Primer pair combination, kit and method for multi-cell species identification and cross contamination detection

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a primer pair combination, a kit and a method for multi-cell species identification and cross contamination detection. The primer pair combination comprises forward primers and reverse primers, the forward primers are used for specifically amplifying a pig COXI gene, a human COXI gene, a cat gene COXI gene, a Chinese hamster CYTB gene, a rhesus monkey COXI gene, an African green monkey COXI gene, a rat COXI gene, a dog COXI gene, a mouse COXI gene and a cattle COXI gene respectively, the sequences of the forward primers are shown as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 respectively, and the sequences of the reverse primers are shown as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 respectively. By utilizing the primer pair combination, the kit and the detection method, multi-cell species identification and qualitative and quantitative detection of cross contamination can be carried out in a high-specificity, high-sensitivity and high-stability manner.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular, relates to a primer pair combination, a kit and a method for multi-cell species identification and cross-contamination detection. Background Art

[0002] Cell lines are important experimental tools in biomedical research. With the widespread application of cell lines and primary cells in scientific research, drug development, and biotechnology, many cell lines are often misused or cross-contaminated by other cells, especially interspecies and intraspecies cross-contamination of human and animal cells. Although cross-contamination was reported in the 1950s, the misuse and cross-contamination of cell lines in experiments are still common problems.

[0003] In recent years, with the rapid development of therapeutic cell products in multiple fields including stem cell therapy, immune cell therapy and gene-edited cell therapy, the importance of cell line species identification and cross-contamination detection has become increasingly prominent. Cell line species identification and cross-contamination detection are not only key links in cell quality control, but also important measures to ensure research accuracy, clinical safety and patient rights.

[0004] In cell line identification and cross-contamination detection, the most commonly used methods include short tandem repeat (STR) analysis, single nucleotide polymorphism (SNP) analysis, and high-throughput sequencing. These methods are usually costly and require extensive database support to ensure the accuracy of the results. In addition, data processing and analysis are time-consuming and not suitable for situations where rapid results are required. Multiplex PCR is a polymerase chain reaction (PCR) method that simultaneously amplifies multiple target DNA fragments in a single reaction system. This technology allows the simultaneous amplification of multiple DNA sequences in a single reaction tube by using multiple specific primer pairs, thereby achieving the detection of multiple target sequences and effectively improving detection efficiency.

[0005] In the current methods for identifying cell lines and cross-contamination based on multiplex PCR technology, cell samples need to be digested with trypsin and then multiple steps are performed to extract and purify cell DNA. In the multi-step process, each step may result in partial loss of the sample. For example, during cell lysis, DNA extraction and purification, the recovery rate of DNA in the sample may be reduced due to improper operation, reagent quality problems or equipment errors. In addition, multi-step operations increase the possibility of contamination, and the risk of introducing exogenous DNA or other contaminants in each step also increases. These contaminants may not only affect the specificity and sensitivity of PCR amplification, but may also lead to false positive results, seriously affecting the accuracy and reliability of the final results. Summary of the invention

[0006] The purpose of the present invention is to provide a primer pair combination, a kit and a method for multicellular species identification and cross-contamination detection, which are used for multicellular species identification and cross-contamination detection and have the characteristics of high sensitivity, high specificity and high stability.

[0007] To this end, the present invention provides the following technical solutions.

[0008] The first aspect of the present invention provides a primer pair combination for multi-cell species identification and cross-contamination detection, the primer pair combination comprising:

[0009] (1) Primer pair for specific amplification of porcine COXI gene:

[0010] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 1,

[0011] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 2;

[0012] (2) Primer pair for specific amplification of human COXI gene:

[0013] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 3,

[0014] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 4;

[0015] (3) Primer pair for specific amplification of cat COXI gene:

[0016] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 5,

[0017] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 6;

[0018] (4) Primer pair for specific amplification of the Chinese hamster CYTB gene:

[0019] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 7,

[0020] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 8;

