CN112941201B - Mixed primer for multi-cell species identification and cross contamination detection and use method thereof - Google Patents

Mixed primer for multi-cell species identification and cross contamination detection and use method thereof Download PDF

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CN112941201B
CN112941201B CN202110223240.8A CN202110223240A CN112941201B CN 112941201 B CN112941201 B CN 112941201B CN 202110223240 A CN202110223240 A CN 202110223240A CN 112941201 B CN112941201 B CN 112941201B
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臧照星
幸晓莹
徐国东
刘愈杰
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Canvest Wuhan Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of molecular genetics, and particularly provides a mixed primer for multi-cell species identification and cross contamination detection and a using method thereof. The mixed primer comprises 11 pairs of primers, each primer is designed based on the difference of standard sequences of Cytb and CO I genes of MDBK, BALB/c-3T3, MDCK, Walker-256, BHK-21, Hela, FRhK-4, Vero, CH0-K1, PG-4 and PK-15 cells, the cross pairing and interference probability among the primers is greatly reduced, the specificity and sensitivity of detection are improved, species sources of various cells can be simultaneously identified only by extracting DNA of the cells, whether cross contamination of the cells among different species exists in the culture process can be detected, and the mixed primer has the advantages of simple operation, short time, high efficiency, low price and the like.

Description

Mixed primer for multi-cell species identification and cross contamination detection and use method thereof
Technical Field
The invention belongs to the field of molecular genetics, and particularly relates to a mixed primer for multi-cell species identification and cross contamination detection and a using method thereof.
Background
With the increasing maturity of in vitro mammalian cell culture techniques and the closer approach of biological properties of their expression products to natural products of human body, mammalian cells are receiving more and more attention from researchers and are often used in pathological mechanism research, drug screening, and biological product production, and statistically, about 70% of therapeutic recombinant protein drugs are produced by mammalian cells. However, mammals have similar nutritional requirements and culture environment conditions, which are susceptible to intercellular cross-contamination during culture passages. At least 18% of the 252 cell lines identified by the researchers at the DSMZ Collection, Germany, were cross-contaminated. Of the 360 cell lines published in the international journal of cancer 2010, most of them were cross-contaminated intraspecies, and 9% of them were cross-contaminated interspecies.
The intercellular cross contamination not only affects the accuracy and repeatability of experimental results, resulting in failure of drug screening, but also is a potential threat to the production of biological products. At present, STR (short tandem repeat) spectrum analysis is a method which is commonly used for cell identification and cross contamination detection, but the method is suitable for intra-species cell identification and cross contamination detection of cells of few species of human sources and mouse sources. In addition, karyotype analysis and isozyme techniques can also be used for cell identification and cross-contamination detection, however, such detection methods have the disadvantages of large cell usage, complex operation, time consumption, high cost, and the like, and have extremely low detection efficiency. In order to improve the detection efficiency, a sensitive, rapid and stable method for multi-cell identification and species cross contamination detection is urgently needed.
Mitochondrial genes are genetic molecular units independent of genomes, have the characteristics of maternal inheritance, relative conservation of gene sequences, low mutation rate and the like, and are often used for species classification, population genetics, evolutionary biology and other aspects of analysis by molecular geneticists. The method is characterized in that a plurality of primers for amplifying different fragment lengths are designed based on the gene sequence difference of two relatively conserved genes cytochrome b (Cytb) and cytochrome C oxidase I (CO I) on eleven species cell mitochondria, a set of mixed primers with strong specificity and high sensitivity is finally determined through primer specificity screening, primer combination inspection and sensitivity inspection, and a multiple PCR detection method for multi-cell species identification and cross contamination detection is established.
Disclosure of Invention
The invention aims to solve the problems of large cell consumption, complex operation, low detection efficiency, high cost and the like in the prior detection technology.
