CN113322252A - Swab sample preserving fluid - Google Patents
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Abstract
The invention discloses a swab sample preservation solution. The components of the swab sample preservation solution are added with isosorbide dimethyl ether, sucrose fatty acid ester and polyether NPE-108. The preservation solution is a swab sample preservation solution which can rapidly inactivate viruses and has a good preservation effect, can preserve viral nucleic acids for 16 days at the normal temperature of 37 ℃ and preserve nucleic acids for 60 days at the temperature of 2-8 ℃, can effectively protect the nucleic acids from degradation, does not influence the subsequent extraction and detection of the nucleic acids, and is safe and effective. In addition, the preservation solution can quickly inactivate the preserved virus sample within 10min, and can effectively prevent secondary infection of operators.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection. And more particularly, to a swab specimen preservation solution.
Background
Swab sampling is a common means for pathogen detection and sampling, and for example, detection and screening of an infected person under a novel coronavirus pneumonia situation requires collection of a large number of swab samples for detection. The swab sample preservation solution, namely the virus preservation solution, is suitable for collection, preservation and transportation of common virus samples such as novel coronavirus, influenza virus and hand-foot-and-mouth virus, is a liquid which is used for immersing a detected substance of the sampled swab virus sample in a sampling tube and protecting the virus, can preserve collected throat swabs, nose swabs or specific part tissue samples, and can be used for subsequent molecular biology experiments such as nucleic acid extraction, enzyme digestion and PCR.
Chinese patent CN 111925941a discloses a virus preservation solution in which virus nucleic acid can be preserved for 21 days at the maximum, but the preservation solution does not have a virus inactivation function and is not effective in preventing secondary infection of operators, and its application. Chinese patent CN 111718908A discloses a virus sample preservation solution and a preparation method and application thereof, the virus sample preservation solution can inactivate virus within 10min, the preservation time of virus nucleic acid is 7-14 days at 37 ℃, the preservation time is 1-2 months at 2-8 ℃, and long-term preservation can be realized at-20 ℃, but the preservation solution is not used for preserving COVID-19, and the inactivation and preservation effects of the preservation solution on new coronavirus are unknown.
In view of the background of the current new crown epidemic prevention, the swab sample preservation solution which can quickly inactivate viruses and has a good preservation effect is provided, and the method has important significance.
Disclosure of Invention
The invention mainly solves the technical problem of providing the preserving fluid which can quickly inactivate viruses and can keep the virus DNA and RNA complete for a long time in order to further optimize the defects of the existing preserving fluid products. After the sample is collected, the sample and the preservation solution are mixed for 10min to inactivate viruses, virus nucleic acid can be preserved for 16 days at 37 ℃ without being degraded, and molecular biological experiments can be carried out after nucleic acid extraction, so that the method is simple and convenient to operate, safe to use and good in preservation effect.
The invention aims to provide a swab sample preserving fluid.
The above purpose of the invention is realized by the following technical scheme:
in general, a virus sample preservation solution needs to contain a protein lysate and an enzyme inhibitor, so that the virus is inactivated and nucleic acid is protected from degradation. After multiple experiments, the invention discovers that the addition of isosorbide dimethyl ether, sucrose fatty acid ester and polyether NPE-108 in the preservation solution can quickly and effectively inactivate viruses, can protect virus nucleic acid from being degraded within 16 days, and is safe, effective and good in preservation effect.
Preferably, the concentration of the isosorbide dimethyl ether in the preservation solution is 0.1-10% (v/v).
Preferably, the concentration of sucrose fatty acid ester in the preservation solution is 1-15% (w/w).
Preferably, the concentration of the polyether NPE-108 in the preservation solution is 1-10% (w/w).
Preferably, the pH value of the preservation solution is 6-9.
Further preferably, the pH value of the preservation solution is 6.5-7.5.
Preferably, the preservation solution also contains guanidine hydrochloride, NP40, Proclin300 and NaCl.
