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CN113855092A - Saliva collector for disease detection - Google Patents

Saliva collector for disease detection Download PDF

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CN113855092A
CN113855092A CN202111335968.6A CN202111335968A CN113855092A CN 113855092 A CN113855092 A CN 113855092A CN 202111335968 A CN202111335968 A CN 202111335968A CN 113855092 A CN113855092 A CN 113855092A
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saliva
dna
preservation solution
disease detection
sample collection
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巩赞华
巩赞博
巩赞斌
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Huachenyang Shenzhen Technology Co ltd
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
    • A61B10/0045Devices for taking samples of body liquids
    • A61B10/0051Devices for taking samples of body liquids for taking saliva or sputum samples
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA

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Abstract

本申请涉及DNA采集检测的领域,具体公开了一种疾病检测用唾液采集器,包括唾液采集机构和保存液储存机构,唾液保存机构包括样本收集管和可拆卸连接在样本收集管上的采样漏斗,采样漏斗内壁上设置有消泡涂层;保存液储存机构包括存储有DNA保存液的存液管以及可拆卸设置在存液管上的管帽,管帽规格与样本收集管管口规格适配。DNA保存液原料包括固定剂50%‑60%(V/V)、抗凝剂0.5%‑1%(W/V)、缓冲液5‑15mM、离子强度维持剂0.5%‑1%(W/V)、抗菌剂0.3%‑3%(W/V)以及余量的无菌水,且DNA保存液体系pH范围为7.0‑8.0。本申请具有提升采集体积精度的效果。

Figure 202111335968

The present application relates to the field of DNA collection and detection, and specifically discloses a saliva collector for disease detection, comprising a saliva collection mechanism and a preservation solution storage mechanism, and the saliva preservation mechanism includes a sample collection tube and a sampling funnel detachably connected to the sample collection tube , the inner wall of the sampling funnel is provided with a defoaming coating; the preservation solution storage mechanism includes a storage tube storing the DNA preservation solution and a cap detachably arranged on the storage tube. The specifications of the cap and the mouth of the sample collection tube are suitable match. The raw materials of DNA preservation solution include fixative 50%-60% (V/V), anticoagulant 0.5%-1% (W/V), buffer 5-15mM, ionic strength maintenance agent 0.5%-1% (W/ V), antibacterial agent 0.3%-3% (W/V) and the remaining amount of sterile water, and the pH range of the DNA preservation solution is 7.0-8.0. The present application has the effect of improving the accuracy of the collected volume.

