A primer combination for cell species identification and cross-contamination detection and its usage method

Abstract

This application relates to the field of molecular genetics, specifically to a primer set for cell species identification and cross-contamination detection, and its method of use. The primer set includes primer pairs Pig-COX I, Human-COX I, Cat-COX I, Sheep-COX I, Horse-COX I, Monkey-COX I, Rat-COX I, Mouse-COX I, Goat-COX I, and Cow-COX I. This primer set exhibits high specificity; in a single PCR system, using nucleic acids from a sample of test cells containing 10 different cell species as templates, it can amplify 10 corresponding target bands. Furthermore, it demonstrates high sensitivity, is simple and efficient to apply, and therefore possesses good scalability. It can be used as a cell quality control method for the production of cell matrix and therapeutic cell products.

Description

Technical Field

[0001] This application relates to the field of molecular genetics, specifically to a primer combination for cell species identification and cross-contamination detection, and a method for using it. Background Technology

[0002] In recent years, my country's therapeutic cell products have developed rapidly in both basic research and clinical application. Species identification and cross-contamination detection of cell lines are crucial for cell quality control. According to the Chinese Pharmacopoeia, the main methods for cell species identification and cross-contamination analysis include chromosome analysis, STR mapping, and isoenzyme mapping. However, these methods suffer from drawbacks such as being time-consuming, costly, requiring large cell volumes, and having a narrow range of detectable species.

[0003] Therefore, there is an urgent need to develop a method that is highly effective, specific, and can be rapidly and flexibly applied to cell species identification and cross-contamination detection. Summary of the Invention

[0004] This application addresses at least one of the problems of the related technology in the following aspects.

[0005] Therefore, a first aspect of this application provides a primer combination for cell species identification and cross-contamination detection, wherein the primer combination comprises the following primer pairs:

[0006] (1) Primer pair Pig-COX I for specific amplification of the porcine COX I gene:

[0007] Forward primer: 5'-GTCTGATCAGTACTAATCACAGCCGT-3'

[0008] Reverse primer: 5'-GAGCTCATAGTATTGCGGGTGATCAT-3';

[0009] (2) Primer pair for specific amplification of the human COX I gene: Human-COX I:

[0010] Forward primer: 5'-TAGACATCGTACTACACGACACG-3'

[0011] Reverse primer: 5'-TCCAGGTTTATGGAGGGTTC-3';

[0012] (3) Primer pair for specific amplification of the feline COX I gene: Cat-COX I:

[0013] Forward primer: 5'-TATTGCCATTCCTACCGGGGTG-3'

[0014] Reverse primer: 5'-ACGTTATATTGACTCCTACAAACATAATC-3';

[0015] (4) Primer pair for specific amplification of the sheep COX I gene: Sheep-COX I:

[0016] Forward primer: 5'-CTATAATTATCGCCATCCCAACAGGAG-3'

[0017] Reverse primer: 5'-AGGGGAAATCAATGTACAAATCCTCCTA-3';

[0018] (5) Primer pair Horse-COX I for specific amplification of the equine COX I gene:

[0019] Forward primer: 5'-TGGGACCCTACTAGGAGATGATCAGA-3'

[0020] Reverse primer: 5'-CCTGTTCCGGCACCTGCTTCA-3';

[0021] (6) Primer pair for specific amplification of the green monkey COX I gene: Monkey-COX I:

[0022] Forward primer: 5'-CCTCCTTCCTGCTGCTAATGGC-3'

[0023] Reverse primer: 5'-TTTGATACTGGGATATGGCG-3';

[0024] (7) Primer pair Rat-COX I for specific amplification of the rat COX I gene:

[0025] Forward primer: 5'-GTACATCTTAATTCTTCCAGGGTTTGGA-3'

[0026] Reverse primer: 5'-CGGGTGTCTACATCTAGGCCTACTG-3';

[0027] (8) Primer sequence for specific amplification of the mouse COX I gene: Mouse-COX I:

[0028] Forward primer: 5'-AACAGACCGCAACCTAAACACAACT-3'

[0029] Reverse primer: 5'-ATGTGAAATAATTCCAAATCCTGGGAGG-3';

[0030] (9) Primer pair Goat-COX I for specific amplification of the goat COX I gene:

[0031] Forward primer: 5'-ATATCAATCGGGTTTCTAGGATTTATT-3'

[0032] Reverse primer: 5'-AGTTGGGATAGCGATAATTATGGTAGC-3'; and

[0033] (10) Primer sequence for specific amplification of the bovine COX I gene: Cow-COX I:

[0034] Forward primer: 5'-GCTATTCCAACCGGGGTAAAAGTC-3'

[0035] Reverse primer: 5'-GAAAATAAAGCCTAGGGCTCAC-3'.

[0036] A second aspect of this application provides a reagent for cell species identification and cross-contamination detection comprising the primer combination described in the first aspect of the application.

[0037] In some embodiments, the total molar ratio of each primer pair in the reagent is the same.

[0038] An embodiment of the third aspect of this application provides a kit for cell species identification and cross-contamination detection, wherein the kit comprises the primer combination described in the first aspect embodiment and the reagent described in the second aspect embodiment.

[0039] In some embodiments, the kit further includes auxiliary reagents for PCR amplification.

[0040] In some embodiments, the auxiliary reagents include DNA polymerase and / or positive control.

[0041] The embodiments of the fourth aspect of this application provide the application of the primer combination described in the first aspect embodiment, the reagent described in the second aspect embodiment, and the kit described in the third aspect embodiment in cell species identification and cross-contamination, wherein the cell species is selected from one or more of pigs, humans, cats, sheep, horses, green monkeys, rats, mice, goats, and cattle.

[0042] In some embodiments, the cell species identification is to identify whether the species of the cell sample to be tested is pig, human, cat, sheep, horse, green monkey, rat, mouse, goat or cow.

[0043] In some embodiments, the cross-contamination detection is to detect whether the cell sample to be tested contains cells of one or more species, namely pig, human, cat, sheep, horse, green monkey, rat, mouse, goat or cow.

