CN113604585B - Universal primer and application thereof in multi-cell species identification and cross contamination detection - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and particularly provides a universal primer capable of amplifying COI genes of various mammals and insects simultaneously, and a method for cell species identification and cross contamination detection by using the universal primer. The method provided by the invention has wide coverage, and can carry out species identification and species cross contamination detection on all mammalian and insect cells; the enzyme digestion detection is carried out only after the COI gene of each cell is amplified, and the fragments after 15 minutes of enzyme digestion completely conform to the theory, so that the method has the advantages of simple and accurate operation, high efficiency and the like; the sample usage amount is small, when the marked COI gene of each species is subjected to PCR amplification before enzyme digestion, species identification and cross contamination detection among all mammal and insect species can be realized only by 100ng of DNA, the sample usage amount is saved, and the sensitivity is high; and the fragment after enzyme digestion is subjected to agarose gel electrophoresis analysis, so that the operation is simple, the time consumption is short, and the efficiency is high.

Description

Universal primer and application thereof in multi-cell species identification and cross contamination detection

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a universal primer and application thereof in multi-cell species identification and cross contamination detection.

Background

The biological product industry is an important component of the pharmaceutical industry and has been rapidly developed in recent years. Because of the increasing maturity of mammalian cell in vitro culture techniques and the closer biological properties of their expressed products to natural products of humans, statistically, about 70% of therapeutic recombinant protein drugs are produced by mammalian cell expression. In recent years, with the continuous improvement of insect baculovirus expression systems (BEVS), BEVS is increasingly used for the production of biological products due to its advantages of fast development speed, high yield, low risk and the like. Currently, 7 products are approved, 4 for human use, including vaccines and therapeutic products. Spodoptera frugiperda ovarian cell line and Trichoplusia ni embryonic cells are receptor cells of baculovirus, and are two cell lines which are mostly researched and applied at present.

In the process of in vitro operations such as preparation, propagation, modification and the like, intercellular cross contamination is easy to occur, and not only can the accuracy and repeatability of experimental results be affected by the intercellular cross contamination, but also the yield and quality of biological products can be affected, and even the expression failure of the biological products can be caused. The new cell line/strain, the Main Cell Bank (MCB), the Working Cell Bank (WCB) and the production end cell (EOPC) are definitely specified in the preparation of animal cytoplasm substrate for new pharmacopoeia biological product production assay and quality control, and need to be subjected to identification test so as to confirm that the cell is the cell and has no cross contamination of other cells. At present, methods commonly used for cell identification and cell cross contamination include STR spectroscopy, karyotype analysis, isozyme technique, and the like. Wherein, STR spectrum analysis method is only suitable for identifying cells of a few species and cross-contamination of cells between species; karyotyping and isozyme techniques are complex, time consuming and inefficient to perform. Therefore, a stable and efficient method for identifying cells and detecting cross contamination between cell species is urgently needed to be established.

The mitochondrial gene is a relatively independent genetic molecular unit, has the characteristic of maternal inheritance, has low sequence mutation rate, is conservative compared with a nuclear gene, is an important molecular basis in aspects of species classification, population genetics, evolutionary biology and the like, and comprises 16s rRNA gene, COI gene, Cy b gene and the like. The CAPS labeling technology is a molecular biology technology which is developed more mature, and is also called PCR-RFP technology, corresponding primers are designed according to corresponding genes, a target sequence of the primers is amplified, and the purposes of genotyping, gene localization, variety identification and the like are realized according to the difference of polymorphism of enzyme digestion fragments of PCR amplification products. The invention designs a set of universal primers capable of amplifying COI genes of mammals and insects simultaneously, and verifies the COI genes of the mammals and the insects on 9 types of cells of the mammals and 2 types of cells of the insects which are commonly used in biological products. The method is simple and rapid to operate, and high in stability, and is a perfect technology for multi-cell species identification and cross contamination among species.

Disclosure of Invention

The invention aims to solve the problems of large cell consumption, complex operation, low detection efficiency and high cost in the prior art.

To this end, the invention provides a universal primer based on CAPS markers, the sequence of the universal primer is MI-COI-F: TYATAGTAATACCCATWATAATTGGRGG, respectively; MI-COI-R: CCTCCTATAATAGCAAAWACWGCTCC are provided.

Specifically, the universal primers can amplify mammalian COI genes and insect COI genes simultaneously.

