CN111020001A - Novel saliva preserving fluid and preparation method and application thereof - Google Patents
Novel saliva preserving fluid and preparation method and application thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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Abstract
The invention relates to a novel saliva preservation solution which comprises the following components: EDTA 1-10mmol/L, Tris-HCl 1-50mmol/L, sodium chloride 10-100mmol/L, sodium sorbate 0.1-3% (w/v), sodium diacetate 0.1-3% (w/v), proclin 3000.01-0.1% (v/v), TWEEN 200.1-1% (v/v), NP 400.1-1% (w/v), SDS 0.1-2% (w/v); the saliva preservation solution disclosed by the invention is used for preserving a saliva sample, can effectively inhibit the activity of nuclease, prevents microbial contamination, ensures the integrity and purity of DNA fragments, simplifies the pretreatment step, is suitable for being used in a centrifuge-free and low-temperature environment, can ensure the transportation stability of the sample among various places, and is suitable for large-scale population investigation and research in molecular epidemiology.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a novel saliva preserving fluid composition, and a preparation method, a production method and a use method thereof, wherein the novel saliva preserving fluid composition is suitable for transportation and storage after saliva collection, and is only used for in vitro detection subsequently.
Background
With the progress of society, the application of nucleic acid DNA in the fields of gene detection, disease detection and treatment, forensic identification, etc. is more and more extensive. Usually, the source of the sample DNA for human genetic analysis is blood, tissue, or body fluid. Nucleic acid DNA for PCR amplification and detection is commonly derived from blood samples. The collection of blood samples requires professional skills to complete and causes some trauma to the human body. Blood clots are caused by factors such as overlong blood sample storage time, and inconvenience in extraction is also caused, and a noninvasive, simple, convenient and rapid examination and diagnosis method is generally concerned by researchers.
Compared with blood samples, saliva is safe and convenient to collect, has no wound, has no risk of spreading blood-borne diseases, and patients have no pain to accept, so that the sampling range of gene research can be enlarged to the maximum extent, and the method is particularly suitable for large-scale population investigation of molecular epidemiology.
Due to the presence of many kinds of microorganisms and impurities such as lipid, polysaccharide, mucin and the like in saliva, a large amount of microorganisms are generally grown to cause the decrease in the proportion of human genetic genes and the degradation thereof as the preservation time of a sample increases. On the other hand, improper storage of the solution will cause degradation of the DNA and inhibition of downstream detection.
Generally, saliva preservation solution on the market needs to centrifugally collect saliva cells and then lysate is used for cell lysis, a centrifuge is needed, and pretreatment is complicated. The saliva preservation solution is sprayed on the bottom of the collection tube in advance and dried, the collected saliva sample is cracked in the transportation process, the pretreatment step is simplified, and the saliva preservation solution is suitable for the environment without a centrifuge and at low temperature and can ensure the transportation stability of the sample among various places.
Disclosure of Invention
The invention uses the saliva preservation solution to preserve the saliva sample, cracks cells in the transportation process, inhibits the pollution of microorganisms and protects the integrity of genome DNA.
The invention adopts the following technical scheme: EDTA 1-10mmol/L, Tris-HCl 1-50mmol/L, sodium chloride 10-100mmol/L, sodium sorbate 0.1-3% (w/v), sodium diacetate 0.1-3% (w/v), proclin 3000.01-0.1% (v/v), TWEEN 200.1-1% (v/v), NP 400.1-1% (w/v), SDS 0.1-2% (w/v), and the pH range of the system is 7-8.
The saliva preservation and preparation method specifically comprises the following steps:
(1) taking the raw materials in the proportion;
(2) respectively preparing the EDTA, Tris-HCl and SDS weighed in the step (1) into solutions and storing the solutions; wherein the pH is 6-8.5 Tris-HCl buffer solution.
