CN114134239A

CN114134239A - Kit for rapidly evaluating mammalian cell quality by PCR (polymerase chain reaction) method and detection method thereof

Info

Publication number: CN114134239A
Application number: CN202111417378.8A
Authority: CN
Inventors: 林锦梅; 陈少锐
Original assignee: Guangzhou Yeshan Biotechnology Co ltd
Current assignee: Guangzhou Yeshan Biotechnology Co ltd
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-03-04
Anticipated expiration: 2041-11-25
Also published as: CN114134239B

Abstract

The invention relates to the field of cell identification, in particular to a kit for rapidly evaluating the cell quality of mammals 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 the fragments with higher conservation in the types of the general possibly polluted cells as the primers, can detect the cell species and types, has short detection time, can quickly obtain the result, can obtain the accurate infected bacteria, mycoplasma, cell growth quality or cell cross infection type and sex, is convenient for subsequent experimental treatment or protection, has wide detection range, can detect a plurality of types of the possibly polluted cells, reduces the possibility of pollution or false positive, and can comprehensively identify the cell line.

Description

Kit for rapidly evaluating mammalian cell quality by PCR (polymerase chain reaction) 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 cell quality of mammals by a PCR method and a detection method thereof.

Background

Cells cultured in vitro are increasingly used in scientific research and production. Meanwhile, the use of cross-contaminated cells is getting more and more serious, and the quality of scientific research and the quality of products are seriously influenced. In addition to research, cultured cells are also used for disease diagnosis and production of bioactive substances, and the quality of the cells is particularly important. Therefore, the method attracts the attention of students and puts forward quality requirements. The cell lines currently being proposed for research need to be fully characterized.

The core problems existing in the use of the in vitro cultured cells are: pathogens, microbial contamination, cell cross-contamination or identification errors, and genetic information changes due to over-passaging of cells. The reasons for these changes are mainly: cell culture procedures are not standard, cell sources are not clear, passage numbers are not clear, introduced cell errors, and the like.

Mycoplasma contamination is a problem that is easy to occur, difficult to avoid and difficult to find in the cell culture process. When cells (particularly subcultured cells) are contaminated with mycoplasma, the expression of DNA, RNA and protein in the cells is changed, and the growth rate of the cells is generally not significantly influenced, so that the contamination of the cells with mycoplasma is generally difficult to detect.

Mycoplasma detection generally employs several methods:

1. the culture method comprises the following steps: the method is characterized by comprising the following steps of placing a cell culture in a mycoplasma broth liquid culture medium, culturing at 37 ℃ for 3 days, centrifuging, transferring a precipitate into a mycoplasma solid agar culture medium, culturing under the same condition, and observing that if a similar fried egg-like bacterial colony appears in the culture medium, the cell is polluted by the mycoplasma.

DNA fluorescent staining method: cells and mycoplasma DNA are marked by using a fluorescent reagent Hoechst 33258 and the like, a fluorescent dye is combined to an A-T enrichment area of the DNA, the mycoplasma DNA can be dyed because the A-T content is most, the dye generates yellow green fluorescence under the excitation of ultraviolet light, and whether the mycoplasma exists is observed by using a fluorescence microscope. The normal nucleus region shows fluorescence with clear and regular edges, and the cytoplasm region does not show fluorescence. The mycoplasma contaminated cells can see many fluorescent dots with uniform size on the cell nucleus, the outside of the cell nucleus and the cell membrane, namely mycoplasma DNA, and the mycoplasma contaminated cells are proved.

Method for detecting Mycoplasma by PCR (polymerase Chain reaction): the rRNA gene of mycoplasma, a prokaryotic organism, is formed by interval arrangement of conserved and multiple-variant sequences and can be used as an index for biological classification. PCR uses 4 dNTPs as substrates, and performs extension of a complementary strand at the 3' end of a DNA template in the presence of a primer, and can exponentially amplify a trace amount of template nucleic acid through repeated cycles. The PCR detection method is particularly suitable for detection and identification of mycoplasma which are difficult to culture and time-consuming.