Specifically, the universal primers can simultaneously amplify CHO-K1, BHK-21, Vero, Hela, BALB/c-3T3, Walker-256, PG-4, MDBK, MDCK, sf9 and Hi-5 cells.

The invention also provides a method for cell species identification and cross contamination detection, which comprises the following steps:

(1) preparing a cell sample to be detected;

(2) extracting genome DNA of a cell sample to be detected;

(3) performing PCR amplification by using the universal primer of claim 1 or 2 and using the genomic DNA of a cell sample to be detected as a template;

(4) after the PCR reaction is finished, carrying out double enzyme digestion reaction on the PCR amplification product;

(5) and (3) carrying out electrophoresis detection on the double enzyme digestion reaction product, identifying the cell sample species according to the enzyme digestion site polymorphism and judging whether cross contamination occurs.

Specifically, the reaction system for PCR amplification in step (3) is: 10xBuffer for KOD-Plus-Neo 2.5. mu.l, 10. mu.M MI-COI-F0.5. mu.l, 10. mu.M MI-COI-R0.5. mu.l, 2mM dNTPs 2.5. mu.l, 25mM MgSO₄Mu.l, 100 ng/. mu.l of template DNA 1. mu.l, PCR grade water 15.5. mu.l, 1.0U/. mu.l KOD-Plus-Neo 0.5. mu.l.

Specifically, the reaction procedure of the PCR amplification in the step (3) is as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 15s, and extension at 68 ℃ for 15s, for 5 cycles; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 68 ℃ for 15s, and performing 30 cycles; completely extending for 5min at 72 ℃; storing at 16 ℃.

Specifically, before performing the double enzyme digestion reaction in the step (4), purifying the PCR amplification product.

Specifically, the endonucleases used in the double digestion reaction in the step (4) are BseGI and MvaI endonucleases.

Specifically, the double enzyme digestion reaction system in the step (4) is as follows: 10X

buffer 1.5μl，1％BSA 0.5μl，

enzyme 1 0.5μl，

20.5. mu.l of enzyme, 8. mu.l of 30ng/ul PCR-purified product, and 4. mu.l of sterile water were reacted at 37 ℃ for 15 minutes.

Specifically, the electrophoresis detection in the step (5) is agarose gel electrophoresis detection, specifically, 2 μ l of 10 × Loading Buffer is added to the double-restriction reaction product, and 170V electrophoresis is performed for 30 minutes at 2% agarose concentration.

The invention also provides a kit for cell species identification and cross contamination detection, which is assembled by the universal primer and related reagents, wherein the related reagents can be reagents except the DNA template in the cell species identification and cross contamination detection method provided by the invention, and can also be other conventional reagents suitable for PCR and double enzyme digestion reaction.

The kit provided by the invention can be used for species identification and cross-contamination detection of multi-species cell mixed samples, and can also be used for species identification and cross-contamination detection of single-species cells.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the method provided by the invention has wide coverage, and can carry out species identification and species cross contamination detection on all mammalian and insect cells compared with STR (short tandem repeat) atlas analysis technology.

2. The method provided by the invention only needs to carry out enzyme digestion detection after the COI gene of each cell is amplified, and the fragment after 15 minutes of enzyme digestion completely conforms to the theory.

3. The method provided by the invention has less sample usage amount, only 100ng of DNA is needed to realize species identification and cross contamination detection among all species of mammals and insects when PCR amplification is carried out on the marker COI genes of various species before enzyme digestion, and compared with the RFLP detection technology, the method saves the sample usage amount and has high sensitivity.

4. The method provided by the invention can be used for carrying out agarose gel electrophoresis analysis on the fragments after enzyme digestion, and can save complex enzyme digestion and polyacrylamide gel electrophoresis analysis compared with the conventional AFLP method, and the method is simple in operation, short in time consumption and high in efficiency.

The present invention will be described in further detail below with reference to the accompanying drawings.

Drawings

FIG. 1 shows the results of amplification of 9 kinds of mammalian cells and 2 kinds of insect cells using the universal primer of the present invention; m is DNAmarker DL 2000; 1-11: CHO-K1, BHK-21, Vero, Hela, BALB/c-3T3, Walker-256, PG-4, MDBK, MDCK, sf9 and Hi-5 cells.