(3) According to the total volume of the prepared saliva preservation solution and the content requirement of each component in the system, selecting an EDTA solution with a corresponding volume, a Tris-HCl buffer solution with the pH of 6-8.5 and a water storage solution of SDS, adding corresponding amounts of sodium sorbate, sodium diacetate, sodium chloride, TWEEN20, NP40 and proclin300, adding sterile water to the total volume of the prepared saliva preservation solution, and performing sterilization by adopting a microporous filter membrane filtration mode to obtain the saliva preservation solution, wherein the preservation temperature of the preservation solution is 15-25 ℃.
According to the scheme, the pH value of the system in the step (3) can be adjusted to be in a range of 7-8 by adding HCl or NaOH when needed, wherein the addition amount of HCl or NaOH is small, and the influence on the concentration of each component in the system is negligible.
The method for preserving, producing and using the saliva specifically comprises the following steps:
during production, the saliva preservation solution is stored at the bottom of the saliva collection tube by quantitatively adding the saliva preservation solution into a medicament quantitative sprayer (the model HY100BCA-T, the Macro and profound precision machinery technology in Liuyang city) in advance and spraying at high pressure, after the sample addition is finished, the saliva preservation solution is quickly dried at high pressure by a PTC clean dust-free dryer (PTC clean dust-free dryer, HY100DG-T2) at room temperature (15-25 ℃), and the liquid saliva preservation solution is converted into a solid saliva preservation agent and is enriched at the bottom of the saliva collection tube.
The application method of the saliva preserving fluid of the invention is as follows: mixing 1.5-2.5ml saliva with saliva preserving solution, and preserving at normal temperature.
Mg in EDTA chelate sample in saliva preservation solution2+、Ca2+Divalent cations are equal, so that the activity of nuclease is inhibited, and the integrity of genome DNA fragments is ensured; SDS and TWEEN20 can crack cell membrane and effectively crack protein, lipid and other impurities; the Tris-HCl buffer solution ensures the stable alkalescent environment of the DNA; more mainly proclin300, has a very good broad-spectrum antibacterial system, effectively inhibits bacteria, and better prevents the pollution of microorganisms, thereby achieving betterRequirements for preservation of saliva samples.
The saliva preservation solution of the invention protects the integrity of genomic DNA, inhibits the growth of bacteria, simplifies the pretreatment steps and meets various complex PCR experiments.
Compared with the prior art, the invention has the following beneficial effects because the technology is adopted:
the saliva preservation solution disclosed by the invention is used for preserving saliva samples, is low in cost and convenient to prepare, can effectively inhibit the activity of nuclease, prevents microbial pollution, ensures the integrity of DNA fragments, simplifies the pretreatment steps, is suitable for being used in a centrifuge-free and low-temperature environment, can ensure the transportation stability of the samples in various places, and is suitable for large-scale population investigation and research in molecular epidemiology. Compared with liquid saliva preservative liquid, the solid saliva preservative is more beneficial to the health and safety of users, and effectively prevents children from eating the liquid saliva preservative by mistake.
The results of the related saliva preservation and the genome DNA extraction show that the saliva preservation solution can be effectively preserved for more than 3 months at room temperature.
Drawings
FIG. 1 is an image of DNA gel after saliva samples are preserved for 0 days (1-5) and 30 days (6-10) by the saliva preserving fluid of the present invention.
Detailed Description
The invention is further elucidated with reference to the drawings and the detailed description.
Example 1:
the saliva preserving fluid comprises the following components in percentage by weight: EDTA 10mmol/L, Tris-HCl 20mmol/L, sodium chloride 100mmol/L, sodium diacetate 1% (w/v), proclin 3000.01% (v/v), TWEEN 201% (w/v), SDS 1% (w/v), system pH 8.
The preparation method comprises the following steps:
(1) respectively preparing 0.1mol/L EDTA, Tris-HCl buffer solution with the pH value of 8 and 20% SDS (w/v) storage aqueous solution, and storing at room temperature;
(2) according to the total volume of the prepared saliva for preservation, the components in the system are mixed according to the content requirement of the components in the system according to the following proportion: 10ml of 0.1mol/L EDTA aqueous solution; 20ml of 0.1mol/L Tris-HCl (PH 8) buffer; 5ml of 20% SDS (w/v) aqueous solution; TWEEN 201 ml; proclin 3000.1ml; adding 1g of sodium diacetate and 0.58g of sodium chloride, adding sterile water to 100ml, filtering and sterilizing by a microporous filter membrane, and storing at 15-25 ℃ for later use.