Figure 202111335968

Description

Saliva collector for disease detection
Technical Field
The application relates to the field of DNA (deoxyribonucleic acid) acquisition and detection, in particular to a saliva collector for disease detection.
Background
When genetic disease screening, genome detection and partial disease detection are carried out, DNA samples are required to be collected for gene detection, and the commonly used DNA sample collection method mainly comprises saliva sampling and blood sampling. Because saliva sampling is painless and noninvasive, and compared with blood collection, the saliva sampling is more convenient and free, and people's acceptance is higher, therefore, the saliva is more popular to be collected for DNA detection at present.
Currently, in the ordinary saliva collection, about 2mL of saliva is collected into a sample collection tube through a sampling funnel, then a DNA preservative solution is added into the sample collection tube, then the sample collection tube is closed, and the sample collection tube is shaken to be uniform, so that the saliva collection is completed.
In view of the above-mentioned related technologies, the inventor believes that the saliva collected often contains a lot of foam, which causes a large error between the collected amount read and the actual collected amount, requires re-collection, and increases the collection cost.
Disclosure of Invention
In order to promote saliva collection volume precision, this application provides a disease detects uses saliva collector.
A saliva collector for disease detection comprises a saliva collecting mechanism and a preserving fluid storage mechanism, wherein the saliva preserving mechanism comprises a sample collecting pipe and a sampling funnel detachably connected to the sample collecting pipe, volume scales are arranged on the side wall of the sample collecting pipe, and a defoaming coating is arranged on the inner wall of the sampling funnel; the preservation liquid storage mechanism comprises a liquid storage tube for storing DNA preservation liquid and a tube cap detachably arranged on the liquid storage tube, and the specification of the tube cap is matched with that of the orifice of the sample collection tube.
Through adopting above-mentioned technical scheme, when carrying out saliva collection, the personnel of waiting to gather who prepares spit saliva to the sampling funnel, saliva in the sampling funnel in-process that flows contacts with the defoaming coating, the defoaming coating is acted on the foam in the saliva, make the foam breakage eliminate, then saliva flows in the sample collecting pipe, the volume of collecting saliva is confirmed to the saliva through the scale on the sample collecting pipe, pull down the pipe cap after reaching the numerical value, to deposit DNA in the liquid pipe and pour into the sample collecting pipe, then pull down the sampling funnel, establish the pipe cap lid on the sample collecting pipe, rock the sample collecting pipe, make saliva and DNA preserve liquid intensive mixing, accomplish saliva collection. The sampling funnel of design makes the foam in the saliva get into the sample collection pipe through the defoaming coating before by a large amount of elimination for the error between the volume of gathering and the actual volume of gathering of reading can reduce, has effectively promoted saliva collection volume precision, helps reducing and gathers the volume again, reduces and gathers the cost. In addition, the design enables the sample collecting pipe and the liquid storing pipe to share one pipe cap, so that the device cost is reduced on the premise of not influencing the use.
Optionally, the defoaming coating is made of a food grade polyether defoamer or a silicone based defoamer.
By adopting the technical scheme, the food-grade polyether defoaming agent or silicone defoaming agent is used as the defoaming coating raw material, so that on one hand, the potential safety hazard caused by mistakenly touching the defoaming coating in the sampling process can be effectively reduced, and the safety of the sampling process is improved; on the other hand, neither the polyether defoaming agent nor the silicone-based defoaming agent is easy to dissolve in saliva, and the influence on the DNA collection of the saliva is reduced.
Optionally, a filtering partition plate is arranged at the liquid outlet end of the sampling funnel.
Through adopting above-mentioned technical scheme, the debris in the saliva of the filter baffle of design on the one hand filters, and on the other hand helps delaying the speed that the saliva breaks away from the sampling funnel, prolongs the contact time of saliva and defoaming coating, abundant defoaming.
Optionally, the raw materials of the DNA preservation solution include 50% -60% (V/V) of a fixative, 0.5% -1% (W/V) of an anticoagulant, 5-15mM of a buffer solution, 0.5% -1% (W/V) of an ionic strength maintaining agent, 0.3% -3% (W/V) of an antibacterial agent, and the balance of sterile water, and the pH range of the DNA preservation solution system is 7.0-8.0. The fixing agent is preferably any one of methanol, ethanol or isopropanol, and the ionic strength maintaining agent is preferably NaCl or KCl.
