CN107299049B - Kit for extracting DNA from oral cells of infant - Google Patents
Kit for extracting DNA from oral cells of infant Download PDFInfo
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- CN107299049B CN107299049B CN201710547778.8A CN201710547778A CN107299049B CN 107299049 B CN107299049 B CN 107299049B CN 201710547778 A CN201710547778 A CN 201710547778A CN 107299049 B CN107299049 B CN 107299049B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/02—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by impregnation, e.g. using swabs or loops
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses an oral cell DNA extraction kit for infants, which comprises an oral cell collection swab, a heat preservation component and a packaging component; the oral cell collection swab and the heat preservation component are packaged in the packaging component, and the kit also relates to a method for extracting the DNA of the oral cell. The invention has the beneficial effects that: the oral cell collecting swab is safe, comfortable and efficient; after sampling, the low temperature can be continuously kept to avoid the microbial pollution in the sending process; the operation is simple, and the exposure time of the swab is reduced; the yield is high, the oral cell DNA extraction kit in the current market can only extract 3ug of genome from one sampling at most, and if a large amount of gene operation is required subsequently, the extraction can be satisfied by sampling and extracting for multiple times; the invention can obtain 50ug genome under the condition of the same sampling amount; the kit is a special kit for extracting the DNA of the oral cells of the infants, and can efficiently finish the steps of sampling, storing, extracting and the like in consideration of various difficulties of sampling and amplifying the infants.
Description
Technical Field
The invention relates to an oral cavity cell DNA extraction kit for infants.
Background
With the rapid increase of the requirements of infant gene preservation, susceptibility gene detection and prenatal diagnosis in recent years, the requirements of oral cell DNA extraction kits are also increasing year by year.
However, the oral cell DNA extraction kit currently on the market has the following significant drawbacks: (1) the sampling swab is simple and crude, uses coarse cotton swab or coarse chemical fibre scraper bar usually, and sampling efficiency is not high, is difficult to judge the sample effect. (2) After sampling, the swab is easy to decay and deteriorate in the process of sending the swab to a detection unit, and subsequent operation cannot be carried out. (3) The existing kit usually requires the swab to be dried in the air for preventing the sample swab from being rotten, and the operation of the existing kit is very easy to cause sample pollution. (4) At present, the oral cell DNA extraction kit on the market can only extract 3ug of genome from one sampling at most, and can only be satisfied by sampling and extracting for multiple times if a large amount of gene operation is required subsequently. (5) No oral cell DNA extraction kit specially aiming at infants exists in the market at present, because the sampling of the oral cells of the infants is difficult, the sampling amount is extremely small, and the required gene amount is difficult to obtain if the oral cell DNA extraction kit commonly used in the market at present is used for operation.
The invention patent with the publication number of CN 1943517B discloses a kit (combined with gene biotechnology (Shanghai) limited) for oral mucosa cell self-collection, nucleic acid pre-extraction and stable storage, which is an oral cell collection and storage kit, prevents putrefaction by immediately cracking cells and inactivating proteins after sampling, and prolongs the storage time of cell samples. Although the technical scheme makes a certain breakthrough, the problems of low sampling efficiency, low yield, complex operation and the like are not solved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an oral cavity cell DNA extraction kit for infants.
The invention is realized by the following technical scheme:
in a first aspect, the present invention provides an oral cell collection swab comprising a sampling head and a swab shaft; the sampling head is arranged in the sampling tube, and the sampling head is arranged in the sampling tube; the cover body is provided with a hole matched with the outer diameter of the swab rod, and the swab rod can freely pass through the hole; the cover body comprises an inner cover and an outer cover which are hinged together, the outer diameter of the inner cover is matched with the inner diameter of the orifice of the storage tube, and the outer diameter of the outer cover is matched with the diameter of the hole.
Preferably, the material of the preservation pipe is polyethylene, and the preservation pipe has better rigidity and strength.
Preferably, the sampling head is an octagonal sheet-like structure. Further, the octagon is a long octagon, i.e., one set of opposing sides parallel to the wiper bar is longer than the other three sets of opposing sides.