[0021] (5) Primer pair for specific amplification of rhesus monkey COXI gene:

[0022] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 9,

[0023] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 10;

[0024] (6) Primer pair for specific amplification of African green monkey COXI gene:

[0025] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 11,

[0026] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 12;

[0027] (7) Primer pair for specific amplification of rat COXI gene:

[0028] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 13,

[0029] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 14;

[0030] (8) Primer pair for specific amplification of canine COXI gene:

[0031] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 15,

[0032] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 16;

[0033] (9) Primer pair for specific amplification of mouse COXI gene:

[0034] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 17,

[0035] Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 18;

[0036] (10) Primer pair for specific amplification of bovine COXI gene:

[0037] Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 19,

[0038] Reverse primer sequence: The nucleotide sequence is shown in SEQ ID NO: 20.

[0039] A second aspect of the present invention provides a kit for multi-cell species identification and cross-contamination detection, the kit comprising the primer pair combination as described above.

[0040] As a preferred embodiment of the present invention, the kit further comprises: positive quality control, cell direct amplification lysis solution, multiplex PCR reaction solution, DEPC water, DL500 DNA Marker, and 6×Loading Buffer.

[0041] As a preferred embodiment of the present invention, the positive quality control is genomic DNA of cells of ten species, namely, pig, human, cat, Chinese hamster, rhesus monkey, African green monkey, rat, dog, mouse, and cattle, and the concentration ratio of the genomic DNA of the ten species of cells is 2:1:1:2:0.5:1:1:1:1:2.

[0042] As a preferred embodiment of the present invention, the composition of the cell direct expansion lysis solution includes: 50mM Tris-HCl pH7.8, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, and 100μg / mL Proteinase K.

[0043] As a preferred embodiment of the present invention, the composition of the multiplex PCR reaction solution includes: novel antibody-modified Taq DNA polymerase, PCR Buffer, dNTPs, Mg 2+ .

[0044] The third aspect of the present invention provides a method for detecting multicellular species identification and cross-contamination for purposes other than disease diagnosis or treatment, using the kit as described above for detection, the method comprising the following steps:

[0045] 1) Lysing the cell sample to be tested to obtain a cell lysate;

[0046] 2) performing a multiplex PCR amplification reaction on the cell lysate obtained above to obtain an amplification reaction product;

[0047] 3) Detecting the amplification reaction product of step 2) and making a determination based on the detection result.

[0048] As a preferred embodiment of the present invention, in step 1), a cell direct expansion lysis solution is used to lyse the cell sample to be tested to obtain a cell lysate.

[0049] As a preferred embodiment of the present invention, in step 2), the multiplex PCR reaction system includes the following components with a total volume of 25 μL: 10 μL of multiplex PCR reaction solution, 7 μL of primer MIX, and 8 μL of cell lysis solution.

[0050] As a preferred embodiment of the present invention, the primer MIX includes primer pairs of the above ten species: pig, human, cat, Chinese hamster, rhesus monkey, African green monkey, rat, dog, mouse, and cattle.

[0051] As a preferred embodiment of the present invention, the concentration ratios of primer pairs for ten species, namely pig, human, cat, Chinese hamster, rhesus monkey, African green monkey, rat, dog, mouse and cattle, in the primer MIX are 1:2:4:3:1:2:3:1:2:3 respectively.

[0052] As a preferred embodiment of the present invention, in step 2), the multiplex PCR reaction conditions are: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 90 sec, 30 cycles; and optimized extension at 72°C for 5 min.

[0053] As a preferred embodiment of the present invention, in step 3), the method for detecting the amplification reaction product includes gel electrophoresis.

[0054] As a preferred embodiment of the present invention, the specific operation steps of the gel electrophoresis method are: first, prepare an agarose gel with a gel concentration of 2.5%, mix the obtained amplification reaction product and DL500 DNA Marker with 6×Loading Buffer in a volume ratio of 1:5, then spot the sample, and perform electrophoresis at a constant voltage of 110V until the bromophenol blue band runs to the edge of the gel and pauses to obtain an electrophoresis band.