4. A fluorescent quantitative PCR method: the fluorescent quantitative PCR is realized by adding a fluorescent probe or a corresponding fluorescent dye on the basis of the conventional PCR. As the PCR reaction proceeds, the PCR reaction products are accumulated continuously, and the intensity of the fluorescence signal is increased in equal proportion. After each cycle, the fluorescence signal is collected, so that the change of the product amount can be monitored through the change of the fluorescence intensity, and a fluorescence amplification curve is obtained. The Ct value in real-time fluorescent quantitative PCR technology refers to the number of cycles that the fluorescent signal in each reaction tube undergoes when it reaches a set threshold. The Ct value of each template has a linear relation with the logarithm of the initial copy number of the template, and the more the initial copy number is, the smaller the Ct value is. A standard curve can be made using a standard with a known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value. Therefore, once the Ct value of an unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.

Bacterial contamination is a common contamination in laboratory cell cultures. Attention is paid to prevent pollution from beginning, otherwise, the pollution can be abandoned, time is wasted, manpower and material resources are wasted, and even loss which cannot be compensated is caused. Even if antibiotics (generally in prophylactic doses) are added to the cell culture fluid, contamination can result from inadvertent handling. The most common are gram-positive bacteria such as Bacillus subtilis, and gram-negative bacteria such as Escherichia coli and Pseudomonas, and the most common is Staphylococcus albus.

After the cultured cells are contaminated by bacteria, the culture solution becomes turbid, and the pH changes to become yellow. The visual observation of some culture solutions has little change, and the contamination can only be known by the thalli found under a mirror. Therefore, careful observation should be made every day. After pollution, cells are pathologically changed, and intracellular particles are increased and thickened, and finally become round and fall off to die, so that test failure and cell strain (line) loss are caused. Bacterial contamination generally progresses rapidly and can be observed visually after about one or two days. Bacteria detection generally employs several methods:

1. and (4) visual observation: bacterial and fungal contamination often occurs after open operations such as passage, liquid change, sample addition and the like, and proliferation is rapid, and if contamination exists, the contamination can be obviously observed within 48 hours.

2. And (3) inoculation observation: contamination can also be detected by inoculating with a normal broth or by inoculating with a culture medium without the double-resistant drug.

3. And (3) observing under a microscope: a large amount of spherical particles in the culture solution can be seen to float under a high power microscope of an inverted microscope, namely bacterial pollution is obtained; if there are filamentous, tubular, dendritic or ovoid material between the cells, fungal contamination is common.

4. Sequencing and verifying: taking a proper amount of culture solution to extract genome and sequencing, and then analyzing a sequencing result.

Cell cross-infection detection generally employs several methods:

cell culture procedures are not technically standardized, cell sources are not clear, passage numbers are not clear, introduced cells misuse cross-contaminated or misidentified cells resulting in erroneous findings, unrepeatable results, catastrophic consequences of clinical cell therapy, which wastes a lot of time, effort and money.

Therefore, in recent years, NIH, ATCC, Nature, Science and the like have called for many times, researchers are required to identify cells, and for example, 2011 the national standard institute of the national standards of cell STR identification has specially issued; journal of Science of 12 months in 2014 and 2 months in 2015 respectively issues a special article to illustrate the severity of cell cross contamination and misidentification; nature notification of month 4 in 2015: nature J.Gen.will require the authors, starting from month 5, to identify the cell lines used in the paper; in 2015, 6 months, it was reported that a scientist withdrawn the Nature paper due to the use of wrong cell lines.

1. Isozyme spectrum detection method: because cells from different species have difference in isozyme spectrum 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 cell cross contamination. The isozyme spectrum detection method is suitable for detecting cross contamination of cells among species. When the isozyme spectrum detection method is adopted to detect the cross contamination of cells, the detection of glucose-6-phosphate dehydrogenase (G6PD), Lactate Dehydrogenase (LDH), Malate Dehydrogenase (MD) and Nuclear Phosphorylase (NP) is very important. If the isozyme mobility 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 intergeneric cell cross contamination.

