A biological sample extraction solution, its application and usage method

Abstract

The present invention provides a biological sample extract and its application and use method. The biological sample extract includes a surfactant and salt ions, wherein the surfactant is selected from Triton X-100 or Tween-20 and a high concentration of salt ions. The biological sample extract of the present invention not only improves the detection sensitivity of immunochromatographic antigen detection products, but also enhances the extract's ability to inactivate various viruses, including the novel coronavirus, playing an important role in preventing secondary contamination and protecting the public.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a virus sample preservation solution, a use method and an application. Background Art

[0002] A virus is a small, simple, non-cellular organism that must parasitize and replicate within living cells. It consists of a protein shell and an inner envelope of genetic material (DNA or RNA). Viruses cannot survive independently and must live within the cells of other organisms.

[0003] Currently, virus detection typically uses nucleic acid testing or antigen-antibody testing. Viruses in a sample are broken down using an extraction solution. The resulting nucleic acid is then analyzed using nucleic acid testing, while the resulting protein is analyzed using antigen testing to determine whether the virus is present.

[0004] Take the current novel coronavirus (SARS-CoV-2) test as an example. A swab is used to collect samples from specific parts of the mouth, nose, etc., and then the swab with the collected sample is immersed in a virus extract for subsequent testing and analysis.

[0005] After testing is complete, there's often some residual extract containing the virus sample. During the processing of this residual extract, the complete inactivation of the collected sample presents a significant risk. Incompletely inactivated virus samples pose a risk of secondary infection to personnel during transport and handling. This is particularly true for highly contagious and pathogenic viruses like the novel coronavirus, requiring effective inactivation of the virus in the sample to prevent the spread of the epidemic caused by secondary infection.

[0006] One current method for inactivating viral samples is to directly add inactivating agents such as guanidine salts to the extract. However, guanidine salts can cause protein denaturation and are not suitable for antigen detection methods that use proteins as the detection target.

[0007] Currently, novel coronavirus antigen testing, due to its simple operation and rapid results, has been promoted and used in the community, enabling rapid screening through self-testing by the community. However, developers of viral antigen testing reagents need to further research how to quickly and effectively inactivate the remaining samples collected and tested by non-professionals to prevent exposure to these infectious and pathogenic virus samples. Developing an extract that can effectively inactivate viral samples is crucial. Summary of the Invention

[0008] In order to solve the above problems in the prior art, the present invention provides an extract of a biological sample (virus), comprising a surfactant and salt ions, wherein the surfactant is selected from Triton X-100 or Tween-20.

[0009] Furthermore, the concentration of the surfactant is 0.5% to 2% (w / V).

[0010] Furthermore, the salt ion concentration is 0.9%-5% (w / V).

[0011] Furthermore, the salt ion is selected from NaCl or KCl.

[0012] Furthermore, the pH value of the extract is 8.0-9.0.

[0013] The present invention also provides application of the virus extract in extracting virus protein samples.

[0014] Furthermore, the biological sample is a virus, and further, the virus is selected from the new coronavirus.

[0015] The present invention also provides a method for using the biological sample extract, comprising the following steps: directly adding a swab sample or a liquid sample to a certain volume of virus extract, incubating and extracting, dropping the extracted sample onto a reagent plate, and interpreting the result.

[0016] Furthermore, the swab sample or liquid sample is incubated in the virus extract for 30 seconds to 1 minute.

[0017] Furthermore, the reagent plate includes an upper cover and a lower plate, and the test strip is assembled between the upper cover and the lower plate.

[0018] The biological sample extract described in this invention not only improves the sensitivity of immunochromatographic antigen detection products but also enhances its ability to inactivate viruses, such as the novel coronavirus (SARS-CoV-2). This facilitates the rapid and effective inactivation of samples collected by non-professionals, preventing exposure to infectious and pathogenic viruses, and plays a crucial role in preventing secondary contamination and protecting the public. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Microscopic photograph of the negative control in Example 2.

[0020] Figure 2 Microscopic photograph of the positive control in Example 2.

[0021] Figure 3 Microscopic photos of the experimental group in Example 2.

[0022] Figure 4 Place the extract in an extraction tube.

[0023] Figure 5 Reagent plates housed in extraction tubes.