[0044] The fifth aspect of this application provides a method for cell species identification and cross-contamination detection, comprising:

[0045] Step S1. Perform PCR amplification of the nucleic acid in the cell sample to be tested using the primer combination described in claim 1; and

[0046] Step S2. Characterize the DNA fragments in the PCR amplification product, and determine whether the cell sample to be tested contains cells of one or more species, namely pig, human, cat, sheep, horse, green monkey, rat, mouse, goat or cow, based on the characterization results.

[0047] In some embodiments, the nucleic acid is total DNA.

[0048] In some embodiments, determining whether the cell sample to be tested contains cells of one or more species of pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, or cow based on the characterization results includes:

[0049] When the PCR amplification product does not contain a 472bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species pig; when the PCR amplification product contains a 472bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species pig.

[0050] When the PCR amplification product does not contain a 425bp DNA fragment, it is determined that the cell sample to be tested does not contain human cells; when the PCR amplification product contains a 425bp DNA fragment, it is determined that the cell sample to be tested contains human cells.

[0051] When the PCR amplification product does not contain a 341bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species cat; when the PCR amplification product contains a 341bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species cat.

[0052] When the PCR amplification product does not contain a 271bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species sheep; when the PCR amplification product contains a 271bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species sheep.

[0053] When the PCR amplification product does not contain a 243bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species horse; when the PCR amplification product contains a 243bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species horse.

[0054] When the PCR amplification product does not contain a 222bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species green monkey; when the PCR amplification product contains a 222bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species green monkey.

[0055] When the PCR amplification product does not contain a 177bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species rat; when the PCR amplification product contains a 177bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species rat.

[0056] When the PCR amplification product does not contain a 139bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species mouse; when the PCR amplification product contains a 139bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species mouse.

[0057] When the PCR amplification product does not contain a 117bp DNA fragment, the cell sample to be tested is determined not to contain cells of the species goat; when the PCR amplification product contains a 117bp DNA fragment, the cell sample to be tested is determined to contain cells of the species goat.

[0058] When the PCR amplification product does not contain a 102bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the bovine species; when the PCR amplification product contains a 102bp DNA fragment, it is determined that the cell sample to be tested contains cells of the bovine species.

[0059] In some embodiments, the characterization is achieved by performing agarose gel electrophoresis on the amplification products.

[0060] In some embodiments, the total molar ratio of each primer pair contained in the PCR amplification system is the same.

[0061] In some embodiments, the PCR amplification system contains a final concentration of 10... -3 Up to 50 ng / μL of template.

[0062] In some embodiments, the PCR amplification system contains a template at a final concentration of 0.1 to 10 ng / μL.

[0063] In some embodiments, the PCR amplification system comprises a primer combination with a final concentration of 5 to 50 μM.

[0064] In some embodiments, the PCR amplification system comprises a primer combination with a final concentration of 10 to 30 μM.

[0065] In some embodiments, the PCR amplification procedure includes:

[0066] (i) React at 97 to 99°C for 2 to 4 minutes;

[0067] (ii) Cyclic reactions; and

[0068] (iii) Full extension at 70 to 74°C for 8 to 12 minutes.

[0069] The cyclic reaction includes a reaction at 97 to 99°C for 8 to 12 seconds; a reaction at 59 to 63°C for 28 to 32 seconds; and a reaction at 68 to 74°C for 28 to 32 seconds, wherein the cyclic reaction is repeated 32 to 38 times.

[0070] In some embodiments, the PCR amplification procedure includes:

[0071] (i) React at 98℃ for 3 min;

[0072] (ii) Cyclic reactions; and

[0073] (iii) Extend completely at 72°C for 10 minutes.

[0074] The cyclic reaction includes a reaction at 98°C for 10 seconds; a reaction at 61°C for 30 seconds; and a reaction at 72°C for 30 seconds, wherein the cyclic reaction is repeated 35 times.

[0075] The embodiments of this application achieve the following beneficial effects:

[0076] The primer combinations provided in this application are highly specific and can accurately identify cell species from different sources; they are highly sensitive, with a detection limit of 0.1%; they have a wide detection range, covering ten common mammalian cell types; and they are simple and efficient to use, simultaneously and accurately determining whether the sample contains cells from ten different sources: pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, and cow. Therefore, they have good scalability and can be used as a cell quality control method for the quality control of cell matrix and therapeutic cell products. Attached Figure Description

[0077] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0078] Figure 1 This is a schematic diagram of the process for cell species identification and cross-contamination detection using the primer combinations provided in the embodiments of this application.

[0079] Figure 2 The results of cell species identification and cross-contamination detection using primer combinations in Example 1 of this application are shown. Lane M is a 100bp DNA Ladder, and the templates corresponding to lanes 1 to 13 are, in order: pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, cow, sample 1 to be tested, sample 2 to be tested, and sample 3 to be tested.

[0080] Figure 3 The results of cell species identification and cross-contamination detection using primer combinations in Example 2 of this application are shown. Lane M is a 100bp DNA Ladder, and the templates corresponding to lanes 1 to 13 are, in order: pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, cow, sample 1 to be tested, sample 2 to be tested, and sample 3 to be tested.

[0081] Figure 4 The results of cell species identification and cross-contamination detection using primer combinations in Example 3 of this application are shown. Lane M is a 100bp DNA Ladder, and the template corresponding to lane 1 is a mixed template of 10 plasmids containing the COX I gene.

[0082] Figure 5 The results show the detection of primer combination sensitivity in Example 4 of this application, where lane M is a 100bp DNA ladder, and lanes 1 to 9 correspond to the human:green monkey DNA ratio in the template as follows: 10:0, 9.99:0.01, 9.9:0.1, 9:1, 1:1, 1:9, 0.1:9.9, 0.01:9.99, and 0:10, respectively.