HLA genotyping assay: since HLA is a stable genetic marker that is most representative of individual specificity due to its high polymorphism and that accompanies the life of an individual, the probability that HLA types are identical between unrelated individuals is extremely low, and thus, the presence or absence of cross contamination of cells can be determined by detecting the HLA genotype of the cells. Since the HLA genotype exists only in human body, the HLA genotyping detection method is suitable for the detection of cross contamination between different human cells (within species). The HLA genotype detection method preferably adopts sequence specific primers for PCR amplification and combines with the second generation sequencing technology for HLA high-resolution typing detection. If the HLA genotyping result of the cell sample to be detected is obviously different from that of the target cell, the existence of cell cross contamination in the species of the human cells can be judged.

3. And (3) cell morphology analysis: the cell morphology detection method generally detects the characteristics of cell growth morphology, growth characteristics and the like, and preliminarily judges whether cell cross contamination occurs. The cell morphology detection method is suitable for detecting cross contamination of cells growing in an adherent manner, and is not suitable for cells growing in a suspension manner. The detection indexes of the cell morphology detection method generally comprise cell morphology (such as round shape, fusiform shape and the like), cell diameter and cell volume. Due to the low detection accuracy, if the cells are detected to be possibly cross-contaminated by a cell morphology detection method, the selection of other detection methods for further verification and confirmation is very important.

PCR assay: the PCR detection method is a cell cross-contamination detection method established based on evolutionary conservative differences among species, and has high sensitivity and specificity. At present, conserved genes such as cytochrome b (CYTB) and cytochrome c oxidase 1(cytochrome coxasebunit 1, Col) have large differences between different species, and have been used for detection of cell cross contamination. The PCR detection method is mainly suitable for detecting cross contamination of cells among species at present.

STR genotyping assay: the STR genotyping detection method is characterized in that an STR map is obtained by simultaneously amplifying and detecting a plurality of STR loci according to the polymorphism of cells from different individual sources at STR loci, whether the cells have cross contamination or not is detected, and the STR genotyping detection method has the characteristics of high sensitivity, high identification capability, standardized automatic typing and the like. The STR genotyping detection method is suitable for detecting cross contamination of cells in 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, D16S 53: TPOX, D7S820, CSF 1PO, vWA and sex determination site Amelogenin (AMEIJ).

6. Immunofluorescence staining assay: some cells have specific antigen or receptor markers (such as specific proteins), and the immunofluorescent staining detection method can observe and detect the biological characteristics of the cells under a fluorescent microscope after the fluorescent pigment which does not influence the activity of antigen antibodies is marked on the antibodies (or antigens) and is combined with the corresponding antigens (or antibodies). The immunofluorescence staining detection method is suitable for detecting the special characteristics of cells; part of the species cells have specific antibodies, and the immunofluorescence staining is also suitable for detecting cross contamination of part of the intergeneric cells. When performing immunofluorescence staining detection, selecting an antigen or receptor marker with typicality and specificity is very important for effectively identifying the specific characteristics of cells.

However, the above detection is not complete, and it is necessary to determine by the experience of the operator which contamination is to be detected by selecting the corresponding method. In addition, various experimental methods have corresponding disadvantages.

And (3) detecting telomerase:

telomerase is an enzyme that synthesizes telomeres at the ends of chromosomes. Telomeres (telomere) are natural ends of chromosomes of eukaryotic cells, are composed of thousands of 6-base repeat sequences (TTAGGG), are essential genetic components of the cells, and have the function of maintaining the telomeres to have enough length and ensuring the accuracy of gene information in a replication process so as to prevent the loss of genetic information at the ends of the chromosomes. Under normal conditions, the telomeres become shorter and shorter in each cell division cycle, and when the telomeres are short to a certain extent, a signal is sent out, and the cell division is stopped. The number of passages is large, and telomeres are shortened due to poor cell growth state. In the process of cell chromosome replication, self RNA is taken as a template, and a repetitive sequence is synthesized at the 3' end of a chromosome to maintain the length of telomeres. Telomerase activity is inhibited in normal somatic cells, but extensive data indicate expression in some malignancies as high as 85%. Because telomerase is specifically expressed in a variety of tumor cells and is required for the continued division of most tumor cells.