Therefore, the invention provides a mixed primer for multi-cell species identification and cross contamination detection, which comprises the following primer pairs:
(1) primer pair I for specific amplification of Cytb gene of bovine MDBK cells:
a forward primer: 5'-AGCCCTAGTAGCAGACCTATTG-3'
Reverse primer: 5'-TTTGTTTTCGATTGTGCCGGCC-3', respectively;
(2) and (2) a primer pair II for specifically amplifying the Cytb gene of the BALB/c-3T3 cell of the mouse:
a forward primer: 5'-ACATACGAAAAACACACCCAT-3'
Reverse primer: 5'-TACTGATGAAAAGGCTGTTATT-3', respectively;
(3) primer pair iii for specific amplification of the Cytb gene of dog MDCK cells:
a forward primer: 5'-ATCCTTACTAGGAGTATGCTTG-3'
Reverse primer: 5'-CAGGTGAAAATAAAACTAGTGA-3', respectively;
(4) and (3) a primer pair IV for specifically amplifying Cytb genes of rat Walker-256 cells:
a forward primer: 5'-ATCAATCCTAATCTTAGCCTTC-3'
Reverse primer: 5'-GCTACTAGGATTCAGTAAAGGA-3', respectively;
(5) primer pair v for amplification of the Cytb gene from syrian hamster BHK-21 cells:
a forward primer: 5'-AGTTATAGCTACAGCATTCGTA-3'
Reverse primer: 5'-GTTATGAGGGCAATCAATAGTAG-3', respectively;
(6) and (3) a primer pair VI for specifically amplifying the Cytb gene of the Vero cell of the African green monkey:
a forward primer: 5'-TGAATCTGAGGTGGGTACTCCATTGG-3'
Reverse primer: 5'-GGTTGTAATTAGGAATCAGAACAGG-3', respectively;
(7) the primer pair VII for specifically amplifying the CO I gene of Chinese hamster CH0-K1 cells:
a forward primer: 5'-TTGGGAATTGATTAGTGCCTT-3'
Reverse primer: 5'-TGGTGTTTGGTATTGTGTAACA-3', respectively;
(8) and a primer pair VIII for specifically amplifying the CO I gene of the feline PG-4 cell:
a forward primer: 5'-TCTCAGGATATACCCTTGACAAC-3'
Reverse primer: 5'-GATGCGAAAGCTTCTCACACT-3', respectively;
(9) a primer pair IX:
a forward primer: 5'-ACGGGATCAAACAACCCCCTA-3'
Reverse primer: 5'-TCCGATTCAGGTTAGAATGAGG-3', respectively;
(10) a primer pair X for specifically amplifying the CO I gene of porcine PK-15 cells:
a forward primer: 5'-ACCGTAGGAATAGACGTAGATACC-3'
Reverse primer: 5'-ATGCTGTGTATGCGTCAGGATAA-3', respectively;
(11) a primer pair XI for specific amplification of the Cytb gene of the rhesus monkey FRhK-4 cells:
a forward primer: 5'-CCCTTACCTGAATTGGAAGCG-3'
Reverse primer: 5'-TTGTTTTCGATTAGGGAGGCC-3' are provided.
Specifically, in the mixed primer, the primer pair i, the primer pair ii, the primer pair iii, the primer pair iv, the primer pair v, the primer pair vi, the primer pair vii, the primer pair viii, the primer pair ix, the primer pair x, and the primer pair ix are mixed in equal proportion.
The invention also provides a method for multi-cell species identification and cross contamination detection, which comprises the following steps:
(1) preparing a cell sample to be detected;
(2) extracting the genome DNA of the cell to be detected;
(3) taking the genome DNA of a cell to be detected as a template, and carrying out PCR amplification by using the mixed primer for multi-cell species identification and cross contamination detection;
(4) and after the PCR reaction is finished, carrying out agarose gel electrophoresis detection on the PCR product, and judging the source cells of the sample according to the size of the DNA fragment of the amplified product.
Specifically, the reaction system for PCR amplification in step (3) is: 2.5. mu.l of 10xPCR buffer, 0.5. mu.l of DNA polymerase, 1. mu.l of DNA template, 0.5. mu.l of dNTP Mix, 13. mu.l of mixed primer, and 7.5. mu.l of deionized water.
Specifically, the reaction procedure of the PCR amplification in the step (3) is as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 1min, and performing 35 cycles; completely extending for 5min at 72 ℃; storing at 16 ℃.
Specifically, the determination criterion in the step (4) is as follows:
the size of a DNA fragment amplified by the primer pair of the bovine MDBK cell is 148 bp;
the size of a DNA fragment amplified by the primer pair of the mouse BALB/c-3T3 cell is 191 bp;
the size of a DNA fragment amplified by a primer pair of dog MDCK cells is 641 bp;
the size of a DNA fragment amplified by a primer pair of rat Walker-256 cells is 102 bp;
the size of a DNA fragment amplified by a primer pair of Syrian hamster BHK-21 cells is 357 bp;
the size of a DNA fragment amplified by the primer pair of the African green monkey Vero cell is 507 bp;
the size of a DNA fragment amplified by the primer pair of the Chinese hamster CH0-K1 cell is 314 bp;
the size of a DNA fragment amplified by the primer pair of the cat PG-4 cell is 235 bp;
the size of a DNA fragment amplified by the primer pair of the human Hela cell is 411 bp;
the size of a DNA fragment amplified by the primer pair of the porcine PK-15 cell is 469 bp;
the size of a DNA fragment amplified by the primer pair of the rhesus monkey FRhK-4 cell is 124 bp;
and comparing the DNA fragment in the agarose gel electrophoresis detection result with the fragment to determine the corresponding cell.