Preferably, the solvent of the preservation solution is ddH2O。
Preferably, the concentration of the isosorbide dimethyl ether in the preservation solution is 0.1-10% (v/v), the concentration of the sucrose fatty acid ester is 1-15% (w/w), the concentration of the polyether NPE-108 is 1-10% (w/w), the concentration of the guanidine hydrochloride is 20-60% (w/w), the concentration of NP40 is 0.1-10% (v/v), the concentration of Proclin300 is 0.1-3% (v/v), and the concentration of NaCl is 2-24% (w/w)
Further preferably, the concentration of the isosorbide dimethyl ether in the preservation solution is 0.1-2% (v/v), the concentration of the sucrose fatty acid ester is 2-6% (w/w), the concentration of the polyether NPE-108 is 2-4% (w/w), the concentration of the guanidine hydrochloride is 30-48% (w/w), the concentration of the NP40 is 1-2% (v/v), the concentration of the Proclin300 is 0.3-1% (v/v), and the concentration of the NaCl is 5-12% (w/w).
Still more preferably, the concentration of dimethyl isosorbide in the preservation solution is 1% (v/v), the concentration of sucrose fatty acid ester is 4% (w/w), the concentration of polyether NPE-108 is 3% (w/w), the concentration of guanidine hydrochloride is 40% (w/w), the concentration of NP40 is 1.5% (v/v), the concentration of Proclin300 is 0.8% (v/v), and the concentration of NaCl is 8% (w/w).
The invention has the following beneficial effects:
the preservation solution is inactivated, added with lysis salt and surfactant with proper concentration, can rapidly and efficiently inactivate virus protein in a sample to be detected, can rapidly inactivate the preserved virus sample within 10min, and can effectively prevent secondary infection of operators.
Meanwhile, the preservation solution also contains an enzyme denaturant, so that the nucleic acid can be protected from being degraded; therefore, the protein can be cracked to inactivate the virus and protect the nucleic acid from being degraded, and the method can be used for extracting and detecting the subsequent nucleic acid, is safe and effective and has good preservation effect.
The preservation solution has reasonable and stable compatibility, can quickly inactivate viruses, can preserve virus nucleic acid at 37 ℃ for 16 days and preserve the virus nucleic acid at 2-8 ℃ for 60 days, protects the nucleic acid from being degraded, does not influence the extraction and detection of subsequent nucleic acid, is safe and effective, and has good preservation effect.
Drawings
FIG. 1 shows the dilution of the new coronavirus with the preservation solution to 104The results of detection of viral nucleic acids were extracted after 0 day of storage at 37 ℃ after copies/mL.
FIG. 2 shows the new coronavirus diluted to 10 with the preservation solution4The results of detection of viral nucleic acids were extracted after 8 days of preservation at 37 ℃ after copies/mL.
FIG. 3 shows the new coronavirus diluted to 10 with the preservation solution3Extracting viral nucleic acid after preservation at 37 ℃ for 0 day after copies/mLThe detection result of (1).
FIG. 4 shows the new coronavirus diluted to 10 with the preservation solution3The results of detection of viral nucleic acids were extracted after 8 days of preservation at 37 ℃ after copies/mL.
FIG. 5 shows the results of detection of viral nucleic acid extracted from a new coronavirus which was diluted to 100copies/mL in a storage solution and stored at 37 ℃ for 0 day.
FIG. 6 shows the results of detection of viral nucleic acid extracted from a novel coronaviruse diluted to 100copies/mL in a storage solution and stored at 37 ℃ for 8 days.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
In the following examples, guanidine hydrochloride, polyether NPE-108, NaCl, sucrose fatty acid ester as solid components of the preservative solution were used in the concentration unit w/w; proclin300, NP40 and isosorbide dimethyl ether are liquid, and the concentration unit is v/v.
EXAMPLE 1 preparation of preservative fluid
1. After a great deal of exploration and attempt, the preservation solution formula with good virus inactivation effect and long nucleic acid preservation time is obtained, and the formula is used as a formal formula.
The formula of the preservation solution 1 is shown in Table 1 (formal formula), and the components are dissolved in ddH2And O, metering the volume to 1L, shaking and uniformly mixing, wherein the pH value of the preservation solution is 7 +/-0.5.
TABLE 1 preserving fluid 1 (formal formulation, 1000mL)
Note: the concentration was calculated as w/w solids and v/v liquids.
In addition, the partial failure formulations are shown below for comparison.
Preservative fluid 2 formulation (as table 2, failure formulation): the formula is different from the formal formula in that polyether NPE-108, sucrose fatty acid ester and isosorbide dimethyl ether are replaced by triton, SDS and sorbitol, and the preparation methods are the same.
