A swab sample preservation solution

Abstract

This invention discloses a swab sample preservation solution. The solution contains isosorbide dimethyl ether, sucrose fatty acid ester, and polyether NPE-108. This preservation solution is a swab sample preservation solution that can rapidly inactivate viruses and has good preservation effects. It can preserve viral nucleic acid for 16 days at room temperature (37°C) and for 60 days at 2–8°C, effectively protecting the nucleic acid from degradation without affecting subsequent nucleic acid extraction and detection, making it safe and effective. Furthermore, this preservation solution can rapidly inactivate the preserved virus sample in just 10 minutes, effectively preventing secondary infection of operators.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology. More specifically, it relates to a swab sample preservation solution. Background Technology

[0002] Swab sampling is a common method for pathogen detection, such as the detection and screening of patients infected with the novel coronavirus, which requires the collection of a large number of swab samples. The preservation and transportation of swab samples require swab sample preservation solution, also known as virus preservation solution. This solution is suitable for the collection, preservation, and transportation of common viral samples such as the novel coronavirus, influenza virus, and hand-foot-and-mouth disease virus. It is a liquid that protects the virus being tested by immersing the sampled swab in the sampling tube. It can preserve pharyngeal swabs, nasal swabs, or tissue samples from specific sites. The stored samples can be used for subsequent molecular biology experiments such as nucleic acid extraction, enzyme digestion, and PCR.

[0003] Chinese patent CN 111925941A discloses a virus preservation solution and its application. While viral nucleic acid can be preserved in this solution for up to 21 days, it lacks virus inactivation capabilities and cannot effectively prevent secondary infection of operators. Chinese patent CN 111718908A discloses a virus sample preservation solution, its preparation method, and its application. This solution can inactivate viruses in 10 minutes, preserve viral nucleic acid for 7–14 days at 37°C, for 1–2 months at 2–8°C, and achieve long-term preservation at -20°C. However, this solution has not been used for COVID-19 preservation, and its effectiveness in inactivating and preserving the novel coronavirus is unknown.

[0004] Given the current context of COVID-19 prevention and control, it is of great significance to provide a swab sample preservation solution that can rapidly inactivate the virus and has good preservation effects. Summary of the Invention

[0005] The main technical problem addressed by this invention is to further optimize the shortcomings of existing preservation solutions by providing a preservation solution that can rapidly inactivate viruses and maintain the integrity of viral DNA and RNA for a long period. After sample collection, mixing the sample with the preservation solution for 10 minutes inactivates the virus. Viral nucleic acid can be stored at 37°C for 16 days without degradation. After nucleic acid extraction, molecular biology experiments can be performed. The procedure is simple, safe, and provides excellent preservation results.

[0006] The purpose of this invention is to provide a swab sample preservation solution.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] Typically, virus sample preservation solutions need to contain protein lysis buffer and enzyme inhibitors to inactivate the virus while protecting the nucleic acid from degradation. After numerous experiments, this invention has found that adding isosorbide dimethyl ether, sucrose fatty acid ester, and polyether NPE-108 to the preservation solution can rapidly and effectively inactivate the virus and protect the viral nucleic acid from degradation for 16 days, demonstrating safety, effectiveness, and good preservation results.

[0009] Preferably, the concentration of isosorbide dimethyl ether in the preservation solution is 0.1% to 10% (v / v).

[0010] Preferably, the concentration of sucrose fatty acid esters in the preservation solution is 1% to 15% (w / w).

[0011] Preferably, the concentration of polyether NPE-108 in the preservation solution is 1% to 10% (w / w).

[0012] Preferably, the pH value of the preservation solution is 6 to 9.

[0013] More preferably, the pH value of the preservation solution is 6.5 to 7.5.

[0014] Preferably, the preservation solution also contains guanidine hydrochloride, NP40, Proclin300, and NaCl.

[0015] Preferably, the solvent of the preservation solution is ddH2O.

[0016] Preferably, the preservation solution contains 0.1%–10% (v / v) isosorbide dimethyl ether, 1%–15% (w / w) sucrose fatty acid ester, 1%–10% (w / w) polyether NPE-108, 20%–60% (w / w) guanidine hydrochloride, 0.1%–10% (v / v) NP40, 0.1%–3% (v / v) Proclin 300, and 2%–24% (w / w) NaCl.