The production and use method of the saliva preservation solution comprises the following steps:
quantitatively adding 200ul of saliva preservation solution into a quantitative sprayer (Hongyuan mechanical science and technology, model HY100BCA-T) in advance, spraying at high pressure (0.3Mpa) to store at the bottom of the saliva collection tube, and rapidly drying with a PTC clean dust-free dryer (PTC clean dust-free dryer, HY100DG-T2) at room temperature (15-25 deg.C) under high pressure after sample addition.
The specific method for testing the preservation effect of the saliva preservation solution comprises the following steps:
before saliva collection, subjects rinse their mouths sufficiently 30 minutes in advance until they do not eat, drink/drink, smoke, chew gum, brush their teeth or apply lipstick before collection. When collecting saliva, the tongue and the two cheek are scraped lightly with teeth for 10 times, and the attention is moderate. The saliva was then spitted a small number of times into a collection funnel and flowed along the inner wall into the collection tube to a level (non-foaming) of saliva about to the 2ml collection line. When the amount of saliva is small, the tongue may be used against the palate or rotated in the mouth to promote saliva secretion. The collection funnel is unscrewed and the collection tube cap is screwed down. The collection tube was inverted 5 times to mix the sample and the bottom dose.
Example 2
(1) The saliva sample preservation solution of the invention produced in example 1 was used for stability testing experiments, 2.5ml of saliva was collected and mixed by inversion, and saliva samples of 5 subjects were collected for use.
(2) After the saliva sample stored in the saliva storage solution is placed for 0 day, 7 days, 14 days, 21 days and 30 days at room temperature, 450ul of the saliva sample is taken, Nanjing Shenyou nucleic acid extraction and purification kit (magnetic bead method) -type I is used, and the genome DNA of the saliva sample is extracted, wherein the total volume of the DNA is 80 ul. 3ul samples of saliva DNA placed for 0 day and 30 days were applied to a 1.0% agarose gel electrophoresis for detection and labeled with a DL15000Marker, as shown in FIG. 1 (samples 1-5 are saliva sample DNA placed for 0 day, and samples 6-10 are saliva sample DNA placed for 30 days); taking 2ul of saliva DNA, and detecting the concentration (unit is ng/ul) of the saliva DNA by using a Qubit, which is specifically shown in a table 1; 2ul of saliva DNA was taken and tested for purity by NanoDrop, see Table 2. The data in table 1 show that the concentration of saliva samples after being placed for 0 day, 7 days, 14 days, 21 days and 30 days at room temperature has small change degree, which indicates that the saliva preservation solution of the present invention effectively protects genomic DNA, and the electrophoresis chart in fig. 1 indicates that the saliva preservation solution of the present invention effectively inhibits the degradation of genome, and the result is consistent with the result in table 1. Wherein, the A260/280 of the samples 1-5 are all between 1.7 and 2.0, and the A260/230 are all above 1.0, which indicates that the purity of the extracted saliva DNA is better.
