CN118667972A - Kit for identifying animal cell species and application method thereof - Google Patents

Abstract

The invention relates to the technical field of cytology, and particularly discloses a kit for identifying animal cell species and a use method thereof, wherein the kit comprises the following components: DNA extraction reagent, DNA purification reagent, species identification primer, PCR reaction reagent, control DNA, 2% agarose solution, electrophoresis buffer solution and staining solution, wherein the DNA extraction reagent comprises: 0.25% pancreatin solution, PBS, protease, elution buffer, DNA purification reagents including: magnetic beads, binding buffer, washing buffer, TE buffer, PCR reagents include: dNTP mixture, PCR reaction buffer solution and DNA polymerase; the kit provided by the invention utilizes the gene bar code sequence of the animal cell mitochondrial CoI gene conservation region to carry out PCR amplification and electrophoresis imaging to detect the species of animal cells, can obtain clear target gene strips, has high primer specificity and detection sensitivity, can detect very trace DNA templates after purification, meets the requirements of amplifying and detecting a plurality of species cells, and improves the efficiency and flux of species identification.

Description

A kit for animal cell species identification and its use method

Technical Field

The invention belongs to the technical field of cytology, and specifically relates to a kit for animal cell species identification and a method for using the kit.

Background Art

In recent years, animal cell culture technology has been widely used in many fields. In vitro cell culture technology can be used to produce protein biological products such as viral vaccines, interferon, monoclonal antibodies, etc.; obtain a large number of own skin cells for skin grafting; detect toxic substances; and be used for physiological, pathological, and pharmacological research to provide a theoretical basis for the treatment and prevention of diseases. However, with the extensive application of in vitro cultured cells in basic research and clinical application research, cross-contamination of cells between different species during in vitro culture has become increasingly serious. Therefore, the origin of cell species has become one of the important contents in cell quality control.

The microgene identification system, which is to perform biological classification diagnosis through the analysis of a small fragment of the genome, is a reliable biological classification method. At present, the DNA-based identification system has been extended to protists such as bacteria and viruses, as well as more advanced organisms. The DNA sequences used for DNA barcode analysis include cytoplasmic mitochondrial DNA, chloroplast DNA and nuclear DNA. Studies have shown that the mitochondrial gene of animals is a better target gene for analysis than nuclear genes. Because it has no introns, rarely recombine, and is inherited in haploid form. The CoI gene is a protein-coding gene located in mitochondrial DNA, with few insertions and deletions. It has two major advantages as a target gene: ① The universal primer of this gene can cover the 5' end of most animal groups; ② The mitochondrial CoI gene has a larger phylogenetic signal range than other mitochondrial genes. In addition, among the 13 protein-coding genes of mitochondrial DNA, the CoI gene has a total length of about 1542bp, encodes 513 amino acids, has a suitable sequence length, and the gene sequence evolution rate is slow. Many researchers use the mitochondrial CoI gene for species identification, which provides new ideas for overcoming the limitations of cell matrix quality control detection.

Therefore, it is necessary to propose a kit for animal cell species identification and a method for using the kit to at least partially solve the problems existing in the prior art.

The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to a person of ordinary skill in the art.

Summary of the invention

The object of the present invention is to provide a kit for animal cell species identification and a method of using the kit to solve the problems raised in the above background technology.

To achieve the above object, the present invention provides the following technical solutions:

A kit for animal cell species identification comprises: a DNA extraction reagent, a DNA purification reagent, species identification primers, a PCR reaction reagent, a control DNA, a 2% agarose solution, an electrophoresis buffer, and a staining solution. The DNA extraction reagent comprises: a 0.25% trypsin solution, PBS, a protease, and an elution buffer. The DNA purification reagent comprises: magnetic beads, a binding buffer, a washing buffer, and a TE buffer. The PCR reaction reagent comprises: a dNTP mixture, a PCR reaction buffer, and a DNA polymerase.

Preferably, the kit and the reagents therein are packaged and stored with storage and transportation-resistant materials to ensure the stability and shelf life of the reagents, and easy-to-use operating instructions are designed to facilitate users to quickly get started and operate.