[0083] Figure 6 This is the detection result of primer combination sensitivity in Example 5 of this application, where lane M is a 100bp DNA ladder and lane 1 corresponds to the mixed template diluted to 10. -1 The detection results at 10 times showed that lane 2 corresponded to a mixed template diluted to 10. -2 The detection results at 10 times showed that lane 3 corresponded to a mixed template diluted to 10. -3 The detection results at 10 times showed that lane 4 corresponded to a mixed template diluted to 10.-4 The test results at times.

[0084] Figure 7 This is the test result of test 1 in Comparative Example 1 of this application.

[0085] Figure 8 This is the test result of test 2 in Comparative Example 1 of this application. Detailed Implementation

[0086] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0087] This application is based on the inventor's following understanding:

[0088] In the fields of animal molecular taxonomy and systematic evolution, mitochondrial DNA is abundant in cells, easily amplified, highly conserved, has short intergenic regions without introns, and exhibits a high mutation rate. It also displays high diversity in natural populations and can effectively reflect signals of population evolutionary history. The most commonly used mitochondrial markers are the cytochrome c oxidase subunit I gene (COX I), the cytochrome b gene (Cytb), and the NADH dehydrogenase subunit gene.

[0089] In related technologies, primer combinations used for cell species identification often select different genes in mitochondrial markers as templates to design primers. Although they can identify the species of samples from a single source, they are difficult to comprehensively cover common species at the same time, resulting in low detection efficiency.

[0090] In response, the inventors conducted extensive research and selected the COX I gene, which has a higher evolutionary rate, from the genes of cytochrome c oxidase subunit I (COX I), cytochrome b (Cytb), and NADH dehydrogenase subunit, as a template to obtain a set of primer combinations with high specificity, sensitivity, and broad coverage.

[0091] The primer combination developed in this application is simple and efficient, and can simultaneously and accurately determine whether the sample to be tested contains cells from ten different sources: pig, human, cat, sheep, horse, green monkey, rat, mouse, goat and cow. It effectively solves the problems of limited species coverage, low detection sensitivity and cross-influence between primer pairs in existing primer combinations.

[0092] The first aspect of this application provides a primer combination for cell species identification and cross-contamination detection.

[0093] The second aspect of this application provides a reagent comprising the primer combination provided in the first aspect of the embodiment described above.

[0094] In some embodiments, the total molar ratio of each primer pair in the reagent is the same.

[0095] An embodiment of the third aspect of this application provides a kit comprising the primer combination provided in the first aspect embodiment or the reagent provided in the second aspect embodiment.

[0096] In some embodiments, the kit also includes auxiliary reagents for PCR amplification.

[0097] In some embodiments, the auxiliary reagents include DNA polymerase and / or positive control.

[0098] The fourth aspect of this application provides the application of the primer combination provided in the first aspect embodiment, the reagent provided in the second aspect embodiment, or the kit provided in the third aspect embodiment in cell species identification and cross-contamination detection, wherein the cell species is selected from one or more of pigs, humans, cats, sheep, horses, green monkeys, rats, mice, goats, and cattle.

[0099] In some embodiments, cell species identification is used to identify whether the species of the cell sample to be tested is pig, human, cat, sheep, horse, green monkey, rat, mouse, goat or cow.

[0100] In some embodiments, cross-contamination detection is used to detect whether the cell sample to be tested contains cells of one or more species, namely pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, or cow.

[0101] An embodiment of the fifth aspect of this application provides a method for cell species identification and cross-contamination detection.

[0102] Figure 1 This application provides methods for cell species identification and cross-contamination detection in its embodiments. For example... Figure 1 As shown, the cell species identification and cross-contamination detection method may include the following steps: S1-S2.

[0103] Step S1. Use primer combinations to perform PCR amplification of the nucleic acid in the cell sample to be tested.

[0104] The primer combinations provided in this application are based on the COX I gene sequences from ten different animal cells listed in Table 1 below:

[0105] Table 1

[0106]

[0107]

[0108]

[0109] The primer combinations provided in this application include the following primer pairs:

[0110] (1) Primer pair Pig-COX I for specific amplification of the porcine COX I gene:

[0111] Forward primer: 5'-GTCTGATCAGTACTAATCACAGCCGT-3' (SEQ ID NO.11)

[0112] Reverse primer: 5'-GAGCTCATAGTATTGCGGGTGATCAT-3' (SEQ ID NO.12);

[0113] (2) Primer pair for specific amplification of the human COX I gene: Human-COX I:

[0114] Forward primer: 5'-TAGACATCGTACTACACGACACG-3' (SEQ ID NO.13)

[0115] Reverse primer: 5'-TCCAGGTTTATGGAGGGTTC-3' (SEQ ID NO.14);

[0116] (3) Primer pair for specific amplification of the feline COX I gene: Cat-COX I:

[0117] Forward primer: 5'-TATTGCCATTCCTACCGGGGTG-3' (SEQ ID NO.15)

[0118] Reverse primer: 5'-ACGTTATATTGACTCCTACAAACATAATC-3' (SEQ ID NO.16);

[0119] (4) Primer pair for specific amplification of the sheep COX I gene: Sheep-COX I:

[0120] Forward primer: 5'-CTATAATTATCGCCATCCCAACAGGAG-3' (SEQ ID NO.17)

[0121] Reverse primer: 5'-AGGGGAAATCAATGTACAAATCCTCCTA-3' (SEQ ID NO.18);

[0122] (5) Primer pair Horse-COX I for specific amplification of the equine COX I gene:

[0123] Forward primer: 5'-TGGGACCCTACTAGGAGATGATCAGA-3' (SEQ ID NO.19)

[0124] Reverse primer: 5'-CCTGTTCCGGCACCTGCTTCA-3' (SEQ ID NO.20);

[0125] (6) Primer pair for specific amplification of the green monkey COX I gene: Monkey-COX I:

[0126] Forward primer: 5'-CCTCCTTCCTGCTGCTAATGGC-3' (SEQ ID NO.21)

[0127] Reverse primer: 5'-TTTGATACTGGGATATGGCG-3' (SEQ ID NO.22);