Specifically, the step (1) includes the following steps: if the cells to be detected are suspension cells, transferring the cells into a 15ml centrifuge tube, centrifuging for 5min by 450g, discarding the culture solution, adding PBS (phosphate buffer solution) for heavy suspension, transferring into a 1.5ml centrifuge tube, centrifuging for 5min by 450g, discarding the supernatant, and collecting the cells; and if the cells to be detected are adherent cells, adding 1ml of pancreatin, digesting until the cells fall off, adding 2ml of 10% FBS DMEM culture medium, mixing uniformly, and treating according to the suspension cells.
Specifically, the step (2) includes the following steps: collecting cells to be tested into a 1.5ml EP tube, adding 500 mul nuclear lysine solution (Promega) into the EP tube, carrying out water bath at 65 ℃ for 30min, adding 10 mul 20mg/ml proteinase K and 5 mul RNase solution, carrying out water bath at 55 ℃ until the solution becomes clear, taking out the solution, cooling the solution to room temperature, adding 200 mul protein precipitation solution (Promega), quickly reversing and uniformly mixing the solution, standing the solution on ice for 10min, centrifuging the solution at 14000rpm for 4 min, transferring the supernatant into a new EP tube, adding isopropanol with the same volume, slightly reversing the solution to generate white precipitates, centrifuging the solution at 14000rpm for 1min, discarding the supernatant, adding 600 mul 80% ethanol, slightly reversing and washing DNA, repeatedly washing the DNA, standing the solution at room temperature for 5min to volatilize the ethanol, adding 50 mul sterile water or TE buffer solution, and standing the temperature at 4 ℃.
The invention also provides a kit for multi-cell species identification and cross contamination detection, which comprises the mixed primer for multi-cell species identification and cross contamination detection. The mixed primer and related reagents of the invention can be assembled into a kit for convenient use, wherein the related reagents can be reagents in a PCR reaction system except for a DNA template in the method for identifying the multicellular species and detecting the cross contamination, and can also be other conventional reagents suitable for PCR reaction.
The kit provided by the invention can be used for species identification and cross-contamination detection of multi-species cell mixed samples, and can also be used for species identification and cross-contamination detection of single-species cells.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the method provided by the invention can be used for detecting only by extracting the cell DNA, and has the advantages of simple operation, short time, high efficiency, low price and the like.
(2) The mixed primer provided by the invention can realize the amplification of multiple fragments in one PCR reaction system, the sample consumption is less than that of the traditional PCR technology, and species identification and species cross contamination detection can be simultaneously carried out on multiple cells.
(3) The method provided by the invention not only carries out species identification and species cross contamination detection on the currently common mammalian cells, but also adds primers of BHK-21 cells of Syrian hamsters commonly used by biological products and scientific research, thereby realizing the detection of more cell species.
(4) The mixed primer provided by the invention is designed based on the difference of Cytb and CO I gene standard sequences, thereby greatly reducing the cross pairing and interference probability between the primers and improving the specificity and sensitivity of detection.
The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is the specificity verification result of the mixed primers for multi-cell species identification and cross-contamination detection of the present invention; m is DNA marker DL 2000; 1-11 are MDCK, Vero, PK-15, Hela, BHK-21, CHO-K1, PG-4, BALB/c-3T3, MDBK, FRhK-4 and Walker-256 cell primers respectively.
FIG. 2 is the results of cross-pairing and interference rejection experiments between mixed primers for multi-cell species identification and cross-contamination detection of the present invention; m is DNA marker DL 2000; 1-11 are MDCK, Vero, PK-15, Hela, BHK-21, CHO-K1, PG-4, BALB/c-3T3, MDBK, FRhK-4 and Walker-256 cell genome DNA templates respectively.