FIG. 2 shows the result of electrophoresis of COI gene of each cell by restriction endonuclease BseGI after PCR amplification using the universal primer of the present invention; m is DNA marker DL 2000; 1-11: CHO-K1, BHK-21, Vero, Hela, BALB/c-3T3, Walker-256, PG-4, MDBK, MDCK, sf9, Hi-5 cells.

FIG. 3 shows the results of electrophoresis of COI genes of each cell by endonuclease MvaI after PCR amplification using the universal primers of the present invention; m is DNAmarker DL 2000; 1-11: CHO-K1, BHK-21, Vero, Hela, BALB/c-3T3, Walker-256, PG-4, MDBK, MDCK, sf9 and Hi-5 cells.

FIG. 4 shows the results of electrophoresis of COI genes of each cell by simultaneous restriction with endonucleases BseGI and MvaI after PCR amplification using the universal primers of the present invention; m is DNAmarker DL 2000; 1-11: CHO-K1, BHK-21, Vero, Hela, BALB/c-3T3, Walker-256, PG-4, MDBK, MDCK, sf9 and Hi-5 cells.

FIG. 5 is the result of the detection of different degrees of cross contamination between CHO cells and Hela cells using the cell species discrimination and cross contamination detection method provided by the present invention; m is DNAmarker DL 2000; 1: CHO-K1; 2: hela; 3-7: CHO-K1 DNA: hela DNA was cross-contaminated at 50:1, 5:1, 1:5, 1:50, respectively.

Detailed Description

The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which the present invention pertains will appreciate that various modifications and changes can be made to the present invention without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.

Unless otherwise specified, reagents and equipment used in the present invention are commercially available from conventional markets. The reagents used are, unless otherwise specified, formulated according to the molecular cloning guidelines. The primers used in the embodiment of the invention are synthesized by biological engineering corporation and purified by PAGE purification; the DNA polymerase is KOD DNA polymerase (KOD-401, Toyobo).

One, designing common primer capable of amplifying COI gene of various mammals and insects simultaneously

The gene sequences of various mammalian and insect cells are compared by using a nucleic acid sequence database of the National Center for Biotechnology Information (NCBI), sequences (conserved regions) which are highly conserved and have specificity in CO I genes of various cells are selected, Primer design software Primer is used for analyzing and designing, and the Primer sequences are finely adjusted to ensure that excessive pairing does not exist among the primers, no dimer exists, the Tm values of all the primers are ensured to be approximately the same, and a pair of universal primers capable of simultaneously amplifying the COI genes of the mammals and the insects is designed. The general primer sequence is as follows:

MI-COI-F：TYATAGTAATACCCATWATAATTGGRGG；

MI-COI-R：CCTCCTATAATAGCAAAWACWGCTCC。

second, cell species identification and cross contamination detection method

(1) Preparing a cell sample to be detected;

(2) extracting genome DNA of a cell sample to be detected;

(3) performing PCR amplification by using the universal primer of claim 1 or 2 and using the genomic DNA of a cell sample to be detected as a template;

the reaction system of PCR amplification is as follows: 10xBuffer for KOD-Plus-Neo 2.5. mu.l, 10. mu.M MI-COI-F0.5. mu.l, 10. mu.M MI-COI-R0.5. mu.l, 2mM dNTPs 2.5. mu.l, 25mM MgSO₄Mu.l, 100 ng/. mu.l of template DNA 1. mu.l, PCR grade water 15.5. mu.l, 1.0U/. mu.l KOD-Plus-Neo 0.5. mu.l;

the reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 15s, and extension at 68 ℃ for 15s, for 5 cycles; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 68 ℃ for 15s, and performing 30 cycles; completely extending for 5min at 72 ℃; storing at 16 ℃.

(4) After the PCR reaction is finished, carrying out double enzyme digestion reaction on the PCR amplification product;

in order to obtain a COI gene amplification product with high purity and high quality, a PCR amplification product can be purified before double enzyme digestion reaction;

the endonucleases used in the double enzyme digestion reaction are BseGI and MvaI endonucleases with good specificity and strong stability; the double enzyme digestion reaction system is as follows: 10X

buffer 1.5μl，1％BSA 0.5μl，

enzyme 1 0.5μl，

20.5. mu.l of enzyme, 8. mu.l of 30ng/ul PCR purified product, 4. mu.l of sterile water;

(5) and (3) carrying out electrophoresis detection on the double enzyme digestion reaction product, identifying the cell sample species according to the enzyme digestion site polymorphism and judging whether cross contamination occurs.