TABLE 2 preservative solution 2 formulation (failure formulation)
Note: the concentration was calculated as w/w solids and v/v liquids.
Table 3 preservative solution 3 formulation (failure formulation)
Note: the concentration was calculated as w/w solids and v/v liquids.
Preservative fluid 4 formulation (as table 4, failure formulation): the formula is different from the formal formula in that sucrose fatty acid ester and isosorbide dimethyl ether are replaced by sorbitol and urea, and the preparation methods are the same.
Table 4 preservative solution 4 formulation (failure formulation)
Note: the concentration was calculated as w/w solids and v/v liquids.
EXAMPLE 2 examination of preservation duration and preservation Effect of preservation solution for New Corona pseudovirus
1. The four storage liquid formulations in example 1 were used as experimental materials to verify the storage time of the virus.
(1) Examination of the storage time at 37 ℃
Detecting a sample: diluting the new coronavirus to 10 deg.C with the above stock solution4The copies/mL are stored for 0 day, 2 days, 5 days, 8 days, 12 days, 14 days and 16 days at 37 ℃, and then virus nucleic acid is extracted by a Kaemp new type coronary disease 2019-nCoV nucleic acid detection kit (fluorescence PCR method) and detected.
The detection results are shown in table 5, the samples stored for 16 days at 37 ℃ in the preservation solution 1 can still be detected by the new crown kit, and the samples stored for 16 days in the rest preservation solutions can not be detected by the Kaemp new type coronary disease 2019-nCoV nucleic acid detection kit (fluorescence PCR method), which indicates that the formulas can not achieve the effect of long-term stable storage.
TABLE 5
| Standing at 37 | Formulation | 1 | Formulation 2 | |
Formulation 4 |
| Day 0 | + | + | + | + | |
| 2 days | + | + | + | + | |
| 5 days | + | + | + | + | |
| 8 days | + | + | + | + | |
| 12 days | + | - | + | + | |
| 14 days | + | - | - | + | |
| 16 days | + | - | - | - |
Note: + represents detected, -represents undetected.
(2) Examination of storage time at 4 ℃
Detecting a sample: respectively separating new coronavirusDiluting to 10 deg.C with the above stock solution4The copies/mL are stored for 0 day, 5 days, 10 days, 20 days, 30 days and 60 days at the temperature of 4 ℃, and virus nucleic acid is extracted and detected by a Kayp novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method).
The test results are shown in table 6, the sample preserved for 60 days at 4 ℃ in the preservation solution 1 of the present invention can still be detected by the new crown kit, and the samples preserved for 60 days in the rest of the preservation solutions can not be detected by the new crown test kit, which indicates that the formulations can not achieve the effect of long-term stable preservation.
TABLE 6
| Standing at 4 | Formulation | 1 | Formulation 2 | |
Formulation 4 |
| Day 0 | + | + | + | + | |
| 5 days | + | + | + | + | |
| 10 days | + | + | + | + | |
| 20 days | + | + | + | + | |
| 30 days | + | + | + | + | |
| 60 days | + | - | - | - |
Note: + represents detected, -represents undetected.
2. The preservation effect of the preservative fluid 1 on viruses was verified by using the preservative fluid as an experimental material
Diluting the new coronavirus to 10 with preservation solution 1 respectively4copies/mL，103copies/mL and 100copies/mL, storing for 0 and 8 days at 37 deg.C, extracting pseudovirus nucleic acid, and detecting with Kaemp new type coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method).
The detection results are shown in Table 7 and FIGS. 1-6 (the kit for detecting the new crown N gene is a Kaemp new type coronary disease 2019-nCoV nucleic acid detection kit (fluorescence PCR method)); the kit for detecting the new crown ORF1ab gene is a Kaemp new crown disease 2019-nCoV nucleic acid detection kit (fluorescence PCR method)), and the preservation solution 1 is stored for 0 day and 8 days at 37 DEG CThe detection limit of the new crown detection kit in the detection of ORF1ab gene is 103copies/mL; when the N gene is used for detection, the detection limit can reach 100 copies/mL. The preservation solution can meet the requirement of the detection limit of the new crown kit.
