CN113171136A - Pretreatment method and kit for direct PCR amplification of oral exfoliated cells - Google Patents
Pretreatment method and kit for direct PCR amplification of oral exfoliated cells Download PDFInfo
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- CN113171136A CN113171136A CN202110401322.7A CN202110401322A CN113171136A CN 113171136 A CN113171136 A CN 113171136A CN 202110401322 A CN202110401322 A CN 202110401322A CN 113171136 A CN113171136 A CN 113171136A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/02—Instruments for taking cell samples or for biopsy
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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
- C12Q1/6851—Quantitative amplification
Abstract
The invention provides a method for directly carrying out PCR amplification on an oral cavity sample, which comprises the following steps: (1) collecting a sample with an oral sampling swab; and (2) directly performing PCR amplification using the obtained sample as a template; the oral sampling swab comprises a handle and a sampling swab head, the sampling swab head comprising a sampling head body, and protrusions and/or depressions on the sampling head body for increasing surface area, wherein the sampling head body and the protrusions and/or depressions are not covered with other materials on the exterior.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a pretreatment method and a kit for directly carrying out PCR amplification on oral exfoliated cells.
Background
For molecular diagnostics, the most important isA commonly used clinical detection method is the fluorescent quantitative PCR method. PCR (Polymerase Chain Reaction) is a technique for amplifying and amplifying specific DNA (deoxyribonucleic acid) fragments in vitro. PCR generally needs three steps of high-temperature melting reaction, annealing reaction and extension reaction when amplifying DNA, and the number of initial DNA molecules is doubled after the three steps, namely, one cycle is adopted; the multiplied DNA molecules become the template of the next cycle, the cycle is multiplied, and after 30-40 cycles, the number of DNA molecules is amplified to be about 10 of the initial value9-1010And (4) doubling. Thus, PCR can specifically amplify a DNA molecule as an analyte on a large scale for the purpose of analysis and examination, and thus PCR technology can be used for early diagnosis of infectious diseases, genetic diseases, tumors, and the like, and is increasingly used for prenatal examination and forensic identification. Molecular detection methods based on PCR technology typically require sample processing, nucleic acid extraction and PCR reaction system construction, PCR amplification reactions, and signal detection.
The fluorescent quantitative PCR method is generally classified into a probe method and a dye method. The probe method is characterized in that a pair of primers is added and a specific fluorescent probe is added at the same time when PCR amplification is carried out, the probe is an oligonucleotide, and two ends of the oligonucleotide are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; initially, the probe is bound to any single strand of DNA; during PCR amplification, the 5 '-3' -exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the fluorescent signal accumulation and the PCR product formation are completely synchronous. The dye method uses a fluorescent dye capable of binding to nucleic acid. The dyes can be combined on double-stranded DNA, when a template in a system is amplified, the fluorescent dyes can be effectively combined on newly synthesized double strands, along with the progress of PCR, more and more combined fluorescent dyes are combined, and fluorescent signals detected by an instrument are stronger and stronger, so that the quantitative purpose is achieved.
The PCR reaction needs a template, and the sample is obtained after the sample is extracted by a conventional oral exfoliated cell template. Most of the extraction processes of the samples are complicated and time-consuming, and researchers can realize simple extraction through formula adjustment. However, the simple extraction cannot completely remove impurities that inhibit the PCR reaction in the sample, and thus the detection rate is low.
In addition, for the sampling tools frequently used at present, such as cotton swabs, flocked swabs and the like, a large amount of useless protein and impurities can be collected in the sampling process, and the quality of a sample is influenced. If such a sampling tool is used, at least a simple sample handling process is required due to excessive sample contamination.
Therefore, there is still a great need for more efficient sampling tools and simpler and easier methods for performing PCR assays.
Disclosure of Invention
In a first aspect, the present invention provides a method of oral sample collection, the method comprising sample collection using an oral sampling swab comprising a handle and a sampling swab head, the sampling swab head comprising a sampling head body, and projections and/or depressions on the sampling head body for increasing surface area, wherein the sampling head body and the projections and/or depressions are not covered by other materials on the exterior.