[0055] As a preferred embodiment of the present invention, in step 3), a gel imager is used to determine the detection result.

[0056] As a preferred embodiment of the present invention, in step 3), the basis for determining the test result is:

[0057] The size of the amplified DNA fragment product of pigs was 465 bp;

[0058] The size of the human DNA amplification product is 392 bp;

[0059] The size of the amplified DNA fragment product of cat is 355 bp;

[0060] The size of the amplified DNA fragment product of Chinese hamster was 320 bp;

[0061] The size of the DNA amplification product of rhesus monkeys was 295 bp;

[0062] The size of the amplified DNA fragment product of African green monkeys is 263 bp;

[0063] The size of the amplified DNA fragment product of rat was 198 bp;

[0064] The size of the DNA amplification product of dog was 172 bp;

[0065] The size of the amplified DNA fragment product of mouse was 148 bp;

[0066] The size of the amplified DNA fragment of cattle is 93 bp.

[0067] The fourth aspect of the present invention provides a use of the primer pair combination, or the kit, or the detection method as described above in the preparation of reagents for multi-cell species identification and cross-contamination detection.

[0068] By means of the above technical solution, the present invention has at least the following advantages:

[0069] (1) The present invention has carefully optimized and screened the PCR amplification reaction system. During the reaction process, there is no need to perform complex steps such as extraction and purification of the DNA of the cell sample to be tested. The cell lysate obtained after lysis with the cell direct amplification lysis solution can be directly used for PCR amplification. It takes less than 15 minutes to complete the processing of the cell sample, which greatly simplifies the experimental operation, reduces the loss of the sample to be tested, and reduces the risk of contamination of the sample to be tested.

[0070] (2) The primer pair combination provided by the present invention is designed for the mitochondrial genes of ten species, and the best primer pair combination is obtained by optimizing and screening each primer pair. This primer pair combination can efficiently and accurately detect and identify these species, thereby avoiding nonspecific amplification and the generation of false positive results.

[0071] (3) The detection kit provided by the present invention has the advantages of high sensitivity and high accuracy. While maintaining high sensitivity and high accuracy, the lower limit of the detection limit is further reduced. In the detection of samples with low cell counts (cell counts greater than the detection limit), a 100% detection rate can be achieved, ensuring the reliability and consistency of the test results.

[0072] (4) The detection kit provided by the present invention is easy to use and simple to operate. It can be used to detect large quantities of samples. The detection process is simple and fast, and can provide reliable result support for multi-cell species identification and cross-contamination detection control.

[0073] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 A schematic diagram of the process of the kit provided by the present invention for multi-cell species identification and cell cross-contamination detection.

[0075] Figure 2 This is a gel electrophoresis diagram of the results of identifying the species of cells of ten species in Example 1 of the present invention.

[0076] Figure 3 This is a gel electrophoresis diagram of the detection limit results of cell species identification in Example 2 of the present invention.

[0077] Figure 4 This is a gel electrophoresis diagram of the cell cross-contamination detection results in Example 4 of the present invention.

[0078] Figure 5 The gel electrophoresis diagram is a comparison of the test kit provided by the present invention and commercially available test kits. DETAILED DESCRIPTION

[0079] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] Unless otherwise specified, the primers and DEPC water involved in the present invention are synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd. Except for positive quality control, primer MIX, and cell direct amplification lysis buffer, the remaining components in the kit are commercial products from Jiangsu Kangwei Century Biotechnology Co., Ltd. and can also be replaced by other similar commercial products.

[0081] The following examples are mentioned and used:

[0082] 1. Positive quality control

[0083] Genomic DNA from cells of ten species, including pig, human, cat, Chinese hamster, rhesus monkey, African green monkey, rat, dog, mouse, and cattle, and the concentration ratio is 2:1:1:2:0.5:1:1:1:1:2.