Preferably, the diameter of the sampling head is 0.4-0.8 mm, which is far larger than that of a common sampling cotton swab (the diameter is 0.2mm), so that discomfort of the baby during sampling can be greatly reduced, and the sampling efficiency is improved; the diameter refers in particular to the width of the sampling head perpendicular to the direction of the swab rod.
Preferably, the surface of the sampling head is distributed with villi, the density of the villi is fine, oral cells are easily adsorbed, and the sampling success rate is increased; the length of the fluff is less than 0.5 mm.
Preferably, the sampling head is made of soft water-absorbing materials.
Preferably, the swab shaft has a length of 200mm and a diameter of 2 mm.
After sampling, the sampling head is placed in the storage tube, the outer cover is covered tightly, the swab rod is pulled out, the sampling head carries the oral cells and is left in the storage tube, and the hole on the outer cover is covered and sealed by the storage tube cover, so that sampling is finished.
In a second aspect, the invention provides an oral cell collection box, comprising the oral cell collection swab, an insulation component and a packaging component; the oral cell collection swab and the heat preservation component are packaged in the packaging component.
Preferably, the thermal insulation member comprises an ice bag for controlling temperature. Further, the ice bag is selected from common ice bags sold in the market, and the type comprises 65mm multiplied by 65 mm.
Preferably, the packaging component comprises a valve bag. Furthermore, the valve bag is specifically an aluminum foil substrate valve bag, opaque paint is coated outside the valve bag, the paint has a certain heat insulation effect, and the valve bag is a thickened heat insulation valve bag and plays an auxiliary role in heat insulation of the ice bag; the model of the self-sealing bag is 14cm multiplied by 23 cm.
After the oral cell collection box is used for collecting samples, the sampling head with the collected samples is stored in the storage tube, the storage tube and the heat-preservation ice bag are placed into the thickened heat-insulation self-sealing bag together, and the bag opening is sealed, so that the samples can be kept at-20 ℃ for at least 8 h. During this time, the sample is transported back to the test unit for examination.
The structural design of the oral cell collection swab according to the first aspect and the composition design of the oral cell collection box according to the second aspect have the following advantages:
1. the special oral cell sampling swab for the infant is provided, so that the sampling efficiency is greatly improved;
2. the sampling swab has the advantages of fast sampling process, short exposure time, no need of other tools in the whole sampling process, capability of separating the sample from redundant parts, and greatly reduced pollution
3. The integrated low-temperature preservation design prevents the sample from being rotten, a swab does not need to be dried in the air like the like products, and the possibility that microorganisms in the air pollute the sample is reduced.
In a third aspect, the invention provides an oral cavity cell DNA extraction kit, which comprises the oral cavity cell collection box and the oral cavity cell DNA extraction kit.
Preferably, the oral cell DNA extraction kit comprises a suspension Buffer, a lysis solution, Phi29DNA Polymerase (Polymerase) and a Reaction Buffer (Reaction Buffer).
Preferably, the suspension buffer and the lysis solution are stored at normal temperature.
Preferably, the suspension buffer formulation is: 0.1% Tween-20, NaCl 200mmol/L, KCl 2mmol/L, Na2HPO410mmol/L，KH2PO42mmol/L。
Preferably, the formulation of the lysis solution is: DTT 0.1M, Tris-HCl 20mM, guanidine hydrochloride 5M.
Preferably, the Phi29DNA Polymerase (Polymerase) and Reaction Buffer (Reaction Buffer) are stored at-10 ℃ to-30 ℃. Further preferably, the Phi29DNA Polymerase (Polymerase) and Reaction Buffer (Reaction Buffer) are stored at-20 ℃.
In a fourth aspect, the present invention provides a method for oral cell DNA extraction using the kit, comprising:
first, lysis of cells: adding a suspension buffer solution into a storage tube provided with a sampling head, and mixing; adding the mixed solution into a PCR tube, adding a lysis solution, mixing and heating to prepare a cell lysis solution;
second, amplifying genes: adding Phi29DNA Polymerase (Polymerase) and Reaction Buffer (Reaction Buffer) into the cell lysate, incubating to complete DNA amplification, and incubating again to inactivate enzyme.