TABLE 7
The results show that the sample preservation solution is obviously superior to the comparative formula in the aspect of sample preservation, and the preservation time can reach 16 days at 37 ℃; when the new crown N gene is used for detection, the detection limit can reach 100 copies/mL.
EXAMPLE 3 Collection, preservation and detection of swab samples
1. Nasopharyngeal swab collection
1) The patient's head is reclined (about 70 degrees) and remains motionless.
2) The ear root to nostril distance was estimated with a swab stick.
3) The insertion is from the nostril to the direction vertical to the face, and the penetration distance is at least half of the length from the ear lobe part to the nose tip. When resistance is met, the patient reaches the posterior nasopharynx, the patient stays for a few seconds to suck secretion (generally required to be 15-30 seconds), the swab is rotated 3-5 times, the swab is taken out by gentle rotation, and the swab head is immersed into a collecting pipe filled with preservation liquid.
4) The sterile swab rod was broken off at the top end, the tail was discarded, and the cap was screwed and sealed with a sealing membrane.
2. Oral pharynx swab collection
1) The patient is ordered to rinse with normal saline or clear water.
2) The swab was wetted in sterile saline.
3) The patient was noted to sit down, recline his head, open his mouth, and make an "o" sound.
4) The tongue is fixed by a tongue spatula, and the swab crosses the tongue root to the pharyngeal posterior wall, the tonsil recess, the lateral wall and the like.
5) The double-sided pharyngeal tonsils are wiped back and forth by a swab with moderate force for at least 3 times, and then the posterior pharyngeal wall is wiped for at least 3 times, preferably 3-5 times.
6) The swab was removed to avoid touching the tongue, pendants, oral mucosa and saliva.
7) The swab head is immersed in a sample-containing preservation solution.
8) The sterile swab shaft was broken off near the top end, the tail was discarded, and the cap was screwed and sealed with a sealing membrane.
3. Swab sample preservation and detection
(1) With reference to the method of example 2, the sample was changed from a new coronavirus to the swab sample collected as described above.
The test results are shown in table 8.
TABLE 8
| Standing at 37 | Formulation | 1 |
| Day 0 | + | |
| 2 days | + | |
| 5 days | + | |
| 8 days | + | |
| 12 days | + | |
| 16 days | + |
Note: + represents detected, -represents undetected.
The result shows that the storage time of the swab sample under the condition of 37 ℃ can still reach 16 days.
(2) Storing the collected new coronA positive nasopharyngeal swab samples for 16 days at 37 ℃ by using the preservation solution of the invention respectively, then extracting nucleic acid by using A nucleic acid extraction kit (NO-R-AI type by A magnetic bead method) of Kaemp's self-research or A purification reagent by A magnetic bead method, and using the extracted nucleic acid for detecting new coronA viruses; table 9 shows Ct values of internal standards of the Kaemp new type coronary disease 2019-nCoV nucleic acid detection kit (fluorescence PCR method);
TABLE 9
The result shows that the preservation solution 1 has good preservation effect on the new coronavirus at 37 ℃, has no obvious difference between the effect on the 0 th day and the effect on the 16 th day, and is suitable for preserving nasopharyngeal swab samples.
Example 4 optimization of preservative fluid ratio
A number of investigation experiments were carried out on the ratios of the components of the preserving fluid for swab specimens according to the invention, the method being referred to in example 2. The results show that the formula concentrations of the preserving fluid of the invention are shown in table 10:
watch 10
Note: the solvent used is ddH2O。
Note: the concentration was calculated as w/w solids and v/v liquids.
The following shows the results of testing some of the formulations (see tables 11 and 12)
TABLE 11
Note: the concentration was calculated as w/w solids and v/v liquids.
With reference to the method of example 2, the new coronaviruses were stored in the storage solutions of groups 1 to 5 for a predetermined period of time, and then nucleic acids were extracted and detected, as shown in Table 12.
TABLE 12
| Standing at 37 | Group | 1 | Group 2 | |
Group 4 | |
| Day 0 | + | + | + | + | + | |
| 2 days | + | + | + | + | + | |
| 5 days | + | + | + | + | + | |
| 8 days | + | + | + | + | + | |
| 12 days | + | + | + | + | + | |
| 16 days | + | + | + | + | + |
Note: + represents detection.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The swab sample preservation solution is characterized by comprising isosorbide dimethyl ether, sucrose fatty acid ester and polyether NPE-108.