[0084] 2. Cell direct expansion lysis buffer

[0085] The composition is: 50 mM Tris-HCl pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 100 μg / mL Proteinase K.

[0086] 3. Multiplex PCR reaction solution (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.)

[0087] Composition: novel antibody-modified Taq DNA polymerase, PCR Buffer, dNTPs, Mg 2+ .

[0088] Example 1: Design and screening of multiplex PCR primer pair combinations for multi-cell species identification and cross-contamination detection

[0089] 1. Design and screening of primer pair combinations

[0090] In this embodiment, the inventors selected highly conserved sequence regions after multiple sequence alignment using MEGA software based on the sequence information of pig COXI gene, human COXI gene, cat COXI gene, Chinese hamster CYTB gene, rhesus monkey COXI gene, African green monkey COXI gene, rat COXI gene, dog COXI gene, mouse COXI gene and bovine COXI gene, and applied primer design software Primer 5, taking into account the GC content of the nucleotide sequence and the Tm value of the primers, and specifically designed and screened primer pair combinations for the above ten cell species identification and cross-contamination detection. The primer pair combination information is shown in Table 1:

[0091] Table 1 Primer names and sequence information

[0092]

[0093] 2. Optimization of primer pair combinations

[0094] Use the primer pair combination in Table 1 to perform multiple PCR amplification reaction on the test cell standards of ten known species to select and determine the best primer combination. Among them, ten cell species of known cell species, HEK293 (human cells), SP2 / 0 (mouse cells), MDBK (bovine cells), PK15 (pig cells), MDCK (canine cells), Vero (African green monkey cells), CRFK (cat cells), C6 (rat cells), LLC-MK2 (rhesus monkey cells) and CHO (Chinese hamster cells) are used as test cell samples for multiple PCR detection. The specific operation process is as follows: Figure 1As shown: Take the freshly cultured cells to be tested and place them in a 1.5mL centrifuge tube. Centrifuge at 10000rpm for 2min, discard the supernatant, and retain the cell pellet. Add 100μL of cell direct expansion lysis solution to each tube of cell sample to be tested, immediately vortex and shake for 30s to fully mix, then place it in a 95℃ metal bath for 10min, and after cooling to room temperature, obtain the cell lysate, which is directly used as a template for subsequent PCR amplification and stored at -18℃ or below. Take 18μL of DEPC water to a new 1.5mL centrifuge tube, add 2μL of positive quality control, mix thoroughly and prepare it into working solution for use. Add 25μL of reaction system for multiple PCR amplification detection to the 1.5mL EP reaction tube, which includes the reaction system shown in Table 2 below. After the reaction system is prepared, it is placed in a PCR instrument for reaction. The reaction program is shown in Table 3, which is: 95°C pre-denaturation for 10 min; 95°C denaturation for 30 sec, 60°C annealing for 30 sec, 72°C extension for 90 sec, 72°C optimized extension for 5 min, 30 cycles. After the reaction is completed, the amplification reaction product is obtained.

[0095] Table 2 Reaction system for multiplex PCR amplification detection

[0096] Element volume Cell lysis buffer 8μL Multiplex PCR reaction solution 10μL Primer MIX 7μL

[0097] Note: The concentration ratios of primer pairs for ten species in primer MIX, including pig, human, cat, Chinese hamster, rhesus monkey, African green monkey, rat, dog, mouse and cattle, are 1:2:4:3:1:2:3:1:2:3 respectively.

[0098] Table 3 Multiplex PCR reaction program

[0099]

[0100] 3. Determination of results

[0101] The amplification reaction products were detected by gel electrophoresis, and the electrophoresis results were analyzed by gel imaging. The basis for judging the detection results is: the size of the DNA amplification product of pigs is 465bp; the size of the DNA amplification product of humans is 392bp; the size of the DNA amplification product of cats is 355bp; the size of the DNA amplification product of Chinese hamsters is 320bp; the size of the DNA amplification product of rhesus monkeys is 295bp; the size of the DNA amplification product of African green monkeys is 263bp; the size of the DNA amplification product of rats is 198bp; the size of the DNA amplification product of dogs is 172bp; the size of the DNA amplification product of mice is 148bp; the size of the DNA amplification product of cattle is 93bp.