Preferably, in the first step, a volume of suspension buffer is added to the holding tube sufficient to submerge the sampling head and ensure that the volume of liquid in the holding tube is greater than the volume of the sampling head. More preferably, the volume of the suspension buffer added is 1 to 2ml, and particularly preferably 1 ml.
Preferably, in the first step, the time for mixing after adding the suspension buffer is 5-20 s. Preferably 20 s.
Preferably, in the first step, the mixture is added to the PCR tube at an equal volume to the lysate. Preferably 5 uL.
Preferably, in the first step, the time for mixing after adding the lysis solution is 5-20 s. Preferably 20 s.
Preferably, in the first step, the heating condition is 60-75 ℃ for 1-10 min. Preferably 70 deg.C and 5 min. The higher the heating temperature, the shorter the time required, the time required for heating above 70 ℃ is less than 5min, the time required for heating at 70 ℃ is 5min, and the time required for heating at 60 ℃ is 10 min.
Preferably, in the second step, the incubation condition is 30 ℃ and 6-10 h. Preferably 8h, after 8h, the Phi29DNApolymerase enzyme enters the plateau stage basically, the replication is not carried out any more, and the product reaches the maximum concentration.
Preferably, in the second step, the further incubation is carried out at 65 ℃ for 5min to inactivate the enzyme.
The method for extracting the DNA of the oral cells has the following advantages:
1. high efficiency: the MDA technology is introduced into the product of 'extraction of baby oral cells' for the first time, and the product yield is greatly improved.
2. Time saving: the original complex method of extracting first and then amplifying needs manual operation for more than 4 hours. The two-step MDA direct amplification technology provided by the invention only needs manual operation for 20 min.
3. The original complex method of firstly extracting and then expanding needs 20 to 100 ten thousand cells to meet the extraction requirement, and the method only needs to adopt 1000 cells.
The main technology used for amplifying the gene is MDA, the key component is Phi29DNA polymerase, and in order to make the content of the invention clearer, the following is the introduction of the MDA and the Phi29DNA polymerase:
phi29DNA Polymerase is an enzyme found from a recombinant E.coli strain carrying the phage Phi29DNA Polymerase gene (AnE.coli strain which carries the Phi29DNA Polymerase gene from bacterial radiophageph 29) and capable of amplifying DNA at 30 ℃. This enzyme was found to have the ability to amplify DNA by rolling circle when amplifying circular DNA templates such as plasmid or phage DNA and was first reported by Blanco, 1984 (1984, Proc. Natl. Acad. Sci. USA, 81, 5325-5329). The enzyme was first used to amplify circular DNA, a process known as rolling circle amplification. However, it was later discovered that phi29DNA polymerase can also amplify linear DNA (referred to as multiple displacement amplification, MDA). In MDA, phi29DNA polymerase and a random primer resistant to exonuclease activity do not need to undergo a PCR thermal cycle process, and the reaction can be carried out only at a constant temperature of 30 ℃. When the template in the above reaction was replaced with human genomic DNA, it was found that a linear template could also be amplified by a strand displacement reaction. First a random 6 base oligonucleotide as a primer anneals to the genomic template DNA at multiple sites, and then phi29DNA polymerase simultaneously initiates replication at multiple sites of DNA, which synthesizes DNA along the DNA template while displacing the complementary strand of the template. The replaced complementary strand becomes a new template for amplification, and becomes an amplification system with cascade branches. Consequently, a large amount of DNA of high relative molecular mass can be obtained, and thus, this method is called Multiple Displacement Amplification (MDA).