[0128] (7) Primer pair Rat-COX I for specific amplification of the rat COX I gene:

[0129] Forward primer: 5'-GTACATCTTAATTCTTCCAGGGTTTGGA-3' (SEQ ID NO.23)

[0130] Reverse primer: 5'-CGGGTGTCTACATCTAGGCCTACTG-3' (SEQ ID NO.24);

[0131] (8) Primer sequence for specific amplification of the mouse COX I gene: Mouse-COX I:

[0132] Forward primer: 5'-AACAGACCGCAACCTAAACACAACT-3' (SEQ ID NO.25)

[0133] Reverse primer: 5'-ATGTGAAATAATTCCAAATCCTGGGAGG-3' (SEQ ID NO.26);

[0134] (9) Primer pair Goat-COX I for specific amplification of the goat COX I gene:

[0135] Forward primer: 5'-ATATCAATCGGGTTTCTAGGATTTATT-3' (SEQ ID NO.27)

[0136] Reverse primer: 5'-AGTTGGGATAGCGATAATTATGGTAGC-3' (SEQ ID NO.28);

[0137] (10) Primer sequence for specific amplification of the bovine COX I gene: Cow-COX I:

[0138] Forward primer: 5'-GCTATTCCAACCGGGGTAAAAGTC-3' (SEQ ID NO.29)

[0139] Reverse primer: 5'-GAAAATAAAGCCTAGGGCTCAC-3' (SEQ ID NO.30).

[0140] The primer combination provided in this application has high specificity. In a PCR system, using nucleic acids from test cell samples containing 10 different cell species as templates for amplification, it can obtain 10 corresponding target bands. It also has high sensitivity, is simple and efficient to apply, and therefore has good scalability. It can be used as a cell quality control method for the quality control of cell matrix and therapeutic cell products.

[0141] Step S2. Characterize the DNA fragments in the PCR amplification product and determine, based on the characterization results, whether the cell sample to be tested contains cells of one or more species, namely pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, or cow.

[0142] In some embodiments, determining whether the cell sample to be tested contains cells of one or more species of pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, or cow based on the characterization results includes:

[0143] When the PCR amplification product does not contain a 472bp DNA fragment, the cell sample to be tested is determined not to contain cells of the species pig; when the PCR amplification product contains a 472bp DNA fragment, the cell sample to be tested is determined to contain cells of the species pig.

[0144] When the PCR amplification product does not contain a 425bp DNA fragment, the cell sample to be tested is determined not to contain human cells; when the PCR amplification product contains a 425bp DNA fragment, the cell sample to be tested is determined to contain human cells.

[0145] When the PCR amplification product does not contain a 341bp DNA fragment, the cell sample to be tested is determined not to contain cells of the cat species; when the PCR amplification product contains a 341bp DNA fragment, the cell sample to be tested is determined to contain cells of the cat species.

[0146] When the PCR amplification product does not contain a 271bp DNA fragment, the cell sample to be tested is determined not to contain cells of the species sheep; when the PCR amplification product contains a 271bp DNA fragment, the cell sample to be tested is determined to contain cells of the species sheep.

[0147] When the PCR amplification product does not contain a 243bp DNA fragment, the cell sample to be tested is determined not to contain cells of the species horse; when the PCR amplification product contains a 243bp DNA fragment, the cell sample to be tested is determined to contain cells of the species horse.

[0148] When the PCR amplification product does not contain a 222bp DNA fragment, the cell sample to be tested is determined not to contain cells of the species green monkey; when the PCR amplification product contains a 222bp DNA fragment, the cell sample to be tested is determined to contain cells of the species green monkey.

[0149] When the PCR amplification product does not contain a 177bp DNA fragment, the cell sample to be tested is determined not to contain cells of the rat species; when the PCR amplification product contains a 177bp DNA fragment, the cell sample to be tested is determined to contain cells of the rat species.

[0150] When the PCR amplification product does not contain a 139bp DNA fragment, the cell sample to be tested is determined not to contain cells of the mouse species; when the PCR amplification product contains a 139bp DNA fragment, the cell sample to be tested is determined to contain cells of the mouse species.

[0151] When the PCR amplification product does not contain a 117bp DNA fragment, the cell sample being tested is determined not to contain cells of the goat species; when the PCR amplification product contains a 117bp DNA fragment, the cell sample being tested is determined to contain cells of the goat species.

[0152] If the PCR amplification product does not contain a 102bp DNA fragment, the cell sample to be tested is determined not to contain bovine cells; if the PCR amplification product contains a 102bp DNA fragment, the cell sample to be tested is determined to contain bovine cells.

[0153] In some embodiments, the total molar ratio of each primer pair contained in the PCR amplification system is the same.

[0154] In some embodiments, the PCR amplification system contains a final concentration of 10... -3 Up to 50 ng / μL (e.g., 10) -2 ng / μL, 10 -1Templates of ng / μL, 1ng / μL, 3ng / μL, 5ng / μL, 7ng / μL, 9ng / μL, 10ng / μL, 20ng / μL, 30ng / μL, 40ng / μL, preferably 0.1 to 10ng / μL (e.g. 0.5g / μL, 1ng / μL, 2ng / μL, 3ng / μL, 4ng / μL, 5ng / μL, 6ng / μL, 7ng / μL, 8ng / μL, 9ng / μL).

[0155] In some embodiments, the PCR amplification system comprises a primer combination with a final concentration of 5 to 50 μM (e.g., 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM), preferably 10 to 30 μM (e.g., 12.5 μM, 15 μM, 17.5 μM, 20 μM, 22.5 μM, 25 μM, 27.5 μM).

[0156] In some embodiments, the PCR amplification procedure includes:

[0157] (i) React at 97 to 99°C for 2 to 4 minutes;

[0158] (ii) Cyclic reactions; and

[0159] (iii) Full extension at 70 to 74°C for 8 to 12 minutes.