[0102] The specific operation process of gel electrophoresis is as follows: first, prepare an agarose gel with a gel concentration of 2.5% (calculation formula: 2.5% gel (w / v) = (agarose 2.5g / 1×TBE buffer 100mL)×100%). Then mix the amplification reaction product obtained above and DL500DNA Marker with 6×Loading Buffer at a volume ratio of 1:5 and then spot the sample. The sample volume is 6μL (i.e. 1μL of 6×Loading Buffer + 5μL of PCR amplification reaction product / DNA Marker). Use DL500DNA Marker as a marker, run electrophoresis at a constant voltage of 110V, and pause the electrophoresis when the bromophenol blue band runs close to the edge of the gel (the length of the gel is about 60mm). Finally, use a gel imager to analyze the electrophoresis results. See the results. Figure 2 .

[0103] according to Figure 2 The results show that the band sizes of the 10 cell sample species identifications are in line with expectations (respectively: cattle 93bp, mouse 147bp, dog 172bp, rat 200bp, African green monkey 256bp, rhesus monkey 287bp, Chinese hamster 315bp, cat 355bp, human 394bp, pig 464bp), and no non-specific miscellaneous bands are generated. Therefore, the primer pair combination provided in Table 1, the reaction system provided in Table 2, and the reaction procedure provided in Table 3 of the present invention can efficiently and accurately detect and identify the species of the ten cells, and can be used as the best primer pair combination, reaction system, and reaction procedure.

[0104] Example 2: Sensitivity testing of primer pair combinations for multiplex PCR detection of multiple cell species identification and cross contamination

[0105] Using the primer pair combination of Table 1 in Example 1, the reaction system of Table 2 and the reaction program of Table 3, 500, 400, 200 and 100 cells of HEK293 (human cells), SP2 / 0 (mouse cells), MDBK (bovine cells), PK15 (pig cells), MDCK (canine cells), Vero (African green monkey cells), CRFK (cat cells), C6 (rat cells) and LLC-MK2 (rhesus monkey cells) of known species were sampled, and 5000, 4000, 2000 and 1000 cells of CHO (Chinese hamster cells) were sampled as the cell samples to be tested, and a multiplex PCR amplification reaction was performed according to the method described in Part 2 of Example 1. After the reaction, the amplification reaction products were detected according to the method described in Part 3 of Example 1 to determine the sensitivity of the primer pair combination. The results are shown in FIG. Figure 3 .

[0106] according to Figure 3It can be seen from the results that the primer pair combination provided in Table 1, the reaction system provided in Table 2, and the reaction program provided in Table 3 of the present invention can accurately detect 9 different species of cells, including HEK293 (human cells), SP2 / 0 (mouse cells), MDBK (bovine cells), PK15 (pig cells), MDCK (dog cells), Vero (African green monkey cells), CRFK (cat cells), C6 (rat cells), and LLC-MK2 (rhesus monkey cells) when sampling 200 cells, that is, the detection limit of the above 9 species of cells can reach 200 cells; for CHO (Chinese hamster cells), it can be accurately detected when sampling 2000 cells, that is, the detection limit of CHO (Chinese hamster cells) can reach 2000 cells. The detection ratio of the above 10 cells can reach 100%. Therefore, the primer pair combination provided by the present invention has the advantage of high sensitivity.