FIG. 3 is the results of the sensitivity and specificity detection of the method of the invention for multi-cell species identification and cross-contamination detection; m is DNA marker DL 2000; 1: MDCK, Vero, PK-15, Hela, BHK-21, CHO-K1, PG-4, BALB/c-3T3, MDBK, FRhK-4 and Walker-256 cell genome DNA mixed template; 2 and 3 are 2 and 4 dilutions, respectively, of the mixed template of 1.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made thereto without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
Unless otherwise specified, the reagents and equipment used in the present invention are commercially available and the reagents used are formulated according to the molecular cloning protocols. The primers used in the examples of the present invention were synthesized by Biotechnology Ltd and purified by PAGE purification, and the DNA Polymerase used was Champagne Taq DNA Polymerase (P122-d2, Novozan Biotechnology Ltd.).
Mixed primer designed for multi-cell identification and detection of cross contamination between species
Screening and analyzing sequences of MDBK, BALB/c-3T3, MDCK, Walker-256, BHK-21, Vero, CH0-K1, PG-4, Hela, PK-15 and FRhK-4 cells by using a nucleic acid sequence database of the National Center for Biotechnology Information (NCBI); selecting highly conserved and specific sequences (conserved regions) in various Cytb and CO I genes of cells, and comparing and searching the specific sequences to determine the specificity of the sequences in all species and avoid the repetition phenomenon of the sequences; in order to reduce cross pairing and interference probability among primers and improve the specificity and sensitivity of detection, the invention selects primers designed aiming at Cytb gene and CO I gene at the same time; analyzing and designing by using Primer design software Primer, finely adjusting Primer sequences to ensure that the primers are not excessively paired and have no dimer, ensuring that Tm values of all the primers are approximately the same, and designing a plurality of specific amplification primers aiming at each cell Cytb or CO I gene; the designed primer pair is used for amplifying mixed templates containing eleven kinds of cell genome DNA, the specificity of the primer is determined, namely only the genome DNA of the corresponding cell is amplified, other genome DNA is not amplified, then a pair of primers with good specific amplification effect is selected for each cell, and the single genome DNA templates of the eleven kinds of cells are amplified after being mixed in equal proportion, so that the cross pairing and the interference among the primers are eliminated.
The sequences of the primers are as follows:
(1) primer pair I for specific amplification of Cytb gene of bovine MDBK cells:
a forward primer: 5'-AGCCCTAGTAGCAGACCTATTG-3'
Reverse primer: 5'-TTTGTTTTCGATTGTGCCGGCC-3', respectively;
(2) and (2) a primer pair II for specifically amplifying the Cytb gene of the BALB/c-3T3 cell of the mouse:
a forward primer: 5'-ACATACGAAAAACACACCCAT-3'
Reverse primer: 5'-TACTGATGAAAAGGCTGTTATT-3', respectively;
(3) primer pair iii for specific amplification of the Cytb gene of dog MDCK cells:
a forward primer: 5'-ATCCTTACTAGGAGTATGCTTG-3'
Reverse primer: 5'-CAGGTGAAAATAAAACTAGTGA-3', respectively;
(4) and (3) a primer pair IV for specifically amplifying Cytb genes of rat Walker-256 cells:
a forward primer: 5'-ATCAATCCTAATCTTAGCCTTC-3'
Reverse primer: 5'-GCTACTAGGATTCAGTAAAGGA-3', respectively;
(5) primer pair v for amplification of the Cytb gene from syrian hamster BHK-21 cells:
a forward primer: 5'-AGTTATAGCTACAGCATTCGTA-3'
Reverse primer: 5'-GTTATGAGGGCAATCAATAGTAG-3', respectively;
(6) and (3) a primer pair VI for specifically amplifying the Cytb gene of the Vero cell of the African green monkey:
a forward primer: 5'-TGAATCTGAGGTGGGTACTCCATTGG-3'
Reverse primer: 5'-GGTTGTAATTAGGAATCAGAACAGG-3', respectively;
(7) the primer pair VII for specifically amplifying the CO I gene of Chinese hamster CH0-K1 cells:
a forward primer: 5'-TTGGGAATTGATTAGTGCCTT-3'
Reverse primer: 5'-TGGTGTTTGGTATTGTGTAACA-3', respectively;
(8) and a primer pair VIII for specifically amplifying the CO I gene of the feline PG-4 cell:
a forward primer: 5'-TCTCAGGATATACCCTTGACAAC-3'
Reverse primer: 5'-GATGCGAAAGCTTCTCACACT-3', respectively;
(9) a primer pair IX:
a forward primer: 5'-ACGGGATCAAACAACCCCCTA-3'
Reverse primer: 5'-TCCGATTCAGGTTAGAATGAGG-3', respectively;
(10) a primer pair X for specifically amplifying the CO I gene of porcine PK-15 cells:
a forward primer: 5'-ACCGTAGGAATAGACGTAGATACC-3'
Reverse primer: 5'-ATGCTGTGTATGCGTCAGGATAA-3', respectively;
(11) a primer pair XI for specific amplification of the Cytb gene of the rhesus monkey FRhK-4 cells:
a forward primer: 5'-CCCTTACCTGAATTGGAAGCG-3'
Reverse primer: 5'-TTGTTTTCGATTAGGGAGGCC-3'
The genomic DNA of the corresponding source cell was amplified by PCR using the primers, and the sizes of the amplified DNA fragments are shown in Table 1.