[0160] The cyclic reaction includes a reaction at 97 to 99°C for 8 to 12 seconds; a reaction at 59 to 63°C for 28 to 32 seconds; and a reaction at 68 to 74°C for 28 to 32 seconds, and the cyclic reaction is repeated 32 to 38 times.

[0161] In some embodiments, the PCR amplification procedure includes:

[0162] (i) React at 98℃ for 3 min;

[0163] (ii) Cyclic reactions; and

[0164] (iii) Extend completely at 72°C for 10 minutes.

[0165] The cyclic reaction includes a reaction at 98°C for 10 seconds; a reaction at 61°C for 30 seconds; and a reaction at 72°C for 30 seconds, and the cyclic reaction is repeated 35 times.

[0166] It should be noted that the foregoing explanations of the method embodiments also apply to the primer combinations, reagents, and application embodiments described above, and will not be repeated here.

[0167] In this application, the term "comprising" is an open-ended expression, meaning it includes the content specified in this invention, but does not exclude other aspects.

[0168] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0169] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0170] Example 1

[0171] The primer combination provided in this embodiment includes primer pairs Pig-COX I, Human-COX I, Cat-COX I, Sheep-COX I, Horse-COX I, Monkey-COX I, Rat-COX I, Mouse-COX I, Goat-COX I, and Cow-COX I. In the primer combination, the total molar ratio of each primer pair is the same.

[0172] The initial concentration of each primer was 100 μM. There were 20 primers in total, consisting of 10 primer pairs. 5 μL of each primer was taken and mixed to prepare a primer combination with a total volume of 100 μL and a total primer concentration of 100 μM.

[0173] (1) Prepare cell samples

[0174] Prepare human HeLa cells and human umbilical cord mesenchymal stem cells; approximately 1 × 10⁻⁶ samples per group. 6 Centrifuge each cell at 2000 rpm for 5 minutes and discard the supernatant.

[0175] (2) Extracting genomic DNA from cell samples

[0176] Collect the cell samples to be tested into 1.5 mL centrifuge tubes. Add 200 μL of GTL (brand: cwbio) to each cell sample and vortex until the sample is completely suspended. Add 20 μL of Proteinase K, then add 200 μL of Buffer GL, vortex thoroughly, and incubate at 56 °C for 10 minutes. Add 200 μL of anhydrous ethanol and vortex thoroughly. Add the entire solution to the adsorption column already loaded into the collection tube, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Add 500 μL of Buffer GW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Add 500 μL of Buffer GW2 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and transfer the adsorption column to a 1.5 mL centrifuge tube. Add 50 μL of sterile water to the middle of the adsorption column, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃.

[0177] (3) Verify the specificity of the primer combination by PCR.

[0178] Using synthetic plasmids containing COX I genes from green monkeys, cats, goats, rats, sheep, mice, horses, pigs, and cattle as listed in Table 1 above, as well as genomic DNA from human HeLa cells and human umbilical cord mesenchymal stem cells extracted in step (2) above, as templates, amplification was performed using primer combinations containing primer pairs Pig-COX I, Human-COX I, Cat-COX I, Sheep-COX I, Horse-COX I, Monkey-COX I, Rat-COX I, Mouse-COX I, Goat-COX I, and Cow-COX I.

[0179] The PCR amplification system consisted of a total volume of 20 μL, including 10 μL of DNA polymerase, 4 μL of template (with a final plasmid concentration of 0.5 ng / μL and a final concentration of genomic DNA from human HeLa cells and human umbilical cord mesenchymal stem cells of 8 ng / μL), 6 μL of primer combination (final concentration of 30 μM), and 0 μL of sterile enzyme-free water.

[0180] The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 61℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ complete extension for 10 min; and storage at 10℃.

[0181] (4) Verification results

[0182] The target DNA fragment size of primer pair Pig-COX I is 472 bp;

[0183] The target DNA fragment size of primer pair Human-COX I is 425 bp;

[0184] The target DNA fragment size of the primer pair Cat-COX I is 341 bp;

[0185] The target DNA fragment size of the primer pair Sheep-COX I is 271 bp;

[0186] The target DNA fragment size of the primer pair Horse-COX I is 243 bp;

[0187] The target DNA fragment size of the primer pair Monkey-COX I is 222 bp;

[0188] The target DNA fragment size of primer pair Rat-COX I is 177 bp;

[0189] The target DNA fragment size of the primer pair Mouse-COX I is 139 bp;

[0190] The target DNA fragment size of the primer pair Goat-COX I is 117 bp;

[0191] The target DNA fragment size of primer pair Cow-COX I is 102 bp.

[0192] The PCR products obtained in step (3) were subjected to agarose gel electrophoresis. A gel imaging analyzer was used to observe the amplified products to determine the cell species from which the samples originated. Using a 2% agarose gel, each PCR product (5 μL) was mixed with 6×DNA Loading Buffer for electrophoresis (130 v / 35-40 min), photographed, and analyzed.

[0193] Electrophoresis results as follows Figure 2 As shown, lane M is a 100bp DNA marker, and the templates corresponding to lanes 1 to 13 are, in order: pig, human (human HeLa cell genomic DNA), cat, sheep, horse, green monkey, rat, mouse, goat, cow, sample 1 (genomic DNA of human umbilical cord mesenchymal stem cells), sample 2 (genomic DNA of human umbilical cord mesenchymal stem cells), and sample 3 (genomic DNA of human umbilical cord mesenchymal stem cells).

[0194] Electrophoresis results showed that when amplifying a single template using primer combinations, lanes 1-10 showed only a single band, and the band size was consistent with the expected fragment size. This indicates that the primer combinations provided in this application embodiment can specifically amplify the target DNA fragments from their respective species, exhibiting excellent specificity. When using genomic DNA from human umbilical cord mesenchymal stem cells as templates, lanes 11-13 amplified the bands using primer combinations, and the resulting band sizes were consistent with those in lane 2 (human HeLa cell genomic DNA as template). This demonstrates that the primer combinations provided in this application embodiment are widely applicable to the identification of animal cells from the above ten species.