Phi29DNA polymerase amplification has the following advantages:
1. the sample does not need to be purified: there are many types of samples that can be used for genomic DNA amplification, including fresh or frozen whole blood, tissue culture cells, buccal swab cells, inflammatory buffy coat lesions, and the like. In the multiple displacement amplification reaction, a sample does not need to be purified, can be directly used as an initial template for whole genome amplification, and can obtain a high-purity DNA product. The conventional sample processing often requires time-consuming steps, is difficult to automate, and limits the use of high throughput. The cells or whole blood for MDA reaction can be subjected to subsequent amplification only by a simple cracking step, and DNA extraction and various purification steps are omitted, so that possible pollution in the treatment process is effectively avoided, the operation is greatly simplified, and the method is particularly suitable for analysis and detection of clinical samples. 10000 times or more DNA is obtained by MDA amplification, and the purity of the DNA is higher than that of the sample DNA obtained by the traditional extraction method. This is because the efficient activity of phi29DNA polymerase ensures consistent yields of the various templates. On the other hand, if 0.05L of blood is added to the 100IxL MDA reaction system, the blood is diluted 2000 times, and some factors inhibiting the reaction, such as hemoglobin in the blood or EDTA as a sample additive, have almost negligible effect after such dilution. Therefore, the sample which is not purified has higher purity after being amplified by MDA, and can be directly used for subsequent genetic analysis research.
2. The yield is stable: the genome DNA reacts for 4-6 h at 30 ℃, and the MDA amplification is maintained at a stable level after reaching the peak, so that the DNA yield of all reactions is almost equal. In the reaction system of lOO μ L, the yield of amplified DNA was kept at about 20-30 g and was very stable regardless of the difference in the amount of the initial template (100 fg-10 ng). This consistent yield is important in some respects. Because there is no need to quantify the DNA, it allows the use of samples of different starting concentrations or quantities and provides reproducible results for downstream genetic analysis. Whereas the genomic DNA produced by traditional methods differs significantly in both yield and purity, concentrations must be re-measured and adjusted before genetic testing can be performed. And compared with other whole genome amplifications such as PEP method which generate short fragments with different sizes, MDA can amplify concentrated and uniform long fragment products. This is because the DNA polymerase from the bacteriophage phi29 binds very tightly to the DNA template and can amplify approximately 70000 bases at a time without dissociation, thus producing long fragments of DNA product averaging 12kb and up to 100kb in length in the MDA reaction. The long fragment product is beneficial to various applications, including the aspects of restriction fragment length polymorphism analysis, probe design, DNA sequencing and the like.
3. And (3) uniformly amplifying the genome: in many cases, whole genome amplification should provide as complete a genome coverage as possible with minimal site amplification error, so that the product retains the genetic sequence information of the template. Any amplification method will cause a certain sequence deviation, which results in many factors such as primer efficiency, template binding capacity, GC content, distance to telomeres and centromere, etc. Whether a whole genome amplification method is effective depends on the ability to represent the entire genome with minimal amplification bias. These PCR-based methods for WGA, such as DOP and PEP, have limited the use of these methods because of the wide range of variation between different sites. Amplification bias alters the information on the composition of the DNA sequence, making it an unreliable template for diagnostic testing. In some cases, certain regions of the genome are completely deleted, resulting in loss of alleles and occurrence of misdiagnosis. MDA can make the genome DNA amplified uniformly, thus making each locus reach a more complete coverage, ensuring the accuracy of the subsequent genetic analysis.
4. The operation is simple, and is independent of PCR reaction: the method is simple in operation, an original sample (whole blood, plasma, cells and the like) does not need to be purified, DNA is extracted through cell lysis, then the extracted DNA is added into a reaction solution of an Eppendorf tube, the reaction solution comprises dNTP, a hexanucleotide random primer, phi29DNA polymerase and enzyme flushing solution, then the reaction solution is placed under a water bath at 30 ℃ for to carry out multiple displacement amplification reaction for 4-6 h, and finally the activity of the enzyme is inactivated at 65 ℃ for 8 min. After the reaction is finished, a large number of long-fragment uniform and complete whole genome DNA sequences can be generated. Because the reaction is carried out in a water bath instead of a thermal cycler, non-specific amplification products caused by PCR reaction are avoided, and the reaction system can be prepared at will according to the proportion. The method is efficient and simple, and is very suitable for clinical genetic analysis and detection.