TABLE 1 information on the primer pairs
Figure BDA0002955628680000081
Figure BDA0002955628680000091
Method for identifying multi-cell species and detecting cross contamination
1. Preparing a cell sample to be tested: if the cells to be detected are suspension cells, transferring the cells into a 15ml centrifuge tube, centrifuging for 5min by 450g, discarding the culture solution, adding PBS (phosphate buffer solution) for heavy suspension, transferring into a 1.5ml centrifuge tube, centrifuging for 5min by 450g, discarding the supernatant, and collecting the cells; and if the cells to be detected are adherent cells, adding 1ml of pancreatin into the cells, digesting until the cells are detached, adding 2ml of 10% FBS DMEM culture medium, uniformly mixing the cells, transferring the cells into a 15ml centrifuge tube, centrifuging 450g for 5min, discarding the culture solution, adding PBS (phosphate buffer solution), re-suspending, transferring the cells into a 1.5ml centrifuge tube, centrifuging 450g for 5min, discarding the supernatant, and collecting the cells.
2. Extracting the genome DNA of the cell to be detected: collecting cells to be detected into a 1.5ml EP tube, adding 500 mul nuclear Lysis solution (Promega) into the EP tube, carrying out water bath at 65 ℃ for 30min, adding 10 mul 20mg/ml proteinase K and 5 mul RNase solution, carrying out water bath at 55 ℃ until the solution becomes clear, taking out the solution, cooling the solution to room temperature, adding 200 mul protein precipitation solution (Promega), quickly reversing and mixing the solution uniformly, standing the mixture on ice for 10min, centrifuging the mixture at 14000rpm for 4 min, transferring the supernatant into a new EP tube, adding isopropanol with the same volume, slightly reversing the mixture up and down to generate white precipitates, centrifuging the mixture at 14000rpm for 1min, discarding the supernatant, adding 600 mul 80% ethanol, slightly reversing the DNA up and down to wash the DNA, standing the mixture at room temperature for 5min to volatilize the ethanol after repeating the washing once, adding 50 mul sterilized water or TE buffer solution, keeping the temperature at 4 ℃ overnight, and fully dissolving the DNA.
3. Taking the genome DNA of a cell to be detected as a template, and carrying out PCR amplification by using the mixed primer for multi-cell identification and cross contamination detection with species; the reaction system of PCR amplification is as follows: 2.5 mul of 10xPCR buffer solution, 0.5 mul of DNA polymerase, 1 mul of DNA template, 0.5 mul of dNTP Mix, 13 mul of mixed primer and 7.5 mul of deionized water; the reaction procedure for PCR amplification was: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 1min, and performing 35 cycles; completely extending for 5min at 72 ℃; storing at 16 ℃.
4. After the PCR reaction is finished, agarose gel electrophoresis detection is carried out on the PCR amplification product, and the size of the DNA fragment of the amplification product is compared with the size of each amplification fragment in the table 1 to judge the cell species to which the sample belongs.
The effects of the present invention will be examined below with reference to specific examples.
Example 1:
this example demonstrates specificity in order to determine that the designed primers only amplify the genomic DNA of their corresponding cells, but not the genomic DNA of other cells.