[0195] Example 2

[0196] Primers

[0197] The primer combination provided in this embodiment includes primer pairs Pig-COX I, Human-COX I, Cat-COX I, Sheep-COX I, Horse-COX I, Monkey-COX I, Rat-COX I, Mouse-COX I, Goat-COX I, and Cow-COX I. In the primer combination, the total molar ratio of each primer pair is the same.

[0198] The initial concentration of each primer was 100 μM. There were 20 primers in total, consisting of 10 primer pairs. 5 μL of each primer was taken and mixed to prepare a primer combination with a total volume of 100 μL and a total primer concentration of 100 μM.

[0199] template

[0200] Genomic DNA from MPK-1 (pig), Hela (human), PG-3 (cat), CRL-1700 (sheep), CCL-57 (horse), Vero (green monkey), Walker256 (rat), NH3T3 (mouse), IM-C046 (goat), and MDBK (bovine) cells were used as templates.

[0201] Detection

[0202] The genomic DNA extraction method, PCR amplification system, and procedure were the same as in Example 1.

[0203] result

[0204] Electrophoresis results as follows Figure 3 As shown, this embodiment uses genomic DNA from various sources as templates for amplification, and the experimental results are the same as those in Example 1, indicating that the primer combination provided in this embodiment can achieve species identification of the cells to be tested.

[0205] Example 3

[0206] Primers

[0207] The primer combination provided in this embodiment includes primer pairs Pig-COX I, Human-COX I, Cat-COX I, Sheep-COX I, Horse-COX I, Monkey-COX I, Rat-COX I, Mouse-COX I, Goat-COX I, and Cow-COX I. In the primer combination, the total molar ratio of each primer pair is the same.

[0208] The initial concentration of each primer was 100 μM. There were 20 primers in total, consisting of 10 primer pairs. 5 μL of each primer was taken and mixed to prepare a primer combination with a total volume of 100 μL and a total primer concentration of 100 μM.

[0209] template

[0210] Synthetic plasmids containing COX I genes from the ten sources listed in Table 1 were mixed in equal proportions and used as templates.

[0211] Detection

[0212] The amplification procedure is the same as in Example 1.

[0213] The PCR amplification system consisted of a total volume of 20 μL, including 10 μL of DNA polymerase, 3 μL of template (final template concentration of 1.5 ng / μL), 5 μL of primers (final primer concentration of 25 μM), and 2 μL of sterile, enzyme-free water. The working solution concentration of the mixed template containing ten synthetic plasmids (in this paper, the working solution concentration refers to the concentration of the corresponding reagents before being added to the reaction system) was 1 ng / μL for each synthetic plasmid, resulting in a total plasmid working solution concentration of 10 ng / μL.

[0214] result

[0215] Electrophoresis results as follows Figure 4 As shown in the figure, lane 1 represents the amplification reaction results of the template (containing synthetic plasmids from ten sources of COX I genes in Table 1) and primer combination defined in this embodiment. The 2% gel effectively separates the ten target bands of different molecular weights in the amplification system. Each product band is consistent with the molecular weight of the corresponding single-species genomic DNA and the mixed PCR primer amplification reaction product, indicating that the mixed primer PCR amplification reaction system provided in this embodiment can not only identify different species of cells, but also detect the presence of contamination from different species of cells at the same time.

[0216] Example 4

[0217] Primers

[0218] The primer combination provided in this embodiment includes: Human-COX I and Monkey-COX I.

[0219] The sensitivity of Human-COX I and Monkey-COX I was detected using primer pairs.

[0220] In primer combinations, the total molar ratio of each primer pair is the same.

[0221] The initial concentration of each primer was 100 μM. There were 4 primers in total, 2 primer pairs. 5 μL of each primer was taken and mixed to prepare a primer combination with a total volume of 20 μL and a total primer concentration of 100 μM.

[0222] template

[0223] Genomic DNA from human and green monkey cells was mixed in different ratios (10:0, 9.99:0.01, 9.9:0.1, 9:1, 1:1, 1:9, 0.1:9.9, 0.01:9.99, and 0:10) as templates, with a total template DNA content of 10 ng. Amplification was performed using a combination of primer pairs Human-COX I and Monkey-COX I to simulate different levels of cross-species contamination in experimental cells and to determine the detection limit for cell contamination.

[0224] Detection

[0225] The method for extracting genomic DNA from cell samples was the same as in Example 1.

[0226] The PCR amplification system consisted of a total volume of 50 μL, including 25 μL of DNA polymerase, 10 μL of template (final concentration 0.2 ng / μL), 5 μL of primer combination (10 μM), and 10 μL of sterile enzyme-free water.

[0227] The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 61℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ complete extension for 10 min; and storage at 10℃.

[0228] The specific steps of step (3) above are as follows: dilute the extracted HeLa (human) and Vero (green monkey) cell genomic DNA to a working solution concentration of 1 ng / μL, mix human and monkey genomic DNA in different ratios (10:0, 9.99:0.01, 9.9:0.1, 9:1, 1:1, 1:9, 0.1:9.9, 0.01:9.99 and 0:10) as templates, keep the total amount of genomic DNA from the two different sources at 10 ng (the total amount of genomic DNA contained in the working solution of each template), and mix human and green monkey COXⅠ gene primers of equal concentration and volume for PCR amplification reaction.

[0229] result

[0230] The results are as follows Figure 5 As shown, in lanes 1-9, the genomic DNA of human and green monkey cells, depending on the different ratios of the template amount (10:0, 9.99:0.01, 9.9:0.1, 9:1, 1:1, 1:9, 0.1:9.9, 0.01:9.99, and 0:10), decreases in the proportion of human genomic template amount within the total genomic DNA amount, resulting in a decrease in the brightness of the PCR product bands from strong to weak. Conversely, the proportion of green monkey genomic template amount increases, resulting in a increase in the brightness of the PCR product bands from weak to strong. This indicates that the detection limit for cell contamination is reflected in the 0.01:9.99 mixing ratio (i.e., the genomic DNA of the contaminant can be detected even when the final concentration in the PCR system is as low as 0.0002 ng / μL), demonstrating that the sensitivity of the mixed primer system for detecting interspecies cell contamination can reach at least one-thousandth of cell contamination.