Although the above technology and its advantages have been disclosed by the prior art CN101619364A (method for detecting HPV and application of Phi29DNA polymerase in detecting HPV), it is a problem of current application that the DNA of oral cavity cells of infants cannot be used for reference and application, so the invention realizes excellent effect by applying the above technology in the DNA of oral cavity cells of infants, and takes a lot of creative labor.
Compared with the prior art, the invention has the following beneficial effects:
(1) the oral cell collecting swab is safe, comfortable and efficient;
(2) after the oral cell collection swab is sampled, the oral cell collection swab can continuously stop the microbial pollution at low temperature in the process of sending the oral cell collection swab to a detection unit;
(3) the operation is simple and easy, the swab does not need to be dried, and the exposure time of the swab is shortened;
(4) the yield is high, the oral cell DNA extraction kit in the current market can only extract 3ug of genome from one sampling at most, and if a large amount of gene operation is required subsequently, the extraction can be satisfied by sampling and extracting for multiple times; the invention can obtain 50ug genome under the condition of the same sampling amount;
(5) the kit is a special kit for extracting the DNA of the oral cells of the infants, and can efficiently finish the steps of sampling, storing, extracting and the like in consideration of various difficulties of sampling and amplifying the infants.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic diagram of a buccal cell collection swab; wherein 1 is a storage tube, 2 is a sampling head, 3 is a cap body, 31 is an outer cap, 32 is an inner cap, 4 is a swab rod, and 5 is a hole.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
The embodiment provides an oral cell collection swab, the structure of which is shown in figure 1 and comprises a sampling head 2 and a swab rod 4; the sampling device also comprises a storage tube 1 for accommodating the sampling head 2, and a detachable cover body 3 is arranged at the tube opening of the storage tube 1; the cover body 3 is provided with a hole 5 matched with the outer diameter of the swab rod 4, and the swab rod 4 can freely pass through the hole 5; the cover body 3 comprises an inner cover 32 and an outer cover 31 which are hinged together, the outer diameter of the inner cover 32 is matched with the inner diameter of the orifice of the preservation pipe 1, and the outer diameter of the outer cover 31 is matched with the diameter of the hole 5.
In the structure, the storage tube 1 is made of polyethylene, so that the rigidity and the strength are good; the sampling head 2 is of an octagonal sheet structure, has the diameter of 0.8mm and is made of a soft water-absorbing material; the surface of the sampling head 2 is distributed with fine villi, the length of the villi is 0.3mm, oral cells are easy to adsorb, and the sampling success rate is increased; the swab rod 4 is 200mm in length and 2mm in diameter.
In the buccal cell collection swab provided by the invention, the diameter of the sampling head can be any value within 0.4-0.8 mm, and the length of the villus is less than 0.5 mm.
After the sampling by using the oral cell collection swab provided by the embodiment is finished, the sampling head 2 is placed in the storage tube 1, the outer cover 31 is tightly covered, the swab rod 4 is pulled out, the sampling head 2 carries the oral cell sample and remains in the storage tube 1, and the hole 5 on the outer cover 31 is sealed by the inner cover 32.
Example 2
The present embodiment provides an oral cell collection kit, comprising the oral cell collection swab of embodiment 1, a heat preservation component, and an encapsulation component; the oral cell collection swab and the heat preservation component are packaged in the packaging component;
wherein the heat preservation part is a common ice bag sold in the market and is used for controlling the temperature and preventing the sample from being corroded at high temperature; the packaging part is mainly used for containing an ice bag and an oral cell collection swab, a self-sealing bag is generally adopted, and the aluminum foil substrate self-sealing bag coated with opaque paint has a certain heat insulation effect and can assist the ice bag in temperature control.
After the oral cell collection box is used for collecting samples, the storage tube containing the sampling head and the ice bag are placed into the self-sealing bag together, and the bag opening is sealed, so that the samples can be kept at-20 ℃ for at least 8 h. This time is sufficient to transport the sample back to the test unit to be examined.