[0017] More preferably, the concentration of isosorbide dimethyl ether in the preservation solution is 0.1%–2% (v / v), the concentration of sucrose fatty acid ester is 2%–6% (w / w), the concentration of polyether NPE-108 is 2%–4% (w / w), the concentration of guanidine hydrochloride is 30%–48% (w / w), the concentration of NP40 is 1%–2% (v / v), the concentration of Proclin300 is 0.3%–1% (v / v), and the concentration of NaCl is 5%–12% (w / w).

[0018] More preferably, the preservation solution contains 1% (v / v) isosorbide dimethyl ether, 4% (w / w) sucrose fatty acid ester, 3% (w / w) polyether NPE-108, 40% (w / w) guanidine hydrochloride, 1.5% (v / v) NP40, 0.8% (v / v) Proclin300, and 8% (w / w) NaCl.

[0019] The present invention has the following beneficial effects:

[0020] This preservation solution is an inactivating type, containing appropriate concentrations of lysis salts and surfactants. It can rapidly and efficiently lyse and inactivate viral proteins in the sample to be tested. It can quickly inactivate the preserved virus sample in 10 minutes, effectively preventing secondary infection of operators.

[0021] Meanwhile, this preservation solution also contains an enzyme denaturant, which can protect nucleic acids from degradation; therefore, it can lyse proteins to inactivate viruses while protecting nucleic acids from degradation, and can be used for subsequent nucleic acid extraction and detection. It is safe, effective, and has a good preservation effect.

[0022] This preservation solution is rationally formulated and stable, capable of rapidly inactivating viruses and preserving viral nucleic acids for 16 days at 37°C and 60 days at 2–8°C. It also protects nucleic acids from degradation, ensuring that subsequent nucleic acid extraction and detection are not affected. It is safe, effective, and provides excellent preservation results. Attached Figure Description

[0023] Figure 1 The pseudovirus was diluted to 10 with preservation solution. 4 The results of viral nucleic acid extraction were obtained after storing the virus at 37°C for 0 days following the sample collection of copies / mL.

[0024] Figure 2 The pseudovirus was diluted to 10 with preservation solution. 4 The results of viral nucleic acid extraction were obtained after storing the virus at 37°C for 8 days following the sample collection of copies / mL.

[0025] Figure 3 The pseudovirus was diluted to 10 with preservation solution. 3 The results of viral nucleic acid extraction were obtained after storing the virus at 37°C for 0 days following the sample collection of copies / mL.

[0026] Figure 4 The pseudovirus was diluted to 10 with preservation solution. 3 The results of viral nucleic acid extraction were obtained after storing the virus at 37°C for 8 days following the sample collection at 1 copy / mL.

[0027] Figure 5 The results of nucleic acid extraction were obtained after diluting the SARS-CoV-2 pseudovirus to 100 copies / mL with preservation solution and storing it at 37°C for 0 days.

[0028] Figure 6 The results of nucleic acid extraction were obtained after diluting the SARS-CoV-2 pseudovirus to 100 copies / mL with preservation solution and storing it at 37°C for 8 days. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] In the following examples, the components of the preservation solution, guanidine hydrochloride, polyether NPE-108, NaCl, and sucrose fatty acid ester, are solids with concentrations in w / w; Proclin 300, NP40, and isosorbide dimethyl ether are liquids with concentrations in v / v.

[0032] Example 1: Preparation of Preservative Solution

[0033] 1. This invention has undergone extensive exploration and experimentation to obtain a preservation solution formula with good virus inactivation effect and long nucleic acid preservation time, and therefore this formula is used as the official formula.

[0034] The formula for preservation solution 1 is shown in Table 1 (formula). Dissolve each component in ddH2O and bring the volume to 1 L. Shake to mix well. The pH of the preservation solution is 7±0.5.

[0035] Table 1 Preservative Solution 1 (Formula, 1000 mL)

[0036]

[0037] Note: Concentration is calculated as w / w for solids and v / v for liquids.

[0038] In addition, some failed recipes are shown below for comparison.

[0039] Preservative solution 2 formulation (as shown in Table 2, failed formulation): The difference between this formulation and the official formulation is that polyether NPE-108, sucrose fatty acid ester, and isosorbide dimethyl ether are replaced with triathon, SDS, and sorbitol. The preparation methods are the same.

[0040] Table 2. Preservative Solution 2 Formula (Failed Formula)

[0041]

[0042] Note: Concentration is calculated as w / w for solids and v / v for liquids.

[0043] Preservative solution 3 formulation (as shown in Table 3, failed formulation): The difference between this formulation and the official formulation is that polyether NPE-108 and sucrose fatty acid ester are replaced with mannitol and polyhexamethylene biguanide hydrochloride, but the preparation methods are the same.