Example 1:

to determine the versatility of the universal primers, this example was PCR-amplified using the above universal primers against 9 mammalian cells (CHO-K1, BHK-21, Vero, Hela, BALB/c-3T3, Walker-256, PG-4, MDBK, MDCK) and 2 insect cell lines (sf9, Hi-5) commonly used for biologics.

(1) Preparing a cell sample to be detected;

when cells are adherent cells, adding a proper amount of 0.25% pancreatin into a cell culture bottle, digesting until the cells fall off, adding 2 times of a basic culture medium containing 10% FBS, uniformly mixing the cells, transferring the cells into a 15ml centrifuge tube, centrifuging 450g at room temperature for 5-8 minutes, then discarding the culture solution, adding a small amount of PBS (phosphate buffer solution) for heavy suspension, transferring into a 1.5ml centrifuge tube, centrifuging 450g at room temperature for 5-8 minutes, then discarding the supernatant, and collecting the cells;

when the cells are suspension cells, transferring a proper amount of cells into a 15ml centrifuge tube, centrifuging for 5-8 minutes at the room temperature by 450g, then discarding the culture solution, adding PBS (phosphate buffer solution) for heavy suspension, transferring into a 1.5ml centrifuge tube, and centrifuging to collect the cells;

(2) extracting genome DNA of a cell sample to be detected;

collecting cells into 1.5ml or 2ml EP tube, adding 800 μ L extraction buffer (SDS 1%, NaCl 75mmol/L, EDTA 0.1mol/L, Tris-HCl 10mmol/L PH8.0) and 20 μ L (20mg/ml) proteinase K into the centrifuge tube, water bath at 55 deg.C for 30 min, adding 5 μ L RNase solution when the solution becomes clear, digesting at 37 deg.C for 15-20 min, adding appropriate amount of protein precipitation solution (promega), inverting and mixing well, placing on ice for 10 min, centrifuging at 13000rpm for 10 min, collecting supernatant into new EP tube, adding equal volume of isopropanol, inverting several times, placing at-20 deg.C overnight, centrifuging at 13000rpm for 4 min, discarding supernatant, adding about 1ml 80% ethanol, washing DNA up and down, centrifuging at 13000rpm for 1 min, repeating washing twice, standing at room temperature for about 5min to volatilize ethanol, about 50ul of sterile water or TE (Tris-HCl 10mmol/L, EDTA 1mmol/L pH8.0) buffer was added thereto, and the DNA was dissolved sufficiently in a water bath at 55 ℃ for one hour.

(3) Taking the genome DNA of a cell sample to be detected as a template, and carrying out PCR amplification by using a universal primer;

the specific PCR reaction system is shown in Table 1, and the reaction procedure of PCR amplification is as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 15s, and extension at 68 ℃ for 15s, for 5 cycles; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and elongation at 68 ℃ for 15s, and performing 30 cycles; completely extending for 5min at 72 ℃; storing at 16 ℃.

TABLE 1 PCR reaction System

After the PCR reaction is finished, agarose gel electrophoresis detection is carried out on the PCR amplification product, wherein marker is DNA marker DL2000, and the result is shown in figure 1.

Example 2:

in order to verify the effect of the double-restriction system, this example performs a comparison test between single restriction and double restriction.

The PCR product obtained in example 1 was recovered and purified using a gel recovery kit (B518131-0100, Biotech Biotechnology Ltd.). First, 3. mu.l of 10 Xloading Buffer (9157, TAKARA) was added to the above PCR reaction system, and 1% agarose gel was electrophoresed for 15 minutes, the target band was cut out and placed in a 1.5ml EP tube, 600. mu.l of Buffer B2 was added, and then placed in a 55 ℃ water bath for 8 minutes, after cooling to room temperature, the above Solution was collected using an adsorption column, 8000g of the Solution was centrifuged for 30 seconds to pour the liquid in the tube, 500. mu.l of wash Solution, 9000g of the Solution was centrifuged for 30 seconds, the adsorption column was washed twice using wash Solution, 9000g of the Solution was left for 2 minutes, the adsorption column was transferred to a new EP tube, 20. mu.l of sterile water was added, and after standing at room temperature for 1 minute, the DNA Solution was collected by centrifugation.