The basic principle is as follows: by using the characteristic that telomerase can take the template region of RNA of the telomerase as a template in vitro and adds a 6-base repeated sequence at the tail end of an appropriate oligonucleotide chain, the repeated sequence is amplified by adopting a PCR method, and then a ladder band with 6 base differences is displayed by agarose electrophoresis. The TRAP method established by Kim in 1994 has the following main principles: firstly, synthesizing 18nt TS as an upstream primer, combining telomerase with GTT at the end of TS and synthesizing AGGGTTAG, then synthesizing a 6-base repeat sequence of ggttag by transposition once, after inactivating the telomerase, adding CX as a downstream primer, and amplifying a telomerase extension product by multiple times of denaturation, annealing and extension. The PCR telomerase activity detection kit detects the activity of telomerase based on the same principle. Positive results show ladder-like bands separated by 6bp on gel electrophoresis, and the more, less, darker or lighter bands indicate the activity of telomerase. The advantages of high sensitivity, strong specificity and the like of the traditional TRAP method are reserved. Meanwhile, an internal standard substance is added to provide a positive reference substance, the design of a primer is optimized, the PCR effect is further improved, and the activity of telomerase in cell and tissue extracts can be detected with high sensitivity.

And (3) detection of mycoplasma:

1. the culture method is simple and intuitive, most mycoplasma can generate special typical colonies, but the growth of the mycoplasma is slow, about 3-4 hours is needed for one-generation culture, 28 days of culture is generally needed for judging whether the mycoplasma is polluted, the workload is large, and the risk of further expanding the spread of the mycoplasma is caused. The mycoplasma is harsh on culture conditions, the normal growth of the mycoplasma is affected by different medium component qualities or batches, and some mycoplasma cannot grow by a culture method, so the detection result by using the culture method is not accurate, and false negative is easily generated.

And 2, the DNA fluorescent staining sensitivity is low, and the mild mycoplasma pollution of the cells is difficult to detect due to the existence of background fluorescence. Debris from cell lysis death can also be mistaken for mycoplasma staining by fluorescent staining resulting in false positives. The DNA fluorescent staining method shortens the detection time compared with the cell culture method, but has low sensitivity and accuracy and needs a fluorescent microscope for detection. The fluorescent staining method cannot determine which mycoplasma caused the contamination of cells in particular. Fluorescent staining generally requires the culture of non-contaminating indicator cells as controls, increasing the detection effort.

3. Because of the large copy number of PCR products, a very small amount of contamination can cause false positives. The most likely form of contamination of the PCR product is aerosol contamination, which can occur when the reaction tube is shaken vigorously during handling, especially when the reaction tube is opened for electrophoretic spotting. An aerosol particle contains a large number of copies, so that false positives due to contamination by aerosol are a particular concern, and PCR can only detect one indicator at a time.

4. The real-time fluorescent quantitative PCR needs to be matched with a fluorescent quantitative PCR instrument with high price, the equipment maintenance cost is high, the machine setting operation is complex, professional personnel are needed, and the comprehensive popularization is difficult.

STR spectrum analysis method is only suitable for the intraspecific cell identification and cross contamination detection of cells of few species of human and mouse sources.

And (3) detecting bacteria:

1. and (4) visual observation: the method is not objective and accurate enough and is easy to misjudge.

2. And (3) inoculation observation: the culture conditions and operation requirements are high, and the aseptic operation needs to be strictly maintained. Therefore, other strains are easily infected in the operation process, and the result is misjudged.

3. And (4) microscopic observation: the microscopic observation may cause a deviation in the judgment of the result. The method is not rigorous and effective because it cannot accurately judge whether the infected cells are bacteria or not, and no other reason is available.

4. Sequencing and verifying: the requirements for laboratory equipment and technology are high, professional equipment is needed, and the operation is complex.

And (3) detecting cell cross infection:

1. isozyme spectrum detection method: subjective judgment may affect the judgment of the result, and no objective data exists. The sensitivity is low.