By adopting the technical scheme, the cell is fixed by the fixing agent, the cell lysis effect of protease in the cell and the nucleic acid digestion effect of nucleic acid digestive enzyme are avoided, and the integrity of the protein and the nucleic acid is preserved for subsequent analysis; the buffer solution is matched with the fixing solution to keep the shape of the cells complete to the maximum extent so as to ensure that the cells are fully suspended and dispersed; the ionic strength maintaining agent provides a required nutrient environment for the cells; heavy metal ions are introduced into the anticoagulant chelating solution to prevent cells from coagulating; the antibacterial agent is used for inhibiting bacteria, and multiple components are matched together, so that the cell morphology and the integrity of nucleic acid of the preservation solution can be effectively maintained.
Optionally, the antibacterial agent is cetylpyridinium chloride.
By adopting the technical scheme, cetylpyridinium chloride is adopted as an antibacterial agent, and the bacteria in saliva are limited by utilizing the antibacterial effect of cetylpyridinium chloride so as to prolong the storage time; and cetylpyridinium chloride has the effect of reducing surface tension, is beneficial to inhibiting the generation of foam by forming surface tension difference, reduces the stability of the formed foam, and is beneficial to accurately checking the total volume of the DNA preservative fluid and saliva after the DNA preservative fluid is blended with the saliva so as to ensure that a sample does not need to be collected again and reduce the sampling cost.
Optionally, the buffer solution is prepared by compounding a phosphate buffer solution with the pH value of 5.7-8.0 and a Tris-HCl buffer solution with the pH value of 6.5-9.0 in a ratio of 1: 1.
By adopting the technical scheme, the phosphate buffer solution and the Tris-HCl buffer solution are compounded, and the formed buffer system not only accords with the physiological range of the intracellular environment, but also is beneficial to reducing the interference effect of the preservation solution in the DNA detection process, and is suitable for carrying out a transformation test and a DNA reaction in the subsequent detection process.
Optionally, the anticoagulant is EDTA.
By adopting the technical scheme, a TE buffer system can be formed after EDTA is added into a Tris-HCl buffer solution, and metal ions such as calcium, magnesium and the like in a preservation solution can be chelated, so that DNase can be effectively inhibited from playing a role, the stability of DNA can be improved, the long-term preservation is facilitated, and the long-term research can be carried out.
Optionally, the pH range of the DNA preservation solution system is 7.0-7.6.
By adopting the technical scheme, the DNA preservation solution has better preservation effect in the pH range of 7.0-7.6, and is beneficial to further prolonging the preservation period of the DNA preservation solution.
In summary, the present application has the following beneficial effects:
1. because the defoaming coating is arranged on the inner wall of the sampling funnel, foam in saliva is greatly eliminated before the saliva enters the sample collecting tube through the defoaming coating, so that the error between the read collecting amount and the actual collecting amount is reduced, the saliva collecting volume precision is effectively improved, the re-collecting amount is reduced, and the collecting cost is reduced;
2. the DNA preservation solution preferably adopts cetylpyridinium chloride as an antibacterial agent, and utilizes the antibacterial effect of the cetylpyridinium chloride to limit germs in saliva so as to prolong the preservation time; the cetylpyridinium chloride has the effect of reducing the surface tension, is beneficial to inhibiting the generation of foam by forming the surface tension difference, reduces the stability of the formed foam, and is beneficial to accurately checking the total volume of the DNA preserving fluid and the saliva after the DNA preserving fluid and the saliva are blended so as to ensure that a sample does not need to be collected again and reduce the sampling cost;
3. the DNA preservation solution is preferably compounded by a phosphate buffer solution and a Tris-HCl buffer solution, and a formed buffer system not only accords with the physiological range of the intracellular environment, but also is beneficial to reducing the interference effect of the preservation solution in the DNA detection process, and is suitable for carrying out a transformation test and a DNA reaction in the subsequent detection process.
Drawings
Fig. 1 is a schematic overall structure diagram of a saliva collector for disease detection in embodiment 1 of the present application;
fig. 2 is a schematic diagram of the structure of the sampling funnel of fig. 1.
Reference numerals: 1. a saliva collection mechanism; 11. a sample collection tube; 12. a sampling funnel; 121. a defoaming coating; 122. a filtering baffle plate; 2. a preservative fluid storage mechanism; 21. a liquid storage tube; 22. and (4) a pipe cap.