A method for using a kit for animal cell species identification, comprising:

Step 1: Use a DNA extraction reagent to extract DNA from the animal cell sample to be tested, and purify the extracted DNA;

Step 2: Prepare a PCR reaction system in a PCR tube, and use the purified DNA as a template and the species identification primers for amplification;

Step 3: Perform electrophoresis on the PCR amplification product using the prepared agarose electrophoresis film, and perform incubation and staining after the electrophoresis is completed;

Step 4: Prepare positive control and negative control using control DNA, and incubate and stain after electrophoresis under the same conditions;

Step 5: Determine the species of the DNA in the sample to be tested based on the position of the band after staining, and verify it using the results of the control group.

Preferably, in step 1, the extracted DNA is purified using a DNA purification reagent, and then the concentration and purity of the purified DNA are measured using a Nanodrop UV-visible spectrophotometer, and the DNA is diluted to 1 ng/μL.

Use 0.25% trypsin solution to digest the sample cells to be tested, and add the prepared protease to help release DNA, collect the cell digestion solution, add an appropriate amount of isopropyl acetone or ethanol to precipitate proteins and other impurities, let it stand for a while, use a collection tube to pass the mixed solution to make the DNA adhere to the tube wall, wash the collection tube with PBS to further remove residual impurities, dissolve the DNA from the collection tube with elution buffer, collect the elution buffer in a new test tube, and the liquid obtained at this time contains pure DNA, which should be sealed and stored in a 4°C refrigerator for later use.

Preferably, in step 2, the PCR amplification conditions are: 95°C for 2 min, 94°C

30s, 60℃30s, 65℃45s, 25 cycles; 65℃10min, use the purified DNA diluted to 1ng/μL to combine with multiple species identification primers. The species identification primers used are specific primers designed based on the gene barcode sequences of the conserved regions of mitochondrial genes of known target species published by NCBI.

Since the universal primer of the CoI gene can cover the 5' end of most animal groups, multiple specific primers can be combined together based on the universal primer, and multiple PCR amplification can be used to realize the operation of simultaneous detection of multiple species, thereby improving detection efficiency and throughput. Fluorescent markers can be introduced into the universal primers to amplify the information of the PCR products, making the results more intuitive and easy to read.

Preferably, in step three, the prepared agarose electrophoresis film is injected into a gel-making mold using a 2% agarose solution to form a flat film, which is then stored in a 4°C refrigerator for later use. The electrophoresis conditions are: 100V, room temperature, 90min, and electrophoresis is performed using TBE buffer. The agarose electrophoresis film is then transferred to a staining box and stained with a staining solution.

When adding TBE buffer, make sure it completely covers the film. At the same time, pay attention to the temperature of the solution during electrophoresis to prevent the agarose electrophoresis film from melting due to high temperature. After the electrophoresis, take out the agarose electrophoresis film, absorb the excess buffer with filter paper, put it in the staining box, pour the staining solution evenly on the film, let it stand for about 30 minutes, take out the agarose electrophoresis film and remove the residual staining solution on the surface.

Preferably, in step 4, the positive control and the negative control are pre-treated under the same conditions as the sample to be tested, and a sufficient amount of the control group is prepared for multiple repeated operations to reduce the impact of accidental errors.

Preferably, in step five, the stained gel is placed in an ultraviolet transmission imaging system to observe and record the position and intensity of the DNA bands, and the operation process and results of the control group test are recorded in detail to verify the accuracy of the test results of the samples to be tested.

During the PCR amplification, electrophoresis and imaging processes, automated instruments using microfluidic technology are used to achieve partial automation of detection using test kits, reduce human errors, improve detection accuracy, and establish a user feedback collection mechanism to continuously optimize and improve the test kits and usage methods based on user evaluations and suggestions.

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

The kit of the present invention utilizes the gene barcode sequence of the conserved region of the mitochondrial CoI gene of animal cells to perform PCR amplification and electrophoresis imaging to detect the species of animal cells, and can obtain clear target gene bands, with high primer specificity and detection sensitivity, and can detect even a very small amount of purified DNA template. According to the characteristics of the mitochondrial CoI gene, multiple PCR amplification of cells of multiple species can be achieved, meeting the needs of amplifying and detecting cells of multiple species, improving the efficiency and throughput of species identification, and the detection process is simple and highly operable, providing a new idea for effectively judging the species origin of cells.