HLA genotyping assay: and the requirement on typing and identification is high.

3. And (3) cell morphology analysis: suspension cells cannot be detected, and the accuracy is low.

PCR assay: the experimental equipment is high in cost, and the experiment is relatively complex to carry out.

STR genotyping assay: at present, the method is only suitable for detecting the cross infection of human cells.

6. Immunofluorescence staining assay: non-specific staining affects detection sensitivity.

The growth quality of the cells is as follows:

the mechanism of cellular senescence has not been completely elucidated so far. Since most immortalized and tumor cells highly express telomerase activity, it is presently 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 detection cannot be simultaneously performed on infected mycoplasma, bacteria, cell types and cell algebra accurately and qualitatively, the detection cannot be performed on multiple types, the common detection is single, the single detection range is narrow, the cell types cannot be detected, the observed result cannot be judged only by subjectivity, 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 cell quality of mammals by a PCR method and a detection method thereof.

In order to achieve the purpose, the invention adopts the technical scheme that: provides a kit for rapidly evaluating the cell quality of mammals by a PCR method, wherein the kit comprises a nucleotide sequence shown as SEQ ID NO: 1 to 12.

As a preferred embodiment of the kit of the present invention, the kit further comprises Taq Pro Multiplex DNA polymerase.

In a preferred embodiment of the kit of the present invention, the kit further comprises a2 × Multiplex buffer.

In a preferred embodiment of the kit of the present invention, the 2 × Multiplex buffer comprises: enzyme reaction buffer, 2mM dNTPs and 25mM MgSO₄。

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 as SEQ ID NO: 1-12, and carrying out PCR detection.

As a preferred embodiment of the detection method of the present invention, the PCR reaction may employ standard procedures under the following conditions: pre-denaturation at 95 ℃ for 30 sec-5 min; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 90 sec; extension 72 ℃ 60 sec; completely extending at 72 ℃ for 10 min; the number of cycles: 30-34.

As a preferred embodiment of the detection method of the present invention, the PCR reaction can be performed by a rapid procedure under the following conditions: pre-denaturation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 15 sec; annealing at 60 ℃ for 30 sec; extension 72 ℃ 30 sec; completely extending at 72 ℃ for 5 min; the number of cycles: 25-30.

The invention has the beneficial effects that:

1. the detection time is short, and the result can be obtained quickly;

2. the cross infection type or cell growth condition (telomere length) and sex of the infected bacteria, mycoplasma or cells can be accurately obtained, and the subsequent experimental treatment or protection is convenient;

3. the kit is designed, and the experimental detection is simple and convenient;

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. selecting a fragment with high conservation in types which are generally possible to pollute cells as a primer;

7. cell species and type can be detected (optionally with sex determination primers).

Drawings

FIG. 1 is a graph showing the results of detection of Mycoplasma; 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 solution for culturing THP-1 cells; 7: hyclone 1640 medium; 8: hyclone australian fetal bovine serum; 9: blank control.

FIG. 2 is a 1.5% agarose electrophoresis chart of the results of bacterial detection; lane M: DL2000 DNA Marker; lane 1: sample amplification products; lane 2: a positive control amplification product; lane 3: negative control amplification products; lane M: DL2000 DNA Marker.

FIG. 3 is a diagram showing identification 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 (Homo sapiens 391bp, Cricetulus griseus315bp, Macaca mulatta287bp, Cercopticitech aethiops222bp, Rattus norvegicus196bp, Canis familiaris172bp, Mus mululus 150bp, Bos Taurus102bp, IC 70 bp). 1485: the sample is temporarily human.

FIG. 4 is a diagram showing the results of telomere length measurement of cells.

FIG. 5 is a diagram of the positions of primers.

FIG. 6 is a sex determination electrophoretogram.