[0107] Example 3: Precision testing of primer pair combinations for multiplex PCR detection of multiple cell species identification and cross-contamination

[0108] The primer pair combination of Table 1, the reaction system of Table 2 and the reaction conditions of Table 3 in Example 1 were used, and HEK293 (human cells), SP2 / 0 (mouse cells), MDBK (bovine cells), PK15 (pig cells), MDCK (dog cells), Vero (African green monkey cells), CRFK (cat cells), C6 (rat cells), LLC-MK2 (rhesus monkey cells) and CHO (Chinese hamster cells) of known species were used as cell samples to be tested, and samples with high cell counts (500,000 cells) and low cell counts (400 cells, 4000 cells for CHO) were prepared respectively, and multiple PCR amplification reactions were performed according to the method described in Part 2 of Example 1. After the reaction, the amplification reaction products were detected according to the method described in Part 3 of Example 1 to determine the detection rate of the primer pair combination of the present invention for batch samples, and each sample was tested 10 times. The results are shown in Table 4.

[0109] Table 4 Detection rate of primer pair combination of the present invention for different cell numbers

[0110]

[0111] It can be seen from the results in Table 4 that the detection rate of the results of 10 repeated tests was 100% under the conditions of high cell number samples and low cell number samples. It can be seen that the primer pair combination in Table 1, the reaction system in Table 2 and the reaction conditions in Table 3 of the present invention have good precision and can be used for the detection of large batches of samples.

[0112] Example 4: Cell cross-contamination detection based on multiplex PCR detection of multiple cell species identification and cross-contamination primer pair combination

[0113] In this example, in order to verify the accuracy of the primer pair combination in Table 1, the reaction system in Table 2 and the reaction conditions in Table 3 for detecting cell cross contamination, cell cross contamination was set. The specific experimental arrangement is shown in Table 5:

[0114] Table 5 Cell cross contamination settings

[0115]

[0116] The cells set in Table 5 were used as the cell samples to be tested, and a multiplex PCR amplification reaction was performed according to the method described in Part 2 of Example 1. After the reaction was completed, the amplification reaction products were detected according to the method described in Part 3 of Example 1 to determine the detection of cell cross-contamination by the primer pair combination of the present invention. The results are shown in Figure 4 .

[0117] according to Figure 4 It can be seen from the results that the primer pair combination in Table 1, the reaction system in Table 2 and the reaction conditions in Table 3 of the present invention can all detect the contaminating cells present in the main cells in the presence of the main cells.

[0118] Example 5: Comparison with commercially available products

[0119] In this example, in order to verify the excellent performance of the primer pair combination of Table 1, the reaction system of Table 2 and the reaction conditions of Table 3 of the present invention, a commercially available cell species identification detection kit (Huzhou Shenke Biotechnology Co., Ltd., Article No.: 1801940) was compared with the primer pair combination, reaction system and reaction conditions provided by the present invention. Among them, the same number (5000 cells) of HEK293 (human cells), SP2 / 0 (mouse cells), MDBK (bovine cells), PK15 (pig cells), MDCK (canine cells), Vero (African green monkey cells), CRFK (cat cells), C6 (rat cells), LLC-MK2 (rhesus monkey cells) and CHO (Chinese hamster cells) were used as cell samples to be tested, and a multiple PCR amplification reaction was performed according to the method described in Part 2 of Example 1 to obtain an amplification reaction product I.

[0120] The commercially available kit is used to lyse the cell sample to be tested according to its instructions, and then obtain the DNA to be tested after nucleic acid extraction and purification steps. PCR amplification is performed using the DNA to be tested as a template to obtain an amplification reaction product II.

[0121] Finally, the amplification products I and II were detected respectively using the method described in Part 3 of Example 1. The results are shown in FIG. Figure 5 shown.