The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the method for using the kit of the present invention.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

Example:

Please refer to Figure 1, a kit for animal cell species identification, including: DNA extraction reagent, DNA purification reagent, species identification primer, PCR reaction reagent, control DNA, 2% agarose solution, electrophoresis buffer, staining solution, DNA extraction reagent includes: 0.25% trypsin solution, PBS, protease, elution buffer, DNA purification reagent includes: magnetic beads, binding buffer, washing buffer, TE buffer, PCR reaction reagent includes: dNTP mixture, PCR reaction buffer, DNA polymerase; the kit and its internal reagents are packaged and stored with storage and transportation resistant materials to ensure the stability and validity period of the reagents, and easy-to-use operating instructions are designed to facilitate users to quickly get started and operate.

A method for using a kit for animal cell species identification, comprising:

Step 1: Use a DNA extraction reagent to extract DNA from the animal cell sample to be tested, and purify the extracted DNA;

Step 2: Prepare a PCR reaction system in a PCR tube, and use the purified DNA as a template and the species identification primers for amplification;

Step 3: Perform electrophoresis on the PCR amplification product using the prepared agarose electrophoresis film, and perform incubation and staining after the electrophoresis is completed;

Step 4: Prepare positive control and negative control using control DNA, and incubate and stain after electrophoresis under the same conditions;

Step 5: Determine the species of the DNA in the sample to be tested based on the position of the band after staining, and verify it using the results of the control group.

In step 1, the extracted DNA is purified using a DNA purification reagent, and then the concentration and purity of the purified DNA are measured using a Nanodrop UV-visible spectrophotometer, and the DNA is diluted to 1 ng/μL.

The extracted DNA sample was transferred to a centrifuge tube, and binding buffer and magnetic beads were added. After thorough mixing, the particles were washed with washing buffer, and then the DNA was eluted from the particles with TE buffer. The eluate was collected in a new centrifuge tube. The liquid contained purified DNA, and its concentration and purity were measured using a Nanodrop UV-visible spectrophotometer. The DNA was diluted to 1 ng/μL and stored in a -20°C refrigerator for later use.

In step 2, the PCR amplification conditions are: 95°C for 2 min, 94°C for 30 s, 60°C for 30 s, 65°C for 45 s, 25 cycles; 65°C for 10 min, using purified DNA diluted to 1 ng/μL combined with multiple species identification primers. The species identification primers used are specific primers designed based on the gene barcode sequences of the conserved regions of mitochondrial genes of known target species published by NCBI.

Primer specificity test: Specific primers designed for various genera were combined, and the prepared DNA samples of known species were processed as described above, followed by PCR amplification. The amplified products were electrophoresed on a 3500xl genetic analyzer, and the test results were analyzed using GeneMapper IDX software to obtain the results of the primer specificity test. The DNA of a single species was amplified only at the position corresponding to the length of the amplified fragment, and no non-specific amplification occurred at other positions.

In step three, the prepared agarose electrophoresis film is injected into a gel-making mold using 2% agarose solution to form a flat film, which is then stored in a 4°C refrigerator for later use. The electrophoresis conditions are: 100V, room temperature, 90min, and TBE buffer is used for electrophoresis. The agarose electrophoresis film is then transferred to a staining box and stained with a staining solution.

In step 4, the positive control and negative control are pre-treated under the same conditions as the samples to be tested, and a sufficient amount of control group is prepared for multiple repeated operations to reduce the impact of accidental errors.

As a positive control, DNA from animal cell samples with known positive results (i.e., animal cells that have been confirmed to belong to a specific species) are processed according to the processing flow of the samples to be tested. After the test is completed, the known positive sample is observed to see whether it presents the expected positive result. If it is positive, it indicates that the test operation and reagents are reliable. As a negative control, DNA from animal cell samples with known negative results (i.e., animal cells that have been confirmed not to belong to a specific species) are processed together with the above-mentioned positive control according to the processing flow of the samples to be tested. After the test is completed, the known negative sample is observed to see whether it presents the expected negative result. If it is positive, it indicates that the test operation and reagents have not produced a false positive reaction.