Detailed Description
The present application is described in further detail below with reference to figures 1-2 and examples.
Examples
Example 1
The embodiment 1 of the application discloses a disease detection saliva collector, referring to fig. 1 and fig. 2, the disease detection saliva collector comprises a saliva collecting mechanism 1 and a preservation solution storage mechanism 2, the saliva storage mechanism comprises a sample collecting pipe 11 and a sampling funnel 12 in threaded connection with the sample collecting pipe 11, the side wall of the sample collecting pipe 11 is carved with volume scales, the volume scales are not shown in the figure, the inner wall of the sampling funnel 12 is coated with a defoaming coating 121, a filter partition plate 122 is bonded at a liquid outlet at the bottom of the sampling funnel 12, and the filter partition plate 122 is also coated with the defoaming coating 121 in the embodiment; the preservation solution storage mechanism 2 comprises a storage solution tube 21 for storing DNA preservation solution and a tube cap 22 which is connected to the storage solution tube 21 in a threaded manner, the DNA preservation solution is not shown in the figure, the specification of the tube cap 22 is matched with the specification of the tube orifice of the sample collection tube 11, and the tube cap can be covered on the tube orifice of the sample collection tube 11 to seal the sample collection tube.
The defoaming coating 121 is made of a food-grade polyether defoamer, specifically a 2000-type polyether defoamer from Xiamen Ralmann chemical technology Co. The forming method of the defoaming coating 121 comprises the following steps: diluting a polyether defoaming agent into a defoaming agent aqueous solution, and then spraying the defoaming agent aqueous solution onto the inner wall of the sampling funnel 12, wherein the spraying amount takes the condition that the defoaming agent solution cannot flow in a strand as an upper limit; and (3) baking the sampling funnel 12 while spraying, so that a solvent in the defoaming agent solution is evaporated, a solute in the defoaming agent solution is formed into a defoaming agent film on the inner wall of the sampling funnel 12, and the spraying and baking processes are repeated for 3-5 times to form a defoaming coating 121. In other embodiments, the defoaming coating 121 may also be made by the same method using food grade silicone-based defoaming agents.
The implementation principle of the saliva collector for disease detection in embodiment 1 of the application is as follows: the person who wants to collect relaxes the cheek and slightly kneads for 30s to generate saliva; then spit saliva gently into sampling funnel 12 in, saliva flows in sampling funnel 12 in-process and defoaming coating 121 contact, defoaming coating 121 acts on the foam in the saliva, make the foam breakage eliminate, then saliva flows into in sample collection pipe 11, confirm saliva collection volume through the scale on the sample collection pipe 11, detach tube cap 22 after reaching the numerical value, pour the DNA preservative solution in the stock solution pipe 21 into sample collection pipe 11, then detach sampling funnel 12, cover tube cap 22 on sample collection pipe 11, rock sample collection pipe 11, make saliva and DNA preservative solution intensive mixing, accomplish saliva collection.
Use sampling funnel 12 that does not set up defoaming coating 121 as the control, and sampling funnel 12 in this embodiment 1 is repeated under the same condition and is carried out multiunit saliva collection test, finds that the foam volume in the saliva that sampling funnel 12 gathered in adopting this embodiment 1 all obviously reduces, shows that sampling funnel 12 of this application can effectively eliminate the foam in carrying out the saliva through setting up defoaming coating 121, promotes saliva collection volume precision.
In addition, the application example 1 also discloses a DNA preservation solution applied to the saliva collection device, the raw material composition and the dosage of the DNA preservation solution are shown in table 1, and the specific preparation steps are as follows:
step one, respectively preparing EDTA and Tris-HCl buffer solutions, sterilizing at 121 ℃ for 30min, and storing at room temperature;
and secondly, under the aseptic condition, according to the total volume of the pre-prepared composition for preserving the human saliva and in combination with the content requirements of each component in the system, selecting EDTA and ethanol with corresponding volumes, adding NaCl and cetylpyridinium chloride with corresponding amounts, then supplementing sterile water, adding a Tris-HCl buffer solution to adjust the pH, and supplementing the sterile water to the total volume of the pre-prepared DNA preserving solution to obtain the DNA preserving solution with the system pH meeting the requirements.
Examples 2 to 3
A DNA storage solution was different from example 1 in that the composition and the amount of the raw materials used are shown in Table 1.