In a second aspect, the present invention provides a method for directly performing PCR amplification on an oral sample, the method comprising: (1) collecting a sample with an oral sampling swab; and (2) directly performing PCR amplification using the obtained sample as a template; wherein the oral sampling swab comprises a handle and a sampling swab head, the sampling swab head comprising a sampling head body, and protrusions and/or depressions on the sampling head body for increasing surface area, wherein the exterior of the sampling head body and the protrusions and/or depressions are not covered with other materials.
In a third aspect, the invention provides a kit capable of collecting an oral sample and performing PCR amplification directly, the kit comprising an oral sampling swab, and an enzyme, a buffer, dntps, a primer and a probe for PCR amplification, wherein the oral sampling swab comprises a handle and a sampling swab head, the sampling swab head comprises a sampling head body, and protrusions and/or depressions on the sampling head body for increasing the surface area, wherein the exterior of the sampling head body and the protrusions and/or depressions are not covered with other materials.
The method has the advantages that the oral cavity sample is collected by using the oral cavity sampling swab, PCR amplification can be directly carried out, so that a complex and lengthy DNA or RNA extraction process is avoided, and consumables for sample preservation and extraction are saved. In addition, all PCR amplification reagents used in the invention can be stored at normal temperature, and refrigeration and freezing are not required.
Drawings
Fig. 1 is a schematic view of an oral sampling swab according to the present invention, wherein (a), (b), (c), and (d) respectively show specific examples of the oral sampling swab according to the present invention.
Fig. 2 is a partially enlarged schematic view of the swab head of the oral sampling swab of the present invention, wherein (a) (b) (c) (d) (e) (f) (g) (h) (i) respectively show specific examples of the swab head of the oral sampling swab of the present invention.
FIG. 3A shows an amplification curve obtained by directly performing fluorescent quantitative PCR amplification on an oral sample collected by using the oral sampling swab of the present invention shown in FIG. 2 (e).
FIG. 3B shows an amplification curve obtained by directly performing fluorescent quantitative PCR amplification after collecting an oral sample using the oral sampling swab of the present invention shown in FIG. 2 (c).
FIG. 3C shows an amplification curve obtained by directly performing fluorescent quantitative PCR amplification after collecting an oral sample using the oral sampling swab of the present invention shown in FIG. 2 (d).
FIG. 3D shows an amplification curve obtained by directly performing fluorescent quantitative PCR amplification after collecting an oral sample using the oral sampling swab of the present invention shown in FIG. 2 (f).
FIG. 3E shows an amplification curve obtained by directly performing fluorescent quantitative PCR amplification on an oral sample collected by using the oral sampling swab of the present invention shown in FIG. 2 (g).
FIG. 3F shows an amplification curve obtained by directly performing fluorescent quantitative PCR amplification after collecting an oral sample using the oral sampling swab of the present invention shown in FIG. 2 (h).
FIG. 3G shows an amplification curve obtained by directly performing fluorescent quantitative PCR amplification after collecting an oral sample using the oral sampling swab of the present invention shown in FIG. 2 (i).
FIG. 4 shows an amplification curve obtained by direct fluorescent quantitative PCR amplification after collection of an oral sample using a conventional mouth swab stick.
FIG. 5 shows an amplification curve obtained by using a conventional mouth swab sampling stick to collect an oral sample, extracting DNA from the sample using a conventional kit, and then performing fluorescent quantitative PCR amplification.
Detailed Description
The invention provides a method of oral sample collection, the method comprising sample collection using an oral sampling swab comprising a handle and a sampling swab head, the sampling swab head comprising a sampling head body, and protrusions and/or depressions on the sampling head body for increasing surface area, wherein the sampling head body and the protrusions and/or depressions are not covered by other materials on the exterior.
Preferably, the material of the sampling head body and the projections and/or recesses may be polypropylene, polystyrene, nylon, or resin.
In a specific embodiment, the sampling head body is in the shape of a flat cuboid, and 4-25 protrusions, such as 9 protrusions, 12 protrusions, 16 protrusions, etc., are distributed on a horizontal plane of the sampling head body. In particular, the 9 protrusions are distributed in a 3 × 3 array, the 12 protrusions are distributed in a 3 × 4 array, and the 16 protrusions are distributed in a 4 × 4 array.