Dividing the recovered product into three experimental groups for enzyme digestion reaction, specifically, the experimental group 1 and the experimental group 2 both adopt a single enzyme digestion reaction system shown in table 2, wherein the experimental group 1 is

enzyme BseGI, Experimental group 2

The enzyme is endonuclease MvaI; experiment group 3 adopts the double enzyme digestion reaction system shown in Table 3,

enzyme 1 and

enzyme 2 is BseGI and MvaI, respectively. The digestion conditions for the three experimental groups were 37 ℃ for 15 minutes.

TABLE 2 Single enzyme digestion reaction System

TABLE 3 double digestion reaction System

2 mul of 10 XLoading Buffer is added into the three groups of enzyme digestion reaction products respectively, electrophoresis is carried out for 30 minutes at 170V under 2 percent of agarose concentration, the enzyme digestion result of the experimental group 1 is shown in figure 2, the enzyme digestion result of the experimental group 2 is shown in figure 3, the enzyme digestion result of the experimental group 3 is shown in figure 4, and as can be seen from figures 2-4, only a double enzyme digestion system can completely distinguish 11 cell lines.

Example 3:

in order to verify the effect of the method provided by the invention in the detection of cross contamination among species, CHO-K1 cells and Hela cells were mixed according to the proportion of 50:1, 5:1, 1:5 and 1:50, respectively, PCR amplification was performed on the CHO-K1 cells and Hela cells by using the same PCR reaction system and reaction conditions as those in example 1, and after purifying DNA and performing double-restriction enzyme digestion reaction on the amplification product according to the method of example 3, the polymorphism of the restriction enzyme digestion site was analyzed to detect the detection capability of the method of the invention on cross contamination of cells, and as a result, as shown in FIG. 5, the method of the invention was used, and even if only 1/50 cross contamination was detected.

The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.

Claims (5)

1. A method of cell species identification and cross-contamination detection comprising the steps of:

(1) preparing a cell sample to be detected;

(2) extracting genome DNA of a cell sample to be detected;

(3) taking the genome DNA of a cell sample to be detected as a template, and carrying out PCR amplification by using a universal primer, wherein the sequence of the universal primer is MI-COI-F: TYATAGTAATACCCATWATAATTGGRGG, respectively; MI-COI-R: CCTCCTATAATAGCAAAWACWGCTCC, respectively;

(4) after the PCR reaction is finished, carrying out double digestion reaction on the PCR amplification product, wherein the endonucleases used in the double digestion reaction are BseGI and MvaI;

(5) carrying out electrophoresis detection on the double enzyme digestion reaction product, identifying the species of the cell sample according to the polymorphism of the enzyme digestion site and judging whether cross contamination occurs;

the method can be used for species identification and cross-contamination detection of CHO-K1, BHK-21, Vero, Hela, BALB/c-3T3, Walker-256, PG-4, MDBK, MDCK, sf9 and Hi-5 cells.

2. The method for cell species identification and cross-contamination detection according to claim 1, wherein the reaction system of PCR amplification in step (3) is: 10xBuffer for KOD-Plus-Neo 2.5. mu.l, 10. mu.M MI-COI-F0.5. mu.l, 10. mu.M MI-COI-R0.5. mu.l, 2mM dNTPs 2.5. mu.l, 25mM MgSO 42. mu.l, 100 ng/. mu.l of template DNA 1. mu.l, PCR grade water 15.5. mu.l, 1.0U/. mu.l KOD-Plus-Neo 0.5. mu.l.

3. The method for cell species identification and cross-contamination detection according to claim 1, wherein the reaction procedure of the PCR amplification in the step (3) is: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 15s, and extension at 68 ℃ for 15s, for 5 cycles; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and elongation at 68 ℃ for 15s, and performing 30 cycles; completely extending for 5min at 72 ℃; storing at 16 ℃.

4. The method of cell species identification and cross-contamination detection of claim 1, wherein: and (3) purifying a PCR amplification product before carrying out the double enzyme digestion reaction in the step (4).

5. The method for cell species identification and cross-contamination detection according to claim 1, wherein the double enzyme digestion reaction system in step (4) is: 10X

buffer 1.5μl，1％BSA 0.5μl，

enzyme 1 0.5μl，

20.5. mu.l of enzyme, 8. mu.l of 30ng/ul PCR-purified product, and 4. mu.l of sterile water.

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