(1) Preparing single cell samples of MDBK, BALB/c-3T3, MDCK, Walker-256, BHK-21, Vero, CH0-K1, PG-4, Hela, PK-15 and FRhK-4 cells;
(2) extracting genomic DNA of each single cell, which is 1 ng/. mu.l, and mixing them to prepare mixed genomic DNA;
(3) using mixed genomic DNA as a template, and respectively carrying out PCR amplification on mixed genomic DNA of eleven cells by using a single primer pair I-XI;
specific PCR reaction systems are shown in Table 2; the reaction procedure for PCR amplification was: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 1min, and performing 35 cycles; completely extending for 5min at 72 ℃; storing at 16 deg.C;
TABLE 2 PCR reaction System
10x Champagne TaqTM Buffe 2.5μl
Forward primer (10. mu.M) 0.5μl
Reverse primer (10. mu.M) 0.5μl
dNTP Mix 0.5μl
Champagne TaqTM DNA Polymerase 0.5μl
Mixed genomic DNA 1μl
Deionized water 19.5μl
Total amount of 25μl
(4) After the PCR reaction is finished, carrying out agarose gel electrophoresis detection on the PCR amplification product, wherein a marker is DNA marker DL2000, and the result is shown in figure 1, and each single primer amplification product only has a single strip; comparing the amplified fragment sizes in FIG. 1 with those in Table 1, the primers designed only amplify the genomic DNA of the corresponding cells, but not the genomic DNA of other cells, so that the primers designed by the present invention have good specificity.
Example 2:
in order to verify that cross-pairing and interference do not exist between primers in designed primers and that detection can be performed normally, cross-pairing and interference elimination experiments between primers are performed in this example, and the primers used in this round are purified by PAGE and diluted to 10 μ M.
(1) Preparing single cell samples of MDBK, BALB/c-3T3, MDCK, Walker-256, BHK-21, Vero, CH0-K1, PG-4, Hela, PK-15 and FRhK-4 cells;
(2) extracting the genome DNA of each single cell, wherein the concentration of each genome DNA is 50 ng/mu l;
(3) diluting the primers purified by PAGE into 10 mu M, taking single cell genomic DNA as a template, mixing the primer pair I-primer pair XI in equal proportion, and amplifying eleven single cell genomic DNA templates respectively;
specific PCR reaction systems are shown in Table 3; the reaction procedure for PCR amplification was the same as in example 1;
TABLE 3 PCR reaction System
10x Champagne TaqTM Buffe 2.5μl
Mixed primer (10. mu.M) 13μl
dNTP Mix 0.5μl
Champagne TaqTM DNA Polymerase 0.5μl
Genomic DNA 1μl
Deionized water 7.5μl
Total amount of 25μl
(4) After the PCR reaction is finished, carrying out agarose gel electrophoresis detection on the PCR amplification product, wherein the marker is DNA marker DL2000, and the result is shown in figure 2, and each single cell sample amplification product only has a single strip; comparing the amplified fragment size in FIG. 2 with that in Table 1, the designed primers only amplify the genomic DNA of the corresponding cells, and cross pairing and interference do not exist among the primers, so that the detection can be normally carried out.
Example 3:
to demonstrate the sensitivity and specificity of the present invention, this example was performed on mixed genomic DNA templates of different concentrations.
(1) Preparing single cell samples of MDBK, BALB/c-3T3, MDCK, Walker-256, BHK-21, Vero, CH0-K1, PG-4, Hela, PK-15 and FRhK-4 cells;
(2) extracting genomic DNA of each single cell, mixing them to prepare mixed genomic DNA, wherein the concentration of MDBK cell genome DNA is 1 ng/mul, the concentration of BALB/c-3T3 cell genome DNA is 0.5 ng/mul, the concentration of MDCK cell genome DNA is 1 ng/mul, the concentration of Walker-256 cell genome DNA is 0.5 ng/mul, the concentration of BHK-21 cell genome DNA is 1 ng/mul, the concentration of Vero cell genome DNA is 0.5 ng/mul, the concentration of CH0-K1 cell genome DNA is 0.5 ng/mul, the concentration of PG-4 cell genome DNA is 0.5 ng/mul, the concentration of Hela cell genome DNA is 0.5 ng/mul, the concentration of PK-15 cell genome DNA is 0.25 ng/mul, and the concentration of FRhK-4 cell genome DNA is 0.5 ng/mul;
(3) taking mixed genomic DNA as a template, and amplifying the mixed genomic DNA after mixing a primer pair I-a primer pair XI in equal proportion; the PCR reaction system and PCR reaction conditions used in this example were the same as those in example 2;
(4) after diluting the mixed genomic DNA 2 times and 4 times respectively, repeating the step (3);
(5) performing agarose gel electrophoresis detection on the PCR amplification products in the step (3) and the step (4), wherein a marker is DNA marker DL2000, and the result is shown in FIG. 3, wherein eleven-entry bands can be seen in the amplification products of mixed genome DNA with different concentrations; FIG. 3 is a control of the amplified fragment sizes in Table 1, and each primer amplified genomic DNA from its corresponding cell.