In another specific embodiment, the sampling head body is in the shape of a flat cuboid, and 4-25 protrusions, such as 9 protrusions, 12 protrusions, 16 protrusions and the like, are distributed on a horizontal plane of the sampling head body, and each protrusion is provided with a recess. In particular, the 9 protrusions are distributed in a 3 × 3 array, the 12 protrusions are distributed in a 3 × 4 array, and the 16 protrusions are distributed in a 4 × 4 array.
In another embodiment, the sampling head body is in the shape of a cylinder, and 32-128 protrusions, such as 45 protrusions, 72 protrusions, 98 protrusions, etc., are distributed on the side surface of the sampling head body. In particular, the 45 protrusions are distributed in a 5 × 9 array, the 72 protrusions are distributed in a 6 × 12 array, and the 98 protrusions are distributed in a 7 × 14 array.
In another specific embodiment, the sampling head body is in the shape of a cylinder, and 32 to 128 protrusions, such as 45 protrusions, 72 protrusions, 98 protrusions and the like, are distributed on the side surface of the sampling head body, and each protrusion is provided with a recess. In particular, the 45 protrusions are distributed in a 5 × 9 array, the 72 protrusions are distributed in a 6 × 12 array, and the 98 protrusions are distributed in a 7 × 14 array.
In yet another embodiment, the sampling head body is in the shape of a rectangular parallelepiped with a depression in a horizontal plane. In particular, one side of the recess is rectangular, trapezoidal or triangular.
The invention also provides a method for directly carrying out PCR amplification on an oral cavity sample, which comprises the following steps:
(1) collecting a sample with an oral sampling swab; and
(2) directly carrying out PCR amplification by taking the obtained sample as a template;
wherein the oral sampling swab comprises a handle and a sampling swab head, the sampling swab head comprising a sampling head body, and protrusions and/or depressions on the sampling head body for increasing surface area, wherein the exterior of the sampling head body and the protrusions and/or depressions are not covered with other materials.
In a specific embodiment, the PCR is a fluorescent quantitative PCR.
In a particular embodiment, step (2) may be accomplished by: putting the oral sampling swab containing the sample into pure water or buffer solution for rinsing; adding the rinsed liquid into a reaction tube containing a freeze-dried PCR reagent, and uniformly mixing the rinsed liquid and the reaction tube in a shaking way; the resulting reaction tube was placed on an instrument for PCR amplification. Preferably, the buffer solution is TE buffer solution or Tris-HCl buffer solution; the lyophilized PCR reagents comprise enzymes, buffers, dntps, primers and probes for PCR amplification.
The invention also provides a kit capable of collecting an oral sample and directly performing PCR amplification, which comprises an oral sampling swab, and an enzyme, a buffer solution, dNTP, a primer and a probe for PCR amplification, wherein the oral sampling swab comprises a handle and a sampling swab head, the sampling swab head comprises a sampling head body, and a bulge and/or a depression for increasing the surface area on the sampling head body, and the appearance of the sampling head body and the bulge and/or the depression is not covered by other materials.
Preferably, the PCR is a fluorescent quantitative PCR.
Preferably, the enzyme, buffer, dNTP, primer and probe used for PCR amplification are a mixture of lyophilized enzyme, buffer, dNTP, primer and probe.
The following examples are intended to illustrate the invention without limiting it.
Example 1 oral samples were collected using an oral sampling swab of the invention and directly subjected to PCR
1. Test materials and reagents
Note: no. 1 corresponds to fig. 2(e), No. 2 corresponds to fig. 2(c), No. 3 corresponds to fig. 2(d), No. 4 corresponds to fig. 2(f), No. 5 corresponds to fig. 2(g), No. 6 corresponds to fig. 2(h), and No. 7 corresponds to fig. 2 (i).
2. Experimental procedures and results
The target fragments amplified in the PCR experiment are ADH1B and ALDH2 genes.
The detailed information of the primer probe in the PCR reaction system is shown in the following table:
(1) sample collection
Preparing 3 1.5ml centrifuge tubes, and adding 50 mul of pure water into the tubes; using 3 oral sampling swabs of the invention to respectively scrape 3 persons under the inner walls of the oral cavities at the left and right sides; one end of a sampling swab head of each of 3 oral sampling swabs is respectively placed into 3 1.5ml centrifugal tubes, the sampling swab heads are immersed in purified water, the oral sampling swabs are taken out and discarded under the condition of stirring, and 1.5ml centrifugal tube covers are covered to respectively mark a first tube and a second tube to finish sampling.