TABLE 1 detection of DNA concentration (in ng/ul) of saliva samples 1-5 for 0, 7, 14, 21 and 30 days with Qubit
| Sample number | Day 0 | 7 days | 14 days | 21 days | 1 month |
| Sample 1 | 52 | 49 | 45 | 44.7 | 46 |
| |
122 | 100 | 104 | 98 | 86 |
| |
63 | 70 | 54 | 60 | 55 |
| |
88 | 78 | 64 | 70 | 60 |
| |
139 | 120 | 109 | 100 | 95 |
TABLE 2 DNA purity of saliva samples 1-5 tested with NanoDrop for 0, 7, 14, 21, and 30 days
(3) Carrying out PCR amplification on the saliva DNA sample in the step (2), wherein the specific method comprises the following steps:
amplifying the 16SrRNA of the microorganism, wherein an amplification primer and a fluorescent probe are as follows:
16SrRNA-F：5’-AGAGTTTGATCCTGGCTCA-3’(SEQ ID N0：1)
16SrRNA-R：5’-GGTTACCTTGTTACGACTT-3’(SEQ ID N0：2)
16SrRNA-P：5’-CAGCACCTGTGTCCYRGT-3’(SEQ ID N0：3)
RnaseP amplification, amplification primers and fluorescent probes are as follows:
RnaseP-F：5’-CGCCGTAAACGATGATTG-3’(SEQ ID N0：4)
RnaseP-R：5’-CTGGGAACTGCCWTTGTG-3’(SEQ ID N0：5)
RnaseP-P：5’-AATCCCCAACAACCAGTTGA-3’(SEQ ID N0：6)
and (3) fluorescent quantitative detection: taking the saliva DNA sample in the step (2) as a template, placing F and R primers of 16SrRNA gene and RnaseP gene and corresponding P probes into a PCR tube according to an amplification reaction system (see table 3), and simultaneously carrying out PCR amplification by using a real-time fluorescent quantitative PCR instrument (Roche LightCycler 480). The PCR reaction system was premixed with reagents (Takara), primers and probes at 4uM and 2uM, and the PCR amplification procedure was as follows: 3min at 95 ℃; circulating for 45 times at 94 ℃ for 30s and 60 ℃ for 40 s; the samples were cooled at 4 ℃ and the CT values were quantified in real time by fluorescence, as shown in Table 4. The cycle number of the RnaseP peak is basically not increased along with the increase of the preservation time of the saliva sample, the CT value of 16SrRNA is slightly reduced along with the increase of the preservation time of the saliva sample, and the number of microorganisms in the saliva is slightly increased, so that the saliva preservation liquid can effectively prevent the pollution of the microorganisms, protect the genomic DNA of the saliva and has a good overall preservation effect.
TABLE 3 reaction System for amplification
| | 2ul | |
| 10*buffer | 2ul | |
| TaqHS | 0.2ul | |
| 16SrRNA-F | 0.1 | |
| 16SrRNA-R | 0.1 | |
| P | 0.05 | |
| RnaseP-F | 0.1 | |
| RnaseP-R | 0.1 | |
| P | 0.05 | |
| dUTP | 0.2 | |
| UDG | 0.01 | |
| ddH2O | 15 |
TABLE real-time fluorescent quantitation CT values for saliva samples 1-5 at 40, 7, 14, 21, and 30 days
Example 3
The saliva preservation solution produced in example 1 and prepared according to the invention was tested with the BIOG Elite saliva preservation solution according to the following procedure:
(1) taking 10 saliva samples of a subject, shaking and uniformly mixing the saliva samples to be evenly divided into 2 parts, storing 2ml of the saliva sample by using the composition for preserving the saliva, simultaneously preserving 2ml of the saliva sample by using 40ul of BIOG Elite saliva preserving fluid according to the instruction, respectively preserving the preserved saliva sample for 14 days at room temperature, taking 450ul of the saliva sample, and extracting by using a nucleic acid extraction and purification kit (a magnetic bead method) to obtain sample genome DNA, wherein the serial number of the saliva sample of the invention is 1-10, and the serial number of the BIOG Elite saliva preserving fluid sample is 11-20.