[0024] Figure 6 Swab sampling and testing steps. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific examples, which are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0026] The present invention provides an extract having an inactivating effect, comprising a surfactant and salt ions. The surfactant is selected from, but not limited to, Triton X-100 or Tween-20, and the salt ions are selected from, but not limited to, sodium chloride or potassium chloride. In a preferred embodiment, the salt ions in the extract are high-concentration salt ions.

[0027] Example 1

[0028] Experimental Materials:

[0029] 1. Extract formula:

[0030] The surfactant is selected from Triton X-100, Tween-20 or SDS.

[0031] The salt ion is selected from NaCl or KCl.

[0032] 2. New Coronavirus Antigen Detection Kit (New Coronavirus (2019-nCoV) Antigen Detection Kit (latex method) provided by Aikon)

[0033] 3. Recombinant antigen of novel coronavirus N protein

[0034] 4. Novel Coronavirus Strain

[0035] Experimental methods:

[0036] 1. Prepare extracts of varying concentrations by mixing the surfactants Triton X-100, Tween-20, and SDS with the salts NaCl and KCl in varying proportions with purified water. The pH of the extracts should be between 8.0 and 9.0. The specific formulations are shown in Table 1.

[0037] Table 1 Extraction formula

[0038] Recipe Number Surfactant (w / V) Salt (w / v) 1 0.1% Triton X-100 0.5% NaCl 2 0.1% Tween 20 0.5% KCl 3 0.1% SDS 0.5% NaCl 4 0.5% Triton X-100 0.9% NaCl 5 0.5% Tween 20 0.9% KCl 6 0.5% SDS 0.9% NaCl 7 1% Triton X-100 3% NaCl 8 1% Tween 20 3% KCl 9 1% SDS 3% NaCl 10 2% Triton X-100 5% NaCl 11 2% Tween 20 5% KCl 12 2% SDS 5% NaCl

[0039] *(m / V) represents the number of grams of solute dissolved in 100 ml of solvent

[0040] 2. Use the extracts in Table 1 to prepare the same concentrations (low and medium) of the SARS-CoV-2 N protein recombinant antigen quality control products, and perform sensitivity testing using the SARS-CoV-2 Antigen Detection Kit. The low concentration: the concentration of the SARS-CoV-2 N protein recombinant antigen is 20 pg / mL, and the medium concentration: the concentration of the SARS-CoV-2 N protein recombinant antigen is 60 pg / mL. The experimental results are shown in Table 2.

[0041] Table 2: Sensitivity test results

[0042] Sensitivity assessment Low concentration samples Medium concentration samples Recipe 1 L3 L5 Recipe 2 L3 L5 Recipe 3 L1 L2 Recipe 4 L4 L6 Recipe 5 L4 L6 Recipe 6 L1 L2 Recipe 7 L5 L7 Recipe 8 L5 L7 Recipe 9 L2 L3 Recipe 10 L5 L7 Recipe 11 L5 L7 Recipe 12 L2 L3

[0043] *L represents color intensity, ranging from L1 to L10. Higher values indicate stronger color development in the product test line. Low-concentration L values should reach L3, and medium-concentration L values should be as high as possible.

[0044] 3. Use the formula with high sensitivity in step 2 to prepare the extract, which dilutes the new coronavirus strain to a concentration of 1.0*10 6 TCID 50 / mL.

[0045] 4. After incubation at room temperature for 60 minutes, the virus treated with the extract was diluted with culture medium at a ratio of 1:10000 as the experimental group. The extract without virus was diluted with culture medium at a ratio of 1:10000 as the negative control group. 6 TCID 50 The virus at 1:10,000 / mL was directly diluted with culture medium as a positive control group.

[0046] 5. Cultivate the Vero E6 cell line in the culture wells of the culture plate to a density of about 80%, transfer the virus solutions of the experimental group, negative control group, and positive control group in step 4 to the culture wells, and place the culture plate in a 37°C, 5% CO2 incubator for 3 days.

[0047] 6. Observe the production status of cells and evaluate the inactivation effect of the extract.

[0048] Example 2 Inactivation experiment of extract

[0049] Formula 7 of Example 1 was selected as the extract, and a virus inactivation experiment was performed according to the method of Example 1. The experimental results are shown in Table 3 below.

[0050] Table 3 Virus inactivation experimental results

[0051] Microscope photos result Negative control group See Figure 1 N / D Positive control group See Figure 2 CPE Results after inactivation (experimental group) See Figure 3 Non-CPE

[0052] Interpretation of results: N / D means no virus was found; CPE means virus survived; Non-CPE means virus inactivated.