(2) Sample application
Respectively adding 40 mu l of liquid in the third sample tube into a PCR reaction tube containing a freeze-drying reagent; and covering the tube cover, marking and completing sample adding.
(3) Amplification of
The 3 tubes of labeled reaction tubes were placed on the instrument and amplification was performed according to the following pre-set procedure. Results for the oral sampling swabs of the present invention, Nos. 1-7, are shown in FIGS. 3A-3G, respectively. According to the experimental result, the method is simple and convenient to operate, the PCR amplification has an obvious S-shaped curve, and the CT value is less than or equal to 35.
Example 2 oral samples were collected using an existing mouth swab sampling wand and then directly subjected to PCR
1. Test materials and reagents
2. Experimental procedures and results
The target gene amplified in this example, the primers and the probes used were the same as those in example 1.
(1) Sample collection
Preparing 3 centrifugal tubes with the volume of 1.5mL, and adding 500 mu L of purified water into the centrifugal tubes; scraping the inner walls of the oral cavities of the left side and the right side of 3 persons by using 3 mouth swab sampling rods; one end of a sampling head of 3 sampling rods is respectively placed into 3 1.5mL centrifugal tubes, the sampling heads are immersed in purified water, the sampling rods are taken out and discarded under the condition of stirring, and 1.5mL centrifugal tube covers are covered to respectively mark a first tube and a second tube, so that sampling is completed.
(2) Sample application
Respectively adding 40 mu L of liquid in the third sample tube into a PCR reaction tube containing a freeze-drying reagent; and covering the tube cover, marking and completing sample adding.
(3) Amplification of
The 3 tubes of labeled reaction tubes were placed on the instrument and amplification was performed according to the following pre-set procedure, the results of which are shown in FIG. 4.
According to the experimental result, the existing mouth swab sampling rod is used for collecting the oral cavity sample, and then the fluorescence quantitative PCR is directly carried out, so that the obtained amplification S-shaped curve is not obvious, and part of Ct values cannot be interpreted. The results of the experiment were not good.
Example 3 oral samples were collected using an existing mouth swab sampling wand and sample DNA extraction followed by PCR using a conventional kit
1. Test materials and reagents
2. Experimental procedures and results
The target gene amplified in this example, the primers and the probes used were the same as those in example 1.
(1) Sample collection
Preparing 3 centrifugal tubes with the volume of 1.5mL, and adding 500 mu L of purified water into the centrifugal tubes; scraping the inner walls of the oral cavities of the left side and the right side of 3 persons by using 3 mouth swab sampling rods; put 3 sampling head one end of mouth swab sampling stick into 3 respectively in 1.5mL centrifugal tubes, the sampling head submergence is in purified water, stirs under the number, takes out the sampling stick and abandons, covers 1.5mL centrifugal tube and covers and mark (r) No. pipe respectively, accomplishes the sampling.
(2) Sample extraction
DNA extraction was performed on 3 specimens using the Tiangen DP-362 kit.
(3) Sample application
Respectively adding the extracted third sample into a PCR reaction tube containing a freeze-drying reagent; and covering a tube cover on each tube by 40 mu L, and marking to finish sample adding.
(4) Amplification of
3 tubes of labeled reaction tubes were placed on the instrument and amplification was performed according to the following procedure, the results of which are shown in FIG. 5.
According to the experimental result, after the sample is collected by adopting the existing mouth swab sampling rod, the sample is extracted by using the conventional kit, and then the fluorescent quantitative PCR amplification is carried out, so that the obvious S-shaped curve amplification is obtained, the Ct is less than or equal to 35, and the result is better. However, this process involves a sample extraction step, which is cumbersome and complicated.
Claims (14)
1. A method of oral sample collection comprising sample collection using an oral sampling swab comprising a handle and a sampling swab head, the sampling swab head comprising a sampling head body, and protrusions and/or depressions on the sampling head body for increasing surface area, wherein the sampling head body and the protrusions and/or depressions are not covered by other materials on the exterior.