Example 3
This example provides an infant oral cell DNA extraction kit: comprises the oral cell collection box and the oral cell DNA extraction kit in the embodiment 2; the kit for extracting the oral cell DNA comprises a suspension Buffer solution, a lysis solution, Phi29DNA Polymerase (Polymerase) and a Reaction Buffer solution (Reaction Buffer);
the suspension buffer solution formula is as follows: 0.1% Tween-20, NaCl 200mmol/L, KCl 2mmol/L, Na2HPO410mmol/L，KH2PO42 mmol/L; the formula of the lysis solution is as follows: DTT 0.1M, Tris-HCl 20mM, guanidine hydrochloride 5M; in general, the suspension buffer and the lysis solution are stored at normal temperature;
the Phi29DNA Polymerase (Polymerase) and the Reaction Buffer (Reaction Buffer) are preferably stored at-10 ℃ to-30 ℃ and more preferably at-20 ℃.
Example 4
This example provides a method for extracting DNA from infant oral cavity cell DNA using the kit of example 3, the whole process is as follows:
1. distributing the oral cell collection cassette portion to a subject by a sampling person; the buccal cell DNA extraction kit part was stored according to the conditions listed in example 3.
2. An ice bag in the precooled oral cell collection box is used for 10h (-precooling below 20 ℃, preferably-30 ℃) and the baby of 4 months age is sampled.
Scraping the sampling head 2 on the cavity walls of the two sides of the baby mouth for 1-5 times (preferably 5 times or more), putting the sampling head 2 into the storage tube 1, tightly covering the outer cover 31, pulling out the swab rod 4, and tightly covering the inner cover 32. The sampling time is about 5min (preferably within 2 min).
3. Placing the preservation tube 1 provided with the sampling head 2 and a heat-preservation ice bag into a thickened heat-insulation self-sealing bag, sealing the bag opening, conveying the sample to a laboratory, standing at normal temperature for 8-14 h (preferably conveying the sample to a detection laboratory within 8h), and extracting DNA by using an oral cavity cell DNA extraction kit; if the operation cannot be performed immediately, the tube is placed in a freezing layer at-20 deg.C in a refrigerator for storage for one month (preferably within 2 weeks for extraction)
4. After receiving a sample in a laboratory, adding 1mL of suspension buffer solution into a storage tube 1 provided with a sampling head 2, and mixing for 10 s; taking out 5uL, adding 5uL of lysate, mixing for 10s, heating at 70 ℃ for 1-10 min (preferably 5min), and preparing the cell lysate.
5. Adding Phi29DNA Polymerase and reaction buffer solution into 10uL cell lysate, and incubating for 6-10 h (preferably 8h) at 30 ℃ to complete the amplification reaction. Thereafter, the product was incubated at 65 ℃ for 5min to inactivate the enzyme.
6. The concentration of the detected product is 1800ng/uL, and the total concentration is 50 uL. Thus, 90ug of genomic DNA was obtained.
Compared with the prior art, the extraction method provided by the embodiment has the following beneficial effects: (1) the oral cell collecting swab is safe, comfortable and efficient; (2) after the oral cell collection swab is sampled, the oral cell collection swab can continuously stop the microbial pollution at low temperature in the process of sending the oral cell collection swab to a detection unit; (3) the operation is simple and easy, the swab does not need to be dried, and the exposure time of the swab is shortened; (4) the yield is high, the oral cell DNA extraction kit in the current market can only extract 3ug of genome from one sampling at most, and if a large amount of gene operation is required subsequently, the extraction can be satisfied by sampling and extracting for multiple times; the invention can obtain 50ug genome under the condition of the same sampling amount; (5) the kit is a special kit for extracting the DNA of the oral cells of the infants, and can efficiently finish the steps of sampling, storing, extracting and the like in consideration of various difficulties of sampling and amplifying the infants.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. An oral cell collection swab is characterized by comprising a sampling head (2) and a swab rod (4); the sampling device also comprises a storage tube (1) for accommodating the sampling head (2), and a detachable cover body (3) is arranged at the tube opening of the storage tube (1); the cover body (3) is provided with a hole (5) matched with the outer diameter of the swab rod (4), and the swab rod (4) can freely pass through the hole (5); the cover body (3) comprises an inner cover (32) and an outer cover (31) which are hinged together, the outer diameter of the inner cover (32) is matched with the inner diameter of the orifice of the preservation pipe (1), and the outer diameter of the outer cover (31) is matched with the diameter of the hole (5); the sampling head is of an octagonal sheet structure, the octagon is a long octagon, and the length of one group of parallel opposite sides of the swab rod is greater than that of the other three groups of opposite sides; the diameter of the sampling head is 0.4-0.8 mm.