TABLE 1DNA preserving fluid raw material composition and dosage
Figure BDA0003350509100000081
Example 4
A DNA storage solution which is different from that of example 2 in that benzalkonium chloride is used as an antibacterial agent.
Example 5
A DNA preservation solution is different from the DNA preservation solution in the embodiment 2 in that the buffer solution is prepared by compounding phosphate buffer solution with pH7.0 and Tris-HCl buffer solution with pH7.0 in a ratio of 1: 2.
Example 6
A DNA preservation solution is different from the DNA preservation solution in the embodiment 2 in that the buffer solution is prepared by compounding phosphate buffer solution with pH7.0 and Tris-HCl buffer solution with pH7.0 in a ratio of 1: 1.
Example 7
A DNA preservation solution is different from the DNA preservation solution in the embodiment 2 in that the buffer solution is prepared by compounding phosphate buffer solution with pH7.0 and Tris-HCl buffer solution with pH7.0 in a ratio of 2: 1.
Example 8
A DNA preservative solution is different from that in example 6 in that sodium citrate is used as an anticoagulant.
Example 9
A DNA storage solution which is different from that in example 2 in that Tris-HCl (pH6.5) is used for adjusting the pH of the system to 7.0.
Example 10
A DNA storage solution which is different from that in example 2 in that Tris-HCl (pH9.0) is used for adjusting the pH of the system to 8.0.
Comparative example
Comparative example 1
A DNA storage solution which is different from that of example 2 in that it does not contain an antibacterial agent.
Performance test
Collecting saliva of different volunteers, subpackaging into preservation tubes containing the DNA preservation solution of the above examples 1-10 and comparative example 1 under the same and aseptic conditions, mixing uniformly, preserving at room temperature for one year, then performing DNA extraction, and measuring the nucleic acid concentration (ng/μ L) of the extract of each example and comparative example respectively by using a nanodrop spectrophotometer after the extraction is completed; three experiments were performed for each example, and the nucleic acid concentration data for the three experiments were averaged and reported in table 2:
TABLE 2 results table
Group of Nucleic acid concentration (ng/. mu.L)
Example 1 69.3
Example 2 71.1
Example 3 70.1
Example 4 70.6
Example 5 71.6
Example 6 72.9
Example 7 71.8
Example 8 68.4
Example 9 71.8
Example 10 69.8
Comparative example 1 59.9
In addition, according to the experimental observation record, after the collected saliva was dispensed into the storage tubes of the DNA storage solutions of examples 1 to 10 and comparative example 1 and mixed, it was found that many foams with stable forms were generated in the storage tubes using the storage solutions of example 4 and comparative example 1, particularly, the foam was the most generated in the storage tube using the storage solution of comparative example 1, while the foam generated in the storage tubes using the storage solutions of the other examples was less, and the generated foam was unstable in form and rapidly destroyed.
As can be seen by combining examples 1-4 and comparative example 1 and table 2 and experimental observation records, the amount of nucleic acid remaining in saliva preserved by using the DNA preservative solution of comparative example 1 is significantly less than that of saliva preserved by using the DNA preservative solution of examples 1-4, which indicates that the addition of a proper amount of an antibacterial agent in the DNA preservative solution can effectively improve the quality of DNA preservation and prolong the preservation time of DNA; and especially when cetylpyridinium chloride is used as an antibacterial agent, the generation of foam after the saliva and the DNA preservation solution are blended can be effectively inhibited, the observation and reading are convenient to carry out, and the saliva collection can be checked to see whether the saliva collection needs to be carried out again.
It can be seen from the combination of example 2 and examples 5-7 and Table 2 that the best preservation quality can be obtained when the buffer is formulated with phosphate buffer and Tris-HCl buffer 1:1, since the buffer system formed best corresponds to the physiological conditions of the intracellular environment.
As can be seen from the combination of example 2 and example 8 and table 2, the DNA preservation solution in which EDTA is used as the anticoagulant has a better cell preservation effect, because a TE buffer system is formed after EDTA is added to a Tris-HCl buffer solution, and can chelate metal ions such as calcium and magnesium in the preservation solution, thereby effectively inhibiting DNase from playing a role, contributing to the improvement of DNA stability, and facilitating long-term preservation.
As can be seen from the combination of example 2, examples 9 to 10 and Table 2, the DNA preservative solution had a better preservative effect in the pH range of 7.0 to 7.6.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims (8)