2. The method of claim 1, wherein the material of the sampling head body and the projections and/or recesses itself is a polypropylene material, a polystyrene material, nylon, resin.
3. The method of claim 1 or 2, wherein the sampling head body is in the shape of a flat cuboid, wherein 9 protrusions are distributed on one horizontal plane, and the 9 protrusions are distributed in a 3 x 3 array; or 12 bulges are distributed on one horizontal plane of the device, and the 12 bulges are distributed in a 3 multiplied by 4 array; or 16 bulges are distributed on one horizontal plane of the base plate, and the 16 bulges are distributed in a 4 multiplied by 4 array.
4. The method of claim 3, wherein each of said protrusions is provided with a depression therein.
5. The method of claim 1 or 2, wherein the sampling head body is in the shape of a cylinder with 45 protrusions distributed on its side, the 45 protrusions being distributed in a 5 x 9 array; or 72 bulges are distributed on the side surface of the base plate, and the 72 bulges are distributed in a 6 multiplied by 12 array; or 98 bulges are distributed on the side surface of the base plate, and the 98 bulges are distributed in a 7 multiplied by 14 array.
6. The method of claim 5, wherein each of said protrusions is provided with a depression therein.
7. The method of claim 1 or 2, wherein the sampling head body is shaped as a rectangular parallelepiped with a depression in a horizontal plane, one side of the depression being rectangular, trapezoidal or triangular.
8. A method of directly performing PCR amplification on an oral sample, the method comprising:
(1) collecting a sample with an oral sampling swab; and
(2) directly carrying out PCR amplification by taking the obtained sample as a template;
wherein the oral sampling swab is an oral sampling swab for use in the method of any one of claims 1-5.
9. The method of claim 8, wherein the PCR is fluorescent quantitative PCR.
10. The method of claim 8 or 9, wherein step (2) is accomplished by: putting the oral sampling swab containing the sample into pure water or buffer solution for rinsing; adding the rinsed liquid into a reaction tube containing a freeze-dried PCR reagent, and uniformly mixing the rinsed liquid and the reaction tube in a shaking way; the resulting reaction tube was placed on an instrument for PCR amplification.
11. The method of claim 10, wherein the buffer solution is a TE buffer or a Tris-HCl buffer; the lyophilized PCR reagents comprise enzymes, buffers, dntps, primers and probes for PCR amplification.
12. A kit for collecting an oral sample and performing PCR amplification directly, the kit comprising an oral sampling swab and enzymes, buffers, dntps, primers and probes for PCR amplification, wherein the oral sampling swab is used in the method of any one of claims 1-5.
13. The kit of claim 12, wherein the PCR is a fluorescent quantitative PCR.
14. The kit of claim 12, wherein the enzymes, buffers, dntps, primers and probes used for PCR amplification are a mixture of lyophilized enzymes, buffers, dntps, primers and probes.
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| CN202110401322.7A CN113171136A (en) | 2021-04-14 | 2021-04-14 | Pretreatment method and kit for direct PCR amplification of oral exfoliated cells |
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| CN202110401322.7A CN113171136A (en) | 2021-04-14 | 2021-04-14 | Pretreatment method and kit for direct PCR amplification of oral exfoliated cells |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402239A (en) * | 2018-12-29 | 2019-03-01 | 贝南生物科技（厦门）有限公司 | A kind of hands-free cut-off for real-time fluorescence quantitative PCR connects amplifing reagent and its application |
| CN111839606A (en) * | 2020-08-14 | 2020-10-30 | 广州市疾病预防控制中心(广州市卫生检验中心、广州市食品安全风险监测与评估中心、广州医科大学公共卫生研究院) | Sampling swab |
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- 2021-04-14 CN CN202110401322.7A patent/CN113171136A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402239A (en) * | 2018-12-29 | 2019-03-01 | 贝南生物科技（厦门）有限公司 | A kind of hands-free cut-off for real-time fluorescence quantitative PCR connects amplifing reagent and its application |
| CN111839606A (en) * | 2020-08-14 | 2020-10-30 | 广州市疾病预防控制中心(广州市卫生检验中心、广州市食品安全风险监测与评估中心、广州医科大学公共卫生研究院) | Sampling swab |
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