When the mixed primer provided by the invention is used for amplifying genomic DNA containing eleven cells, 0.25ng of MDCK and Vero cell genomic DNA, 0.0625ng of PK-15 cell genomic DNA, 0.125ng of Hela, BHK-21, CHO-K1, PG-4, BALB/c-3T3, MDBK, FRhK-4 and Walker-256 cell genomic DNA can be sensitively detected.
In conclusion, the mixed primers designed based on the difference of Cytb and CO I gene standard sequences greatly reduce the cross pairing and interference probability between the primers, improve the specificity and sensitivity of detection, can simultaneously perform species identification and species cross contamination detection on various cells by only extracting cell DNA, and have the advantages of simple operation, short time, high efficiency, low price and the like.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.

Claims (10)

1. A mixed primer for multi-cell species identification and cross contamination detection is characterized by comprising the following primer pairs:
(1) primer pair I for specific amplification of Cytb gene of bovine MDBK cells:
a forward primer: 5'-AGCCCTAGTAGCAGACCTATTG-3'
Reverse primer: 5'-TTTGTTTTCGATTGTGCCGGCC-3', respectively;
(2) and (2) a primer pair II for specifically amplifying the Cytb gene of the BALB/c-3T3 cell of the mouse:
a forward primer: 5'-ACATACGAAAAACACACCCAT-3'
Reverse primer: 5'-TACTGATGAAAAGGCTGTTATT-3', respectively;
(3) primer pair iii for specific amplification of the Cytb gene of dog MDCK cells:
a forward primer: 5'-ATCCTTACTAGGAGTATGCTTG-3'
Reverse primer: 5'-CAGGTGAAAATAAAACTAGTGA-3', respectively;
(4) and (3) a primer pair IV for specifically amplifying Cytb genes of rat Walker-256 cells:
a forward primer: 5'-ATCAATCCTAATCTTAGCCTTC-3'
Reverse primer: 5'-GCTACTAGGATTCAGTAAAGGA-3', respectively;
(5) primer pair v for amplification of the Cytb gene from syrian hamster BHK-21 cells:
a forward primer: 5'-AGTTATAGCTACAGCATTCGTA-3'
Reverse primer: 5'-GTTATGAGGGCAATCAATAGTAG-3', respectively;
(6) and (3) a primer pair VI for specifically amplifying the Cytb gene of the Vero cell of the African green monkey:
a forward primer: 5'-TGAATCTGAGGTGGGTACTCCATTGG-3'
Reverse primer: 5'-GGTTGTAATTAGGAATCAGAACAGG-3', respectively;
(7) the primer pair VII for specifically amplifying the CO I gene of Chinese hamster CH0-K1 cells:
a forward primer: 5'-TTGGGAATTGATTAGTGCCTT-3'
Reverse primer: 5'-TGGTGTTTGGTATTGTGTAACA-3', respectively;
(8) and a primer pair VIII for specifically amplifying the COI gene of the feline PG-4 cell:
a forward primer: 5'-TCTCAGGATATACCCTTGACAAC-3'
Reverse primer: 5'-GATGCGAAAGCTTCTCACACT-3', respectively;
(9) a primer pair IX:
a forward primer: 5'-ACGGGATCAAACAACCCCCTA-3'
Reverse primer: 5'-TCCGATTCAGGTTAGAATGAGG-3', respectively;
(10) primer pair X for specifically amplifying COI gene of porcine PK-15 cells:
a forward primer: 5'-ACCGTAGGAATAGACGTAGATACC-3'
Reverse primer: 5'-ATGCTGTGTATGCGTCAGGATAA-3', respectively;
(11) a primer pair XI for specific amplification of the Cytb gene of the rhesus monkey FRhK-4 cells:
a forward primer: 5'-CCCTTACCTGAATTGGAAGCG-3'
Reverse primer: 5'-TTGTTTTCGATTAGGGAGGCC-3' are provided.
2. The mixed primer for multi-cell species identification and cross-contamination detection as claimed in claim 1, wherein the primer pair i, the primer pair ii, the primer pair iii, the primer pair iv, the primer pair v, the primer pair vi, the primer pair vii, the primer pair viii, the primer pair ix, the primer pair x and the primer pair xi are mixed in equal proportion in the mixed primer.