[0044] Table 3. Preservative Solution 3 Formulation (Failed Formulation)

[0045]

[0046] Note: Concentration is calculated as w / w for solids and v / v for liquids.

[0047] Preservative solution 4 (as shown in Table 4, failed formula): The difference between this formula and the official formula is that sucrose fatty acid ester and isosorbide dimethyl ether are replaced with sorbitol and urea, but the preparation methods are the same.

[0048] Table 4. Preservative Solution Formulation 4 (Failed Formulation)

[0049]

[0050] Note: Concentration is calculated as w / w for solids and v / v for liquids.

[0051] Example 2: Investigation on the preservation time and preservation effect of preservation solution on SARS-CoV-2 pseudovirus

[0052] 1. The four preservation solution formulations in Example 1 were used as experimental materials to verify their preservation time for viruses.

[0053] (1) Study on storage time at 37℃

[0054] Test samples: Dilute the COVID-19 pseudovirus with the above-mentioned preservation solution to a concentration of 10. 4 The samples were stored at 37°C for 0, 2, 5, 8, 12, 14, and 16 days, and then viral nucleic acid was extracted and detected using the KAP Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method).

[0055] The test results are shown in Table 5. Samples preserved in the preservation solution 1 of this invention for 16 days at 37°C can still be detected by the COVID-19 test kit, while samples preserved in the other preservation solutions for 16 days could not be detected by the KAP Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method), indicating that these formulations do not achieve the effect of long-term stable preservation.

[0056] Table 5

[0057]

[0058] Note: + indicates detected, - indicates not detected.

[0059] (2) Study on storage time at 4℃

[0060] Test samples: Dilute the COVID-19 pseudovirus with the above-mentioned preservation solution to a concentration of 10. 4 The samples were stored at 4℃ for 0, 5, 10, 20, 30 and 60 days, and the viral nucleic acid was extracted and detected using the KAP Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method).

[0061] The test results are shown in Table 6. Samples preserved with the preservation solution 1 of the present invention for 60 days at 4°C can still be detected by the COVID-19 test kit, while samples preserved with the other preservation solutions for 60 days cannot be detected by the COVID-19 test kit, indicating that these formulations do not achieve the effect of long-term stable preservation.

[0062] Table 6

[0063]

[0064] Note: + indicates detected, - indicates not detected.

[0065] 2. Using preservation solution 1 as the experimental material, its preservation effect on the virus was verified.

[0066] The SARS-CoV-2 pseudovirus was diluted to 10⁻⁶ with preservation solution 1. 4 copies / mL, 10 3 The pseudovirus nucleic acid was extracted after storing samples at concentrations of 100 copies / mL and 100 copies / mL at 37℃ for 0 days and 8 days, respectively, and detected using the KAP Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method).

[0067] The test results are shown in Table 7 and Figures 1-6 (The kit used for COVID-19 N gene detection is the KAP Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method)); The kit used for COVID-19 ORF1ab gene detection is the KAP Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method)). As shown, samples preserved in the preservation solution 1 of this invention for 0 days and 8 days at 37°C can be detected by the COVID-19 detection kit, and the detection limit for ORF1ab gene detection is 10. 3 The detection limit is 100 copies / mL; when using the N gene for detection, the detection limit can reach 100 copies / mL. This indicates that the preservation solution of this invention can meet the detection limit requirements of COVID-19 test kits.

[0068] Table 7

[0069]

[0070] The results in summary indicate that, in terms of sample preservation, the sample preservation solution of this invention is significantly superior to the comparative formulation, with a preservation time of up to 16 days at 37°C; and when used for the detection of the COVID-19 N gene, the detection limit can reach 100 copies / mL.

[0071] Example 3: Collection, Preservation, and Detection of Swab Samples

[0072] 1. Nasopharyngeal swab collection

[0073] 1) Tilt the patient's head back (about 70 degrees) and keep it still.

[0074] 2) Use a swab to estimate the distance from the base of the ear to the nostril.

[0075] 3) Insert the swab vertically into the nostril, reaching at least half the distance from the earlobe to the tip of the nose. Once resistance is encountered, it has reached the posterior nasopharynx. Hold the swab for a few seconds to aspirate secretions (generally 15-30 seconds). Rotate the swab 3-5 times, then gently rotate it to remove it. Immerse the swab tip in a collection tube containing preservation fluid.

[0076] 4) Break off the sterile swab rod at the top, discard the tail, tighten the cap and seal it with sealing film.

[0077] 2. Oral and pharyngeal swab collection

[0078] 1) Instruct the patient to rinse their mouth with saline or water first.