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

Example 1

This example provides a method for rapidly evaluating mammalian cell quality by PCR, and the amplification products of the primers selected by the invention are:

the primer sequences used were:

detection method

The culture supernatant of THP-1 cells was aspirated and added to the PCR reaction system. The enzyme used was Taq Pro Multiplex DNA polymerase from Toyobo. The PCR reaction system comprises the following components in percentage by weight: 2 × Multiplex buffer; the PCR reaction conditions are shown in the following table:

standard procedures:

quick program:

note:

a. in many cases, a default annealing temperature may be used. If the amplification effect is not good, the optimal annealing temperature can be found through an annealing temperature gradient experiment; rapid procedures lose part of the amplification yield, and standard procedures are recommended for primary amplification.

b. The pre-denaturation time can be adjusted for different template types. (ii) extracted nucleic acid, with a pre-denaturation time of 30 sec; in the case of direct amplification of whole blood, blood card, etc., the pre-denaturation time may be extended to 5 min.

c. When a low copy template, a long fragment or a large number of amplified fragments are amplified, the annealing time can be properly prolonged to 3min to improve the amplification efficiency.

d. The extension time is based on the longest segment. However, an extension time that is too long leads to increased non-specific amplification, and the amplification specificity can be improved by shortening the extension time.

e. When a trace amount of a sample is amplified, the amount of the amplified product can be increased by increasing the number of cycles. However, too many cycles lead to increased non-specific amplification, and the amplification specificity can be improved by reducing the number of amplification cycles.

Second, the detection result

(1) The detection result of mycoplasma is shown in fig. 1, the primer sequence adopted by the invention can simultaneously identify 12 mycoplasma, and the amplification size is 261-576 (the following list is used for reference, and the specific content is based on the sequencing result):

mycoplasma hominis: 261

Mycoplasma arthritides: 262

Mycoplasma salivarius: 295

Arginine mycoplasma: 300

Mycoplasma orale: 316

Ureaplasma urealyticum: 300

Fermentation of mycoplasma: 391

Mycoplasma neurolysis: 393

Mycoplasma hyopneumoniae: 250

Mycoplasma hyorhinis: 300

Mycoplasma pneumoniae: 576

Acholeplasma leinii: 400

(2) The results of the bacterial tests are shown in FIG. 2, which indicates whether the sample is contaminated with bacteria. The amplification was performed with 16s primers, with the band sample being bacterial contaminated and not.

(3) The cell cross-contamination results are shown in fig. 3, indicating sample species identification. Human: 391, mice: 150.

(4) detecting the length of telomerase: ladder-like bands separated by 6bp are displayed on gel electrophoresis, and the more, less, deeper or lighter bands indicate the size of telomerase activity.

Kim established a sensitive telomerase detection method, Telomere Repeat Amplification (TRAP), by PCR in 1994. The product is a research kit developed by appropriately improving the method. Compared with the common telomerase activity determination method, the product has the following characteristics:

the telomerase extract 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) The results of sex determination are shown in FIG. 4, which shows that the sex product fragments are of different sizes, the Y gene fragment is 218, and the X gene fragment is 106.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Guangzhou Ye-Zhi-Biotechnology Co., Ltd

<120> kit for rapidly evaluating mammalian cell quality by PCR method and detection method thereof

<130> 2021.11.24

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<170> PatentIn version 3.3

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Claims

1. A kit for rapidly evaluating the cell quality of mammals by a PCR method is characterized by comprising a nucleotide sequence shown as SEQ ID NO: 1 to 12.

2. The kit of claim 1, wherein the kit further comprises 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 MgSO₄。

5. A detection method for rapidly evaluating the quality of mammalian cells by a PCR method is characterized in that the detection method adopts a nucleotide sequence shown as SEQ ID NO: 1-12, and carrying out PCR detection.

6. The detection method according to claim 5, wherein the PCR reaction is performed by a standard procedure under the following conditions: pre-denaturation at 95 ℃ for 30 sec-5 min; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 90 sec; extension 72 ℃ 60 sec; completely extending at 72 ℃ for 10 min; the number of cycles: 30-34.

7. The detection method according to claim 5, wherein the PCR reaction is performed by a rapid procedure under the following conditions: pre-denaturation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 15 sec; annealing at 60 ℃ for 30 sec; extension 72 ℃ 30 sec; completely extending at 72 ℃ for 5 min; the number of cycles: 25-30.