3. A method of multi-cell species identification and cross-contamination detection comprising the steps of:
(1) preparing a cell sample to be detected;
(2) extracting the genome DNA of the cell to be detected;
(3) performing PCR amplification by using the mixed primer of claim 1 with the genomic DNA of a cell to be tested as a template;
(4) and after the PCR reaction is finished, carrying out agarose gel electrophoresis detection on the PCR amplification product, and judging the source cells of the sample according to the size of the DNA fragment of the amplification product.
4. The method for multi-cell species identification and cross-contamination detection of claim 3, wherein the reaction system of PCR amplification in step (3) is: 2.5 μ L of 10xPCR buffer solution, 0.5 μ L of DNA polymerase, 1 μ L of DNA template, 0.5 μ L of dNTP Mix, 13 μ L of mixed primer and 7.5 μ L of deionized water.
5. The method for multi-cell species identification and cross-contamination detection of claim 3, wherein the reaction procedure of PCR amplification in step (3) is: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 1min, and performing 35 cycles; completely extending for 5min at 72 ℃; storing at 16 ℃.
6. The method of multi-cell species identification and cross-contamination detection of claim 3, wherein the determination in step (4) is based on:
the size of a DNA fragment amplified by the primer pair of the bovine MDBK cell is 148 bp;
the size of a DNA fragment amplified by the primer pair of the mouse BALB/c-3T3 cell is 191 bp;
the size of a DNA fragment amplified by a primer pair of dog MDCK cells is 641 bp;
the size of a DNA fragment amplified by a primer pair of rat Walker-256 cells is 102 bp;
the size of a DNA fragment amplified by a primer pair of Syrian hamster BHK-21 cells is 357 bp;
the size of a DNA fragment amplified by the primer pair of the African green monkey Vero cell is 507 bp;
the size of a DNA fragment amplified by the primer pair of the Chinese hamster CH0-K1 cell is 314 bp;
the size of a DNA fragment amplified by the primer pair of the cat PG-4 cell is 235 bp;
the size of a DNA fragment amplified by the primer pair of the human Hela cell is 411 bp;
the size of a DNA fragment amplified by the primer pair of the porcine PK-15 cell is 469 bp;
the size of a DNA fragment amplified by the primer pair of the rhesus monkey FRhK-4 cell is 124 bp;
and comparing the DNA fragment in the agarose gel electrophoresis detection result with the fragment to determine the corresponding cell.
7. The method for multi-cell species identification and cross-contamination detection of claim 3, wherein the specific steps of step (1) are: if the cells to be detected are suspension cells, transferring the cells into a 15mL centrifuge tube, centrifuging for 5min by 450g, discarding the culture solution, adding PBS (phosphate buffer solution) for heavy suspension, transferring into a 1.5mL centrifuge tube, centrifuging for 5min by 450g, discarding the supernatant, and collecting the cells; and if the cells to be detected are adherent cells, adding 1mL of pancreatin, digesting until the cells fall off, adding 2mL of 10% FBSDMEM culture medium, mixing uniformly, and treating according to the suspension cells.
8. The method for multi-cell species identification and cross-contamination detection of claim 3, wherein the specific steps of step (2) are: collecting cells to be detected into a 1.5mL EP tube, adding 500 μ L of nuclear Lysis solution, carrying out water bath at 65 ℃ for 30min, adding 10 μ L of 20mg/mL proteinase K and 5 μ L of RNase solution, carrying out water bath at 55 ℃ until the solution becomes clear, taking out the solution, cooling to room temperature, adding 200 μ L of protein precipitation solution, quickly inverting and uniformly mixing, standing on ice for 10min, centrifuging at 14000rpm for 4 min, transferring the supernatant into a new EP tube, adding equal volume of isopropanol, slightly inverting up and down to generate white precipitates, centrifuging at 14000rpm for 1min, discarding the supernatant, adding 600 μ L of 80% ethanol, slightly inverting up and down to wash DNA, repeatedly washing once, standing at room temperature for 5min to volatilize the ethanol, adding 50 μ L of sterilized water or TE buffer, and standing at 4 ℃ overnight.
9. A kit for multi-cell species identification and cross-contamination detection comprising the mixed primer for multi-cell species identification and cross-contamination detection of claim 1.
10. Use of the kit for multicellular species identification and cross-contamination detection as claimed in claim 9 in multicellular species identification and cross-contamination detection.
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