2. The swab specimen preservation solution according to claim 1, wherein the concentration of dimethyl isosorbide is 0.1% to 10% (v/v).
3. The swab specimen preservation solution according to claim 1, wherein the concentration of the sucrose fatty acid ester is 1% to 15% (w/w).
4. The swab specimen preservation solution according to claim 1, wherein the concentration of the polyether NPE-108 is 1% to 10% (w/w).
5. The swab specimen preservation solution according to any one of claims 1 to 4, wherein the pH is 6 to 9.
6. The swab specimen preservation solution according to any one of claims 1 to 4, wherein the preservation solution further comprises guanidine hydrochloride, NP40, Proclin300, and NaCl.
7. The swab specimen preservation solution of claim 6, wherein the solvent is water.
8. The swab specimen preservation solution according to claim 6, wherein the concentration of dimethyl isosorbide is 0.1% to 10% (v/v), the concentration of sucrose fatty acid ester is 1% to 15% (w/w), the concentration of polyether NPE-108 is 1% to 10% (w/w), the concentration of guanidine hydrochloride is 20% to 60% (w/w), the concentration of NP40 is 0.1% to 10% (v/v), the concentration of Proclin300 is 0.1% to 3% (v/v), and the concentration of NaCl is 2% to 24% (w/w).
9. The swab specimen preservation solution according to claim 8, wherein the concentration of dimethyl isosorbide is 0.1% to 2% (v/v), the concentration of sucrose fatty acid ester is 2% to 6% (w/w), the concentration of polyether NPE-108 is 2% to 4% (w/w), the concentration of guanidine hydrochloride is 30% to 48% (w/w), the concentration of NP40 is 1% to 2% (v/v), the concentration of Proclin300 is 0.3% to 1% (v/v), and the concentration of NaCl is 5% to 12% (w/w).
10. The swab specimen preservation solution according to claim 9, wherein the concentration of dimethyl isosorbide is 1% (v/v), the concentration of sucrose fatty acid ester is 4% (w/w), the concentration of polyether NPE-108 is 3% (w/w), the concentration of guanidine hydrochloride is 40% (w/w), the concentration of NP40 is 1.5% (v/v), the concentration of Proclin300 is 0.8% (v/v), and the concentration of NaCl is 8% (w/w).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023179216A1 (en) * | 2022-03-25 | 2023-09-28 | 艾康生物技术(杭州)有限公司 | Biological sample extract solution, and application and use method thereof |
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| US6114108A (en) * | 1995-08-29 | 2000-09-05 | V.I. Technologies, Inc. | Methods and compositions for the selective modification of viral nucleic acids |
| CN110982876A (en) * | 2020-03-04 | 2020-04-10 | 圣湘生物科技股份有限公司 | Pretreatment method, pretreatment liquid, kit and application of virus nucleic acid detection |
| CN111518775A (en) * | 2020-06-18 | 2020-08-11 | 合肥铼科生物科技有限公司 | Preserving fluid for preserving virus sample at normal temperature for long time and application thereof |
| CN111718908A (en) * | 2020-08-11 | 2020-09-29 | 杭州博日科技有限公司 | Virus sample preserving fluid and preparation method and application thereof |
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2020
- 2020-12-28 CN CN202011588126.7A patent/CN113322252A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6114108A (en) * | 1995-08-29 | 2000-09-05 | V.I. Technologies, Inc. | Methods and compositions for the selective modification of viral nucleic acids |
| CN110982876A (en) * | 2020-03-04 | 2020-04-10 | 圣湘生物科技股份有限公司 | Pretreatment method, pretreatment liquid, kit and application of virus nucleic acid detection |
| CN111518775A (en) * | 2020-06-18 | 2020-08-11 | 合肥铼科生物科技有限公司 | Preserving fluid for preserving virus sample at normal temperature for long time and application thereof |
| CN111718908A (en) * | 2020-08-11 | 2020-09-29 | 杭州博日科技有限公司 | Virus sample preserving fluid and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023179216A1 (en) * | 2022-03-25 | 2023-09-28 | 艾康生物技术(杭州)有限公司 | Biological sample extract solution, and application and use method thereof |
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