[0053] Other extracts with high sensitivity were used for inactivation experiments and had the same inactivation effect.

[0054] Example 3

[0055] The experimental steps are as follows: Figure 4 、 Figure 5 and Figure 6 As shown, an extraction tube 2 containing an extract 1 is opened. The extract in the extraction tube uses the formula 7 of Table 1; a sampled swab 3 is inserted into the extraction tube containing the extract, the swab is rotated in the extraction tube for 30 seconds, and then the swab head is squeezed against the tube wall 5 times to ensure that the sample on the swab is fully eluted; the swab head is squeezed along the inner wall of the extraction tube to retain the extract as much as possible in the tube, the swab is removed and discarded, and the processed sample extract is obtained; a dropper 4 is placed in the extraction tube; 4-5 drops of the processed sample extract are dripped into the reagent plate 5 and into the reagent plate sample injection port 7; the test result is interpreted and displayed in the observation window 6 of the reagent plate. The reagent plate includes an upper cover and a lower plate, and a test strip is assembled between the upper cover and the lower plate.

[0056] Samples from patients who tested positive for nucleic acid and those who tested negative were collected using swabs, and the sampling, extraction, and reagent plate loading tests were performed according to the procedures described in Example 4. The test results were consistent with the nucleic acid test results.

[0057] The invention adds 0.5% to 2% (w / v) of a surfactant such as Triton X-100 or Tween 20 and 0.9% to 5% (w / v) of a high-concentration salt ion (such as NaCl or KCl) to the extract. This not only improves the detection sensitivity of the immunochromatographic product but also inactivates the virus within a certain period of time (e.g., within 60 minutes). This prevents secondary contamination of the remaining sample and plays an important role in protecting the public.

Claims

1. An application of a biological sample extract in the preparation of an antigen detection product, characterized in that: The biological sample extract is composed of a surfactant and salt ions, the surfactant is selected from Triton X-100 or Tween-20, and the concentrations of the surfactant and salt ions are respectively selected from one of the following combinations: 1% Triton X-100, 3% NaCl; or 1% Tween 20, 3% KCl; or 2% Triton X-100, 5% NaCl; or 2% Tween 20, 5% KCl, and the antigen is a viral protein.

2. The use of the biological sample extract according to claim 1 in preparing an antigen detection product, characterized in that: The pH value of the extract is 8.0-9.

0.

3. The use of the biological sample extract according to claim 1 in preparing an antigen detection product, characterized in that: The protein is the N protein of the virus.

4. The use of biological sample extract in the extraction of viral protein samples is characterized by: The biological sample extract comprises a surfactant and salt ions, wherein the surfactant is selected from Triton X-100 or Tween-20, and the concentrations of the surfactant and salt ions are respectively selected from one of the following combinations: 1% Triton X-100, 3% NaCl; or 1% Tween 20, 3% KCl; or 2% Triton X-100, 5% NaCl; or 2% Tween 20, 5% KCl.

5. The use according to claim 4, characterized in that The pH value of the extract is 8.0-9.

0.

6. The use according to claim 4, characterized in that The viral protein is the viral N protein.

7. The use according to claim 4, characterized in that The virus is selected from the new coronavirus.

8. A method for using a biological sample extract, characterized in that: The swab sample or liquid sample is directly added to a certain volume of viral extract, incubated and extracted, and the extracted sample is dripped onto a reagent plate to interpret the result. The usage method is a non-disease diagnosis method. The biological sample extract is composed of a surfactant and salt ions. The surfactant is selected from Triton X-100 or Tween-20. The concentrations of the surfactant and salt ions are respectively selected from one of the following combinations: 1% Triton X-100, 3% NaCl; or 1% Tween 20, 3% KCl; or 2% Triton X-100, 5% NaCl; or 2% Tween 20, 5% KCl. The reagent plate is a reagent plate for detecting viral proteins.

9. The method of use according to claim 8, characterized in that: The swab sample or liquid sample is incubated in the virus extract for 30 seconds to 1 minute.

10. The method of use according to claim 8, characterized in that: The reagent plate comprises an upper cover and a lower plate, and a test strip is arranged between the upper cover and the lower plate.

11. The method of use according to claim 8, characterized in that: The pH value of the extract is 8.0-9.

0.

12. The method of use according to claim 8, characterized in that: The viral protein is N protein.

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