[0122] according to Figure 5The results show that, under the same number of cells to be tested, the primer pair combination of Table 1, the reaction system of Table 2 and the reaction conditions of Table 3 of the present invention can detect cells of ten species of HEK293 (human cells), SP2 / 0 (mouse cells), MDBK (cattle cells), PK15 (pig cells), MDCK (dog cells), Vero (African green monkey cells), CRFK (cat cells), C6 (rat cells), LLC-MK2 (rhesus monkey cells) and CHO (Chinese hamster cells), while the commercially available kits do not detect CHO (Chinese hamster cells), Vero (African green monkey cells), MDCK (dog cells), SP2 / 0 (mouse cells) and MDBK (cattle cells). It can be seen that compared with commercially available kit products, the primer pair combination, reaction system and reaction conditions provided by the present invention have higher detection sensitivity and specificity. The kit product of the present invention uses a cell direct amplification lysis solution to lyse the cells to be tested, and uses the lysed cell fluid directly as the DNA template to be tested. This process can avoid complex steps such as nucleic acid extraction and purification, and can effectively reduce the loss of DNA in the cell sample, thereby providing more accurate detection results.

[0123] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A primer pair combination for multi-cell species identification and cross-contamination detection, characterized in that: The primer pair combination comprises: (1) Primer pair for specific amplification of porcine COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 1, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 2; (2) Primer pair for specific amplification of human COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 3, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 4; (3) Primer pair for specific amplification of cat COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 5, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 6; (4) Primer pair for specific amplification of the Chinese hamster CYTB gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 7, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 8; (5) Primer pair for specific amplification of rhesus monkey COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 9, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 10; (6) Primer pair for specific amplification of African green monkey COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 11, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 12; (7) Primer pair for specific amplification of rat COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 13, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 14; (8) Primer pair for specific amplification of canine COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 15, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 16; (9) Primer pair for specific amplification of mouse COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 17, Reverse primer sequence: the nucleotide sequence is shown in SEQ ID NO: 18; (10) Primer pair for specific amplification of bovine COXI gene: Forward primer sequence: The nucleotide sequence is shown in SEQ ID NO: 19, Reverse primer sequence: The nucleotide sequence is shown in SEQ ID NO:

20.

2. A kit for multicellular species identification and cross-contamination detection, characterized in that: The kit comprises the primer pair combination according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes: positive quality control, cell direct amplification lysis solution, multiplex PCR reaction solution, DEPC water, DL500 DNA Marker, and 6×Loading Buffer.

4. The kit according to claim 3, characterized in that The positive quality control is genomic DNA of cells of ten species, namely, pig, human, cat, Chinese hamster, rhesus monkey, African green monkey, rat, dog, mouse and cattle. The concentration ratio of the genomic DNA of cells of the ten species is 2:1:1:2:0.5:1:1:1:1:

2.

5. The kit according to claim 3, characterized in that The cell direct expansion lysis solution comprises: 50mM Tris-HCl pH7.8, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, and 100μg / mL Proteinase K.

6. The kit according to claim 3, characterized in that The composition of the multiplex PCR reaction solution includes: novel antibody-modified Taq DNA polymerase, PCR Buffer, dNTPs, Mg 2+ .

7. A method for detecting multicellular species identification and cross-contamination for purposes other than disease diagnosis or treatment, characterized in that: The detection is performed using the kit according to any one of claims 2 to 6, the method comprising the following steps: 1) Lysing the cell sample to be tested to obtain a cell lysate; 2) performing a multiplex PCR amplification reaction on the cell lysate obtained above to obtain an amplification reaction product; 3) Detecting the amplification reaction product of step 2) and making a determination based on the detection result.

8. The detection method according to claim 7, characterized in that: In step 1), a cell direct expansion lysis solution is used to lyse the cell sample to be tested to obtain a cell lysate.

9. The detection method according to claim 7, characterized in that: In step 2), the multiplex PCR reaction system includes the following components with a total volume of 25 μL: 10 μL of multiplex PCR reaction solution, 7 μL of primer MIX, and 8 μL of cell lysis solution; The multiplex PCR reaction conditions are: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 90 sec, 30 cycles; and optimized extension at 72°C for 5 min.

10. Use of the primer pair combination according to claim 1, or the kit according to any one of claims 2 to 6, or the detection method according to any one of claims 7 to 9 in the preparation of reagents for multicellular species identification and cross-contamination detection.

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