing results are shown as follows):
human mycoplasma: 261
Mycoplasma arthritis: 262
Mycoplasma salivarius: 295
Mycoplasma argininis: 300
Mycoplasma stomatae: 316
Ureaplasma urealyticum: 300
Fermentation of mycoplasma: 391
Mycoplasma neurolytium: 393
Mycoplasma hyopneumoniae: 250
Mycoplasma hyorhinis: 300
Mycoplasma pulmonary disease: 576
Lei's cholesterol-free: 400
(2) The bacterial detection results are shown in FIG. 2, which shows whether the sample is contaminated with bacteria. The amplification was performed with 16s primer, with the banded sample being bacterial contaminated and without bacterial contamination.
(3) The results of cell cross-contamination are shown in FIG. 3, indicating sample species identification. People: 391, mice: 150.
(4) Telomerase length detection: ladder-like bands separated by 6bp are shown on gel electrophoresis, and the number, oligo, deep or shallow of the bands indicates the magnitude of telomerase activity.
Kim established a sensitive telomerase detection method by PCR technology in 1994-telomere repeat fragment amplification (TRAP). The product is a research kit developed by properly improving the method. Compared with the common telomerase activity assay, the product has the following characteristics:
and the telomerase extracting solution is subjected to PCR amplification in the same tube, so that the detection accuracy is improved.
The primer design was optimized to further improve the PCR effect.
(5) As shown in FIG. 4, the sex determination result shows that the fragments of the sex products are different in length, the fragment of the Y gene is 218, and the fragment of the X gene is 106.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Guangzhou light-curing biotechnology Co., ltd
<120> kit for rapidly evaluating quality of mammalian cells by PCR method and detection method thereof
<130> 2021.11.24
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<400> 1
tcgtaacaag gtatccctac 20
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
gcatccacca aatactct 18
<210> 3
<211> 19
<212> DNA
<213> artificial sequence
<400> 3
aactggagga aggtgggga 19
<210> 4
<211> 19
<212> DNA
<213> artificial sequence
<400> 4
aggaggtgat ccaaccgca 19
<210> 5
<211> 23
<212> DNA
<213> artificial sequence
<400> 5
tagacatcgt actacacgac acg 23
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
tccaggttta tggagggttc 20
<210> 7
<211> 20
<212> DNA
<213> artificial sequence
<400> 7
ccagtatgca gtggttcaga 20
<210> 8
<211> 21
<212> DNA
<213> artificial sequence
<400> 8
ctcgatgaca acagagttcc t 21
<210> 9
<211> 21
<212> DNA
<213> artificial sequence
<400> 9
acctcatcct gggcaccctg g 21
<210> 10
<211> 20
<212> DNA
<213> artificial sequence
<400> 10
tccgaactcc ggttggtagt 20
<210> 11
<211> 24
<212> DNA
<213> artificial sequence
<400> 11
ttctctttac taattttgat cact 24
<210> 12
<211> 24
<212> DNA
<213> artificial sequence
<400> 12
tagtctcgaa tttgaccctt cgac 24

Claims (7)

1. A kit for rapidly evaluating the quality of mammalian cells by a PCR method, which is characterized by comprising a nucleotide sequence shown in SEQ ID NO: 1-12.
2. The kit of claim 1, further comprising Taq Pro Multiplex DNA polymerase.
3. The kit of claim 1, further comprising a2 x Multiplex buffer.
4. The kit of claim 3, wherein the 2 x Multiplex buffer comprises: enzyme reaction buffer, 2mM dNTPs and 25mM MgSO 4 。
5. A detection method for rapidly evaluating the quality of mammalian cells by a PCR method for non-disease diagnosis and treatment purposes is characterized in that the detection method adopts a nucleotide sequence shown as SEQ ID NO: 1-12, performing PCR detection on the primer shown in the figure;
wherein, the detection of cell quality is: mycoplasma detection, bacterial detection, cell cross-contamination detection, telomerase length detection, and sex identification.
6. The method of claim 5, wherein the PCR reaction uses standard procedures under the following conditions: pre-denaturation at 95 ℃ for 30 sec-5 min; denaturation at 95℃for 30sec; annealing at 60 ℃ for 90sec; extending at 72 ℃ for 60sec; thoroughly extending at 72 ℃ for 10min; cycle number: 30-34.
7. The method of claim 5, wherein the PCR reaction employs a rapid procedure under the following conditions: pre-denaturation at 95℃for 30sec; denaturation at 95℃for 15sec; annealing at 60 ℃ for 30sec; extending at 72 ℃ for 30sec; thoroughly extending at 72 ℃ for 5min; cycle number: 25-30.
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