In step five, the stained gel is placed in an ultraviolet transmission imaging system to observe and record the position and intensity of the DNA bands, and the operation process and results of the control group test are recorded in detail to verify the accuracy of the test results of the samples to be tested.

As can be seen from the above, the kit of the present invention utilizes the gene barcode sequence of the conserved region of the mitochondrial CoI gene of animal cells to perform PCR amplification and electrophoresis imaging to detect the species of animal cells, and can obtain clear target gene bands, with high primer specificity and detection sensitivity, and even extremely small amounts of purified DNA templates can be detected. According to the characteristics of the mitochondrial CoI gene, multiple PCR amplification of cells of multiple species can be achieved, meeting the needs of amplification and detection of cells of multiple species, improving the efficiency and throughput of species identification, and the detection process is simple and highly operable, providing a new idea for effectively determining the species origin of cells.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

In the drawings of the embodiments disclosed in the present invention, only the structures involved in the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

1. A kit for animal cell species identification comprising: a DNA extraction reagent, a DNA purification reagent, a species identification primer, a PCR reaction reagent, a control DNA, a 2% agarose solution, an electrophoresis buffer, a staining solution, the DNA extraction reagent comprising: 0.25% pancreatin solution, PBS, protease, elution buffer, the DNA purification reagents comprising: magnetic beads, binding buffer, washing buffer, TE buffer, the PCR reaction reagent includes: dNTP mixture, PCR reaction buffer solution and DNA polymerase.

2. The method of using a kit for animal cell species identification according to claim 1, wherein: the kit and the internal reagents thereof are packaged and stored by adopting materials which are durable in storage and transportation, so that the stability and the expiration date of the reagents are ensured, and the operation instruction which is easy to use is designed, so that the user can conveniently and rapidly get up to hand and operate.

3. A method of using a kit for animal cell species identification, comprising:

extracting DNA from a cell sample of an animal to be detected by using a DNA extraction reagent, and purifying the extracted DNA;

step two, preparing a PCR reaction system in a PCR tube, and carrying out amplification by taking purified DNA as a template and combining with a species identification primer;

step three, electrophoresis is carried out on the PCR amplification products by using a prepared agarose electrophoresis film, and incubation dyeing is carried out after the electrophoresis is finished;

Step four, preparing positive control and negative control by using control DNA, and performing electrophoresis under the same conditions, and then incubating and dyeing;

And fifthly, determining the species condition of the DNA in the sample to be detected according to the dyed strip position, and verifying by using the result of the control group.

4. A method of using a kit for animal cell species identification according to claim 3, wherein: in the first step, the extracted DNA is subjected to a purification operation using a DNA purification reagent, and then the purified DNA is measured for its concentration and purity using a Nanodrop ultraviolet-visible spectrophotometer and diluted to 1 ng/. Mu.L.

5. The method of claim 4, wherein the step of using the kit for animal cell species identification is performed by: in the second step, PCR amplification conditions: 95℃2min,94℃30s,60℃30s,65℃45s,25 cycles; the DNA diluted to 1 ng/. Mu.L after purification was used for 10min at 65℃to bind to multiple species identification primers designed specifically based on the gene barcode sequence of the conserved region of the mitochondrial gene of the cell of the known target species published by NCBI.

6. The method of claim 5, wherein the step of using the kit for animal cell species identification is performed by: in the third step, the prepared agarose electrophoresis film is injected into a glue making mould by using 2% agarose solution to prepare a flat film, and the flat film is preserved in a refrigerator at the temperature of 4 ℃ for standby, and electrophoresis conditions are as follows: 100V, room temperature, 90min, using TBE buffer for electrophoresis, then transferring agarose electrophoresis film into a staining box for staining with staining solution.

7. The method of claim 6, wherein the method comprises the steps of: in the fourth step, the positive control and the negative control are pre-treated under the same conditions as the sample to be tested, and a sufficient amount of control groups are prepared for repeated operation for a plurality of times so as to reduce the influence of accidental errors.

8. The method of claim 7, wherein the method comprises the steps of: in the fifth step, the stained gel is placed in an ultraviolet light transmission imaging system, the position and the intensity of the DNA strip are observed and recorded, and the operation process and the result of the detection of the control group are recorded in detail so as to verify the accuracy of the detection result of the sample to be detected.