[0231] Example 5

[0232] Primers

[0233] The primer combination provided in this embodiment includes primer pairs Pig-COX I, Human-COX I, Cat-COX I, Sheep-COX I, Horse-COX I, Monkey-COX I, Rat-COX I, Mouse-COX I, Goat-COX I, and Cow-COX I. In the primer combination, the total molar ratio of each primer pair is the same.

[0234] template

[0235] The mixed template contains: MPK-1 (pig) genomic DNA 5 ng / μL, HeLa (human) genomic DNA 5 ng / μL, PG-3 (cat) genomic DNA 5 ng / μL, CRL-1700 (sheep) genomic DNA 10 ng / μL, CCL-57 (equine) genomic DNA 10 ng / μL, Vero (green monkey) genomic DNA 5 ng / μL, Walker256 (rat) genomic DNA 10 ng / μL, NH3T3 (mouse) genomic DNA 5 ng / μL, IM-C046 (goat) genomic DNA 5 ng / μL, and MDBK (bovine) genomic DNA 5 ng / μL. The mixed template is then divided into 10... -1 ~10 -4 Diluted several times.

[0236] Detection

[0237] The PCR amplification procedure was the same as in Example 1.

[0238] PCR amplification system: The total volume is 20 μL, including 10 μL of DNA polymerase, 4 μL of template (final concentrations of 1.3 ng / μL, 0.13 ng / μL, 0.013 ng / μL and 0.0013 ng / μL), 4 μL of primer combination (final primer concentration of 20 μM), and 2 μL of sterile enzyme-free water.

[0239] result

[0240] The results are as follows Figure 6 As shown, lane 1 is where the mixed template is diluted to 10. -1 At 1.3 ng / μL, lane 2 is where the mixed template is diluted to 10. -2 At a concentration of 1:1 (0.13 ng / μL), lane 3 is where the mixed template is diluted to 10. -3 At a concentration of 0.013 ng / μL, lane 4 is where the mixed template is diluted to 10. -4 When the concentration is 0.0013 ng / μL.

[0241] Comparative Example 1

[0242] Primers

[0243] 1. Primer pair Human-COX I (forward primer: SEQ ID NO.13; reverse primer: SEQ ID NO.14); contrast primer pair 1 (Human-COX I-1); contrast primer pair 2 (Human-COX I-2) and contrast primer pair 4 (Human-COX I-4), wherein...

[0244] Human-COX I-1 contains:

[0245] Forward primer: 5'-TAGACATCGTACTACACGACACG-3' (SEQ ID NO.31)

[0246] Reverse primer: 5'-TCCAGGTTTATGGAGGGTTC-3' (SEQ ID NO.32);

[0247] Human-COX I-2 contains:

[0248] Forward primer: 5'-GGCCTGACTGGCATTGTATTAGC-3' (SEQ ID NO.33)

[0249] Reverse primer: 5'-CTTTTCGCTTCGAAGCGAAGGCTT-3' (SEQ ID NO.34);

[0250] Human-COX I-4 contains:

[0251] Forward primer: 5'-CAGTGCTCTGAGCCCTAGGATTCA-3' (SEQ ID NO.35);

[0252] Reverse primer: 5'-CTATTAGGACTTTTCGCTTCGAAGCGAA-3' (SEQ ID NO.36).

[0253] 2. The primer combination provided in this embodiment includes: primer pairs Pig-COX I, Cat-COX I, Sheep-COX I, Horse-COX I, Monkey-COX I, Rat-COX I, Mouse-COX I, Goat-COX I, and Cow-COX I, as well as control primer pair 1 (Human-COX I-1). In the primer combination, the total molar ratio of each primer pair is the same.

[0254] template

[0255] 1. Using HeLa (human) cell genomic DNA as a single template.

[0256] 2. Genomic DNA from MPK-1 (pig), Hela (human), PG-3 (cat), CRL-1700 (sheep), CCL-57 (horse), Vero (green monkey), Walker256 (rat), NH3T3 (mouse), IM-C046 (goat), and MDBK (bovine) cells were used as single templates and amplified using primer combinations.

[0257] Detection

[0258] 1. The PCR amplification procedure and system (including the final concentrations of enzyme, template and primers) for primer 1 and template 1 were the same as in Example 1. Genomic DNA of HeLa (human) cells was amplified using primer pair Human-COX I; control primer pair 1 (Human-COX I-1); control primer pair 2 (Human-COX I-2); and control primer pair 4 (Human-COX I-4).

[0259] 2. The PCR amplification procedure and system for primer 2 and template 2 are the same as in Example 2.

[0260] result

[0261] The results of test 1 are as follows Figure 7As shown, lane 1 corresponds to the amplification result of primer pair 1 (Human-COX I-1); lane 2 corresponds to the amplification result of primer pair 2 (Human-COX I-2); lane 3 corresponds to the amplification result of primer pair Human-COX I; and lane 4 corresponds to the amplification result of primer pair 4 (Human-COX I-4). Figure 7 The results show that all four primer pairs were able to specifically amplify the target fragment band.

[0262] However, the results in test 2 are as follows Figure 8 As shown, the primer combination provided in this comparative example contains contrast primer pair 1 (Human-COX I-1) instead of Human-COX I in Example 1, resulting in nonspecific bands when using this primer combination to amplify the genomic DNA of HeLa (human) cells (see...). Figure 8 Middle lane 2).

[0263] According to the results of this comparative example, there is mutual interference between primer pairs in primer combinations containing different primer pairs. This results in non-specific bands appearing when a primer with good specificity is included in the primer combination for amplification of the target fragment as a single primer pair.