[0079] 2) Moisten the swab in sterile saline solution.

[0080] 3) Instruct the patient to sit down, tilt their head back, open their mouth wide, and make an "ah" sound.

[0081] 4) Use a tongue depressor to hold the tongue in place, and swab the tongue over the base of the tongue to the posterior pharyngeal wall, tonsillar crypts, and lateral walls.

[0082] 5) First, use a swab to wipe both sides of the pharyngeal tonsils back and forth at least 3 times with moderate force, and then wipe the posterior pharyngeal wall at least 3 times, 3 to 5 times is appropriate.

[0083] 6) Remove the swab, avoiding contact with the tongue, pituitary gland, oral mucosa, and saliva.

[0084] 7) Immerse the swab head in the sample preservation solution.

[0085] 8) Break the sterile swab rod near the top, discard the tail, tighten the cap and seal it with sealing film.

[0086] 3. Preservation and testing of swab samples

[0087] (1) Referring to the method of Example 2, the sample was replaced with the swab sample collected above instead of the COVID-19 pseudovirus.

[0088] The test results are shown in Table 8.

[0089] Table 8

[0090]

[0091] Note: + indicates detected, - indicates not detected.

[0092] The results showed that swab samples could still be preserved for up to 16 days at 37°C.

[0093] (2) The collected nasopharyngeal swab samples that tested positive for COVID-19 were stored at 37°C for 16 days using the preservation solution of this invention. Then, nucleic acid was extracted using the nucleic acid extraction or purification reagent kit developed by KAP using the magnetic bead method (magnetic bead method NO-RA type I). The extracted nucleic acid was used for the detection of COVID-19. Table 9 shows the Ct value of the internal standard of the KAP Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method).

[0094] Table 9

[0095]

[0096] The results showed that preservation solution 1 had a good preservation effect on the novel coronavirus at 37°C, with no significant difference in effect between day 0 and day 16, making it suitable for the preservation of nasopharyngeal swab samples.

[0097] Example 4: Optimization of Preservative Solution Ratio

[0098] We conducted extensive experiments to investigate the proportions of each component in the swab sample preservation solution of this invention, following the methods described in Example 2. The results show that the formulation concentrations of the preservation solution of this invention are as shown in Table 10:

[0099] Table 10

[0100]

[0101] Note: The solvent used is ddH2O.

[0102] Note: Concentration is calculated as w / w for solids and v / v for liquids.

[0103] The following shows the test results for some formulations (as shown in Tables 11 and 12).

[0104] Table 11

[0105]

[0106] Note: Concentration is calculated as w / w for solids and v / v for liquids.

[0107] Following the method in Example 2, the COVID-19 pseudovirus was preserved for a certain period of time using preservation solutions from Groups 1 to 5, and then nucleic acid was extracted for detection, as shown in Table 12.

[0108] Table 12

[0109]

[0110] Note: + indicates detection.

[0111] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A swab sample preservation solution, characterized in that, The preservation solution is composed of isosorbide dimethyl ether, sucrose fatty acid ester, polyether NPE-108, guanidine hydrochloride, NP40, Proclin 300, and NaCl; wherein the concentration of isosorbide dimethyl ether is 0.1%–10% (v / v), the concentration of sucrose fatty acid ester is 1%–15% (w / w), the concentration of polyether NPE-108 is 1%–10% (w / w), the concentration of guanidine hydrochloride is 20%–60% (w / w), the concentration of NP40 is 0.1%–10% (v / v), the concentration of Proclin 300 is 0.1%–3% (v / v), and the concentration of NaCl is 2%–24% (w / w); the solvent is water, and the pH value is 6–9.

2. The swab sample preservation solution according to claim 1, characterized in that, The pH value is 6.5 to 7.

5.

3. The swab sample preservation solution according to claim 1, characterized in that, The solvent is ddH2O.

4. The swab sample preservation solution according to claim 1, characterized in that, The concentrations of isosorbide dimethyl ether, sucrose fatty acid ester, polyether NPE-108, guanidine hydrochloride, NP40, Proclin 300, and NaCl are 0.3%–1% (w / w), respectively.

5. The swab sample preservation solution according to claim 4, characterized in that, The concentrations of isosorbide dimethyl ether, sucrose fatty acid ester, polyether NPE-108, guanidine hydrochloride, NP40, NP40, Proclin 300, and NaCl are 1.5% (v / v), 40% (w / w), 1.5% (v / v), 0.8% (v / v), and 8% (w / w).

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