(2) The concentration of the saliva is detected by using the Qubit, and the specific table 5 shows that the concentration of DNA extracted from the saliva sample stored by the saliva preserving fluid is higher;
TABLE 5 table of concentration test (ng/. mu.l) of saliva samples preserved for inventive products (1-10) and saliva samples preserved for control products (11-20)
| Sample number | The product of the invention detects the concentration | Sample number | Control product test concentration |
| 1 | 57.2 | 11 | 22 |
| 2 | 50 | 12 | 24.9 |
| 3 | 47 | 13 | 20.3 |
| 4 | 28.4 | 14 | 14.4 |
| 5 | 23.8 | 15 | 15.7 |
| 6 | 30.28 | 16 | 18.4 |
| 7 | 16.21 | 17 | 15.3 |
| 8 | 23.61 | 18 | 10.33 |
| 9 | 18.31 | 19 | 15.37 |
| 10 | 45.2 | 20 | 27 |
(3) The purity of the saliva sample is detected by using NanoDrop, wherein the saliva sample of the invention is numbered as 1-10, and the saliva preservation solution sample of BIOG Elite is numbered as 11-20, and the details are shown in Table 6. The DNA product OD extracted from the saliva preservation solution of the present invention260/OD280At 1.7-2.0, OD260/OD230In the range of 1.5-2.0. DNA product, OD, extracted from control saliva stock solution260/OD280At 1.5-2.0, OD260/OD230In the range of 1.0-2.0. The saliva sample extraction product preserved by the saliva preserving fluid has higher purity.
TABLE 6 comparison table of purity tests of saliva samples preserved in accordance with the invention (1-10) and comparative products (11-20)
| Sample number | A260/280 | A260/230 | Sample number | A260/280 | A260/230 |
| 1 | 1.77 | 1.58 | 11 | 1.68 | 0.98 |
| 2 | 1.85 | 2.01 | 12 | 1.72 | 1.56 |
| 3 | 1.69 | 1.72 | 13 | 1.54 | 1.65 |
| 4 | 1.8 | 1.69 | 14 | 1.70 | 1.62 |
| 5 | 1.84 | 1.9 | 15 | 1.74 | 1.80 |
| 6 | 1.84 | 1.53 | 16 | 1.75 | 1.43 |
| 7 | 1.83 | 1.95 | 17 | 1.76 | 1.83 |
| 8 | 1.8 | 1.63 | 18 | 1.82 | 1.20 |
| 9 | 1.83 | 1.61 | 19 | 1.75 | 1.52 |
| 10 | 1.76 | 1.55 | 20 | 1.70 | 1.33 |
(4) Performing RNasep and microbial 16SrRNA amplification fluorescent quantitative amplification on the obtained saliva DNA sample, wherein the saliva sample number is 1-10, the BIOG Elite saliva preservation solution sample number is 11-20, and the Ct value is shown in Table 7:
TABLE 7 Ct values of saliva samples preserved for the inventive products (1-10) and the comparative products (11-20)
The product preserves Ct of saliva sampleRNasepThe cycle number of the saliva sample is lower than that of a control product, which shows that the saliva sample stored by the saliva sample has more human genome DNA, and the saliva sample can effectively protect the saliva genome DNA and prevent degradation. The product preserves Ct of saliva sample16SThe CT value of the saliva preservation solution is slightly higher than that of a control product, and the total DNA of the saliva sample is obviously higher than that of the control product, so that the proportion of microbial genome DNA in the saliva DNA extracted from the saliva sample preserved by the saliva preservation solution in the total DNA is smaller, the number of microbes in the saliva in the control product is larger, the saliva preservation solution can effectively prevent the pollution of the microbes, and the overall preservation effect is better.
The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.
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Claims (9)
1. A novel saliva preservation solution is characterized by comprising the following components: EDTA 1-10mmol/L, Tris-HCl
1-50mmol/L, 10-100mmol/L sodium chloride, 0.1-3% (w/v) sodium sorbate, 0.1-3% (w/v) sodium diacetate, proclin 3000.01-0.1% (v/v), TWEEN 200.1-1% (v/v), NP 400.1-1% (w/v), SDS 0.1-2% (w/v).
2. The novel saliva preserving fluid according to claim 1, wherein: the pH of the saliva preserving fluid is 7-8.
3. A method for producing the saliva preserving fluid as claimed in any of claims 1 to 2, characterized in that the steps are as follows:
(1) taking raw materials in the proportion of any one of claims 1-2;
(2) respectively preparing EDTA, Tris-HCl and SDS weighed in the step (1) into solutions;
(3) mixing an EDTA solution, a Tris-HCl buffer solution and an SDS solution, adding sodium sorbate, sodium diacetate, sodium chloride, TWEEN20, NP40 and proclin300, adding sterile water to the total volume of the prepared saliva preservation solution, sterilizing and preserving to obtain the saliva preservation solution.