1.一种疾病检测用唾液采集器,其特征在于,包括唾液采集机构(1)和保存液储存机构(2),所述唾液保存机构包括样本收集管(11)和可拆卸连接在所述样本收集管(11)上的采样漏斗(12),所述样本收集管(11)侧壁上设置有体积刻度,所述采样漏斗(12)内壁上设置有消泡涂层(121);所述保存液储存机构(2)包括存储有DNA保存液的存液管(21)以及可拆卸设置在所述存液管(21)上的管帽(22),所述管帽(22)规格与样本收集管(11)管口规格适配。1. A saliva collector for disease detection, characterized in that it comprises a saliva collection mechanism (1) and a preservation solution storage mechanism (2), and the saliva preservation mechanism comprises a sample collection tube (11) and is detachably connected to the a sampling funnel (12) on the sample collection tube (11), a volume scale is provided on the side wall of the sample collection tube (11), and a defoaming coating (121) is provided on the inner wall of the sampling funnel (12); The preservation solution storage mechanism (2) comprises a storage tube (21) storing the DNA preservation solution and a cap (22) detachably arranged on the storage tube (21), the cap (22) having a specification of It is adapted to the nozzle specification of the sample collection tube (11). 2.根据权利要求1所述的疾病检测用唾液采集器,其特征在于:所述消泡涂层(121)由食品级的聚醚消泡剂或者硅氧烷基消泡剂制成。2 . The saliva collector for disease detection according to claim 1 , wherein the defoaming coating ( 121 ) is made of food-grade polyether defoaming agent or siloxane-based defoaming agent. 3 . 3.根据权利要求1所述的疾病检测用唾液采集器,其特征在于:所述采样漏斗(12)的出液端设置有过滤隔板(122)。3 . The saliva collector for disease detection according to claim 1 , wherein a filter partition ( 122 ) is provided at the liquid outlet end of the sampling funnel ( 12 ). 4 . 4.根据权利要求1所述的疾病检测用唾液采集器,其特征在于:所述DNA保存液原料包括固定剂50%-60%(V/V)、抗凝剂0.5%-1%(W/V)、缓冲液5-15mM、离子强度维持剂0.5%-1%(W/V)、抗菌剂0.3%-3%(W/V)以及余量的无菌水,且所述DNA保存液体系pH范围为7.0-8.0。4. The saliva collector for disease detection according to claim 1, characterized in that: the DNA preservation solution raw material comprises a fixative 50%-60% (V/V), an anticoagulant 0.5%-1% (W /V), buffer 5-15mM, ionic strength maintenance agent 0.5%-1% (W/V), antibacterial agent 0.3%-3% (W/V) and the balance of sterile water, and the DNA is preserved The pH range of the liquid system is 7.0-8.0. 5.根据权利要求4所述的疾病检测用唾液采集器,其特征在于:所述抗菌剂为西吡氯铵。5 . The saliva collector for disease detection according to claim 4 , wherein the antibacterial agent is cetylpyridinium chloride. 6 . 6.根据权利要求4所述的疾病检测用唾液采集器,其特征在于:所述缓冲液由pH为5.7-8.0的磷酸盐缓冲液和pH为6.5-9.0的Tris-HCl缓冲液1:1复配组成。6. The saliva collector for disease detection according to claim 4, wherein the buffer is composed of a phosphate buffer with pH of 5.7-8.0 and a Tris-HCl buffer with pH of 6.5-9.0 1:1 Compound composition. 7.根据权利要求6所述的疾病检测用唾液采集器,其特征在于:所述抗凝剂为EDTA。7 . The saliva collector for disease detection according to claim 6 , wherein the anticoagulant is EDTA. 8 . 8.根据权利要求7所述的疾病检测用唾液采集器,其特征在于:所述DNA保存液体系pH范围为7.0-7.6。8 . The saliva collector for disease detection according to claim 7 , wherein the pH range of the DNA preservation solution is 7.0-7.6. 9 .
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN115363637A (en) * 2022-08-23 2022-11-22 宁波海尔施基因科技股份有限公司 A saliva sample collection card and a method for collecting saliva samples using the collection card
CN119349012A (en) * 2024-12-26 2025-01-24 潍坊吉涛医学科技有限公司 A detection kit for judging benign or malignant pulmonary nodules and a method for using the same

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Application publication date: 20211231