2. An oral cell collection cartridge comprising the oral cell collection swab of claim 1, an incubation component, and an encapsulation component; the oral cavity cell collecting swab and the heat preservation component are packaged in the packaging component.
3. An oral cell DNA extraction kit, comprising the oral cell collection kit according to claim 2 and an oral cell DNA extraction kit.
4. The kit for extracting DNA of oral cells according to claim 3, wherein the kit for extracting DNA of oral cells comprises a suspension buffer, a lysate, Phi29DNA polymerase and a reaction buffer.
5. A method for extracting DNA from oral cells by using the kit of claim 4, comprising:
first, lysis of cells: adding a suspension buffer solution into a storage tube provided with a sampling head, and mixing; adding the mixed solution into a PCR tube, adding a lysis solution, mixing and heating to prepare a cell lysis solution;
second, amplifying genes: adding Phi29DNA polymerase and reaction buffer solution into the cell lysate, incubating to complete DNA amplification, and incubating again to inactivate enzyme.
6. The method for extracting DNA from buccal cells as claimed in claim 5, wherein the time for mixing after adding the suspension buffer in the first step is 5-20 s.
7. The method for extracting DNA from buccal cells as claimed in claim 5, wherein the time for mixing after adding the lysis solution in the first step is 5-20 s.
8. The method for extracting DNA from buccal cells according to claim 5, wherein the heating condition in the first step is 60-75 ℃ for 5-10 min.
9. The method for extracting DNA from buccal cells as claimed in claim 5, wherein the incubation condition in the second step is 30 ℃ for 6-10 h.
10. The method for extracting DNA from buccal cells as claimed in claim 5, wherein the second step, the incubation condition is 65 ℃ for 5 min.
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| GB2572359A (en) * | 2018-03-27 | 2019-10-02 | Ge Healthcare Uk Ltd | Method and device for collecting, preserving and transferring samples |
| CN109735437B (en) * | 2019-01-28 | 2022-04-19 | 长春长光辰英生物科学仪器有限公司 | Vessel and method for collecting and processing cells after ejection sorting of cells |
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| CN1943517B (en) * | 2005-10-08 | 2010-11-24 | 联合基因生物科技(上海)有限公司 | Reagent kid for self collecting oral mucosa cell and nucleic acid pre-extracting and stablizing storage |
| CN101619364A (en) * | 2008-07-04 | 2010-01-06 | 上海市免疫学研究所 | Method for detecting HPV and application of Phi29DNA polymerase in detecting HPV |
| CN203153809U (en) * | 2013-02-05 | 2013-08-28 | 益善生物技术股份有限公司 | Sampling device |
| CN203473560U (en) * | 2013-09-30 | 2014-03-12 | 崔浩宇 | Sampling and thermal-insulating device for food hygiene inspection |
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Effective date of registration: 20231226 Address after: Building 5, No. 1366 Hongfeng Road, Huzhou City, Zhejiang Province, 313000, 1005-25 Patentee after: Kenabis Pharmaceutical Co.,Ltd. Address before: Room 808, Building C, No. 1888 Daishan Road, Wuxing District, Huzhou City, Zhejiang Province, 313000 Patentee before: ZHEJIANG SENGE BIOTECHNOLOGY CO.,LTD. |