4. The method for preparing a novel saliva preserving fluid according to claim 3, wherein: the pH value of the Tris-HCl buffer solution in the step (2) is 6-8.5.
5. The method for preparing a novel saliva preserving fluid according to claim 3, wherein: the sterilization conditions in the step (3) are as follows: and (4) sterilizing by adopting a microporous filter membrane filtration mode.
6. The method for preparing a novel saliva preserving fluid according to claim 3, wherein: the temperature for storing in the step (3) is 15-25 ℃.
7. The method for preparing a novel saliva preserving fluid according to claim 3, wherein: in the step (3), the pH value of the prepared saliva preservation solution is adjusted, and the steps are as follows: when the pH value of the prepared saliva needs to be adjusted, adding an acid-base regulator, and adjusting the pH value to 7-8.
8. The method for preparing a novel saliva preserving fluid according to claim 7, wherein: the acid-base regulator is hydrochloric acid or sodium hydroxide.
9. A method for producing and using the novel saliva preservation solution prepared according to the claims 1-8, which is characterized by comprising the following steps: a quantitative drug sprayer for saliva is characterized in that the saliva preservation solution of claim 1 or 3 is stored at the bottom of a saliva collection tube, and after sample application is finished, a dryer is used for drying under high pressure to convert the liquid saliva preservation solution into a solid saliva preservation agent.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111518797A (en) * | 2020-04-30 | 2020-08-11 | 上海安五生物科技有限公司 | Normal-temperature protection solution and preparation method and application thereof |
| CN111778239A (en) * | 2020-07-14 | 2020-10-16 | 福建百斯特基因检测有限公司 | Veterinary sampling liquid composition and sampling method |
| CN112438253A (en) * | 2020-12-08 | 2021-03-05 | 武汉吉诺百客医学科技有限公司 | Saliva preserving fluid and preparation method thereof |
| CN113575573A (en) * | 2021-09-07 | 2021-11-02 | 易普森生物科技(深圳)有限公司 | Preservation solution for sputum sample and application thereof |
| CN114467919A (en) * | 2022-01-25 | 2022-05-13 | 力因精准医疗产品（上海）有限公司 | Saliva preserving fluid and using method thereof |
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| US20180235206A1 (en) * | 2017-02-17 | 2018-08-23 | Oasis Diagnostics Corporation | Compositions and methods for stabilizing dna in saliva samples |
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| CN105368812A (en) * | 2014-08-19 | 2016-03-02 | 深圳华大基因股份有限公司 | Saliva preservation solution, preparation method and uses thereof |
| US20180235206A1 (en) * | 2017-02-17 | 2018-08-23 | Oasis Diagnostics Corporation | Compositions and methods for stabilizing dna in saliva samples |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518797A (en) * | 2020-04-30 | 2020-08-11 | 上海安五生物科技有限公司 | Normal-temperature protection solution and preparation method and application thereof |
| CN111778239A (en) * | 2020-07-14 | 2020-10-16 | 福建百斯特基因检测有限公司 | Veterinary sampling liquid composition and sampling method |
| CN111778239B (en) * | 2020-07-14 | 2022-08-05 | 福建省辉腾生物科技有限公司 | Veterinary sampling liquid composition and sampling method |
| CN112438253A (en) * | 2020-12-08 | 2021-03-05 | 武汉吉诺百客医学科技有限公司 | Saliva preserving fluid and preparation method thereof |
| CN113575573A (en) * | 2021-09-07 | 2021-11-02 | 易普森生物科技(深圳)有限公司 | Preservation solution for sputum sample and application thereof |
| CN114467919A (en) * | 2022-01-25 | 2022-05-13 | 力因精准医疗产品（上海）有限公司 | Saliva preserving fluid and using method thereof |
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