[0264] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0265] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for cell species identification and cross-contamination detection, comprising: Step S1. Perform PCR amplification of the nucleic acid in the cell sample to be tested using the following primer combination; and Step S2. Characterize the DNA fragments in the PCR amplification product, and determine, based on the characterization results, whether the cell sample to be tested contains cells from one or more species of pig, human, cat, sheep, horse, green monkey, rat, mouse, goat, or cow. The PCR amplification system contained a final concentration of 10 -3 Up to 50 ng / μL of template, The primer combination described herein has sequences as shown in SEQ ID NO. 11 to SEQ ID NO. 30, and is used to simultaneously identify different cell species and detect the presence of contamination from different cell species in mixed-species cells containing the above ten sources. The nucleic acid mentioned therein is total DNA; The step S2 includes: When the PCR amplification product does not contain a 472 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species pig; when it contains a 472 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species pig. When the PCR amplification product does not contain a 425 bp DNA fragment, the cell sample to be tested is determined not to contain human cells; when it contains a 425 bp DNA fragment, the cell sample to be tested is determined to contain human cells. When the PCR amplification product does not contain a 341 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the cat species; when it contains a 341 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the cat species. When the PCR amplification product does not contain a 271 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species sheep; when it contains a 271 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species sheep. When the PCR amplification product does not contain a 243 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species horse; when it contains a 243 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species horse. When the PCR amplification product does not contain a 222 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species green monkey; when it contains a 222 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species green monkey. When the PCR amplification product does not contain a 177 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species rat; when it contains a 177 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species rat. When the PCR amplification product does not contain a 139 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species mouse; when it contains a 139 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species mouse. When the PCR amplification product does not contain a 117 bp DNA fragment, it is determined that the cell sample to be tested does not contain cells of the species goat; when it contains a 117 bp DNA fragment, it is determined that the cell sample to be tested contains cells of the species goat. and When the PCR amplification product does not contain a 102 bp DNA fragment, the cell sample to be tested is determined not to contain bovine cells; when it contains a 102 bp DNA fragment, the cell sample to be tested is determined to contain bovine cells.

2. The method according to claim 1, characterized in that, The characterization was achieved by performing agarose gel electrophoresis on the amplification products.

3. The method according to claim 1, characterized in that, The PCR amplification procedure includes: (i) React at 97 to 99°C for 2 to 4 minutes; (ii) Cyclic reactions; and (iii) Extend completely at 70 to 74°C for 8 to 12 minutes. The cyclic reaction includes reacting at 97 to 99°C for 8 to 12 s; reacting at 59 to 63°C for 28 to 32 s; and reacting at 68 to 74°C for 28 to 32 s, wherein the cyclic reaction is repeated 32 to 38 times.

4. A primer combination for cell species identification and cross-contamination detection, characterized in that, The primer combination consists of the following primers: (1) For pigs COX I Pig- primer pairs for specific gene amplification COX I : Forward primer: 5'-GTCTGATCAGTACTAATCACAGCCGT-3' Reverse primer: 5'-GAGCTCATAGTATTGCGGGTGATCAT-3'; (2) To people COX I Primer pairs for specific gene amplification Human- COX I : Forward primer: 5'-TAGACATCGTACTACACGACACG-3' Reverse primer: 5'-TCCAGGTTTATGGAGGGTTC-3'; (3) For cats COX I Primer pairs for specific gene amplification Cat- COX I : Forward primer: 5'-TATTGCCATTCCTACCGGGGTG-3' Reverse primer: 5'-ACGTTATATTGACTCCTACAAACATAATC-3'; (4) For sheep COX I Primer pairs for specific gene amplification Sheep- COX I : Forward primer: 5'-CTATAATTATCGCCATCCCAACAGGAG-3' Reverse primer: 5'-AGGGGAAATCAATGTACAAATCCTCCTA-3'; (5) Against the horse COX I Primer pairs for specific gene amplification (Horse- COX I : Forward primer: 5'-TGGGACCCTACTAGGAGATGATCAGA-3' Reverse primer: 5'-CCTGTTCCGGCACCTGCTTCA-3'; (6) Green monkeys COX I Primer pairs for specific gene amplification Monkey- COX I : Forward primer: 5'-CCTCCTTCCTGCTGCTAATGGC-3' Reverse primer: 5'-TTTGATACTGGGATATGGCG-3'; (7) On rats COX I Primer pairs Rat- for specific gene amplification COX I : Forward primer: 5'-GTACATCTTAATTCTTCCAGGGTTTGGA-3' Reverse primer: 5'-CGGGTGTCTACATCTAGGCCTACTG-3'; (8) On mice COX I Primer sequences for specific gene amplification (Mouse-) COX I : Forward primer: 5'-AACAGACCGCAACCTAAACACAACT-3' Reverse primer: 5'-ATGTGAAATAATTCCAAATCCTGGGAGG-3'; (9) Goats COX I Primer pairs for specific gene amplification (Goat-) COX I : Forward primer: 5'-ATATCAATCGGGTTTCTAGGATTTATT-3' Reverse primer: 5'-AGTTGGGATAGCGATAATTATGGTAGC-3'; and (10) On cattle COX I Cow- primer sequences for specific gene amplification COX I : Forward primer: 5'-GCTATTCCAACCGGGGTAAAAGTC-3' Reverse primer: 5'-GAAAATAAAGCCTAGGGCTCAC-3', The primer combination is used to simultaneously identify different species of cells and detect the presence of contamination from different species of cells in mixed-species cells containing the above ten sources.

5. A reagent for cell species identification and cross-contamination detection, said reagent comprising the primer combination of claim 4.

6. The reagent according to claim 5, characterized in that, The total molar ratio of each primer pair in the reagent is the same.

7. A kit for cell species identification and cross-contamination detection, characterized in that, The kit contains the primer combination of claim 4 or the reagent of claim 5 or 6.

8. The application of the primer combination of claim 4, the reagent of claim 5 or 6, or the kit of claim 7 in cell species identification and cross-contamination detection, characterized in that, The cell species are selected from one or more of pigs, humans, cats, sheep, horses, green monkeys, rats, mice, goats, and cattle.

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