Dry-type cervical cancer free sulfydryl detection test paper processing technology and kit

Abstract

The invention provides a dry cervical cancer free sulfydryl detection test paper processing technology which comprises the following steps: preparing a dissolving buffer solution capable of dissolving DTNB, dividing the solution into two parts A and B, adding a DTNB dissolved solution into a component A solution as a detection group for detecting sulfydryl, and adding a DTNB-free solution into a component B solution as a control group; preparing a sodium tungstate solution as a sample adding group; the same specific absorbent paper is selected and divided into three parts, the three parts are soaked in solutions of the detection group, the control group and the sample adding group respectively, the soaked absorbent paper is directly subjected to water filtration and put into an oven, the dried test paper is cut to be suitable for all the installation grooves in the kit respectively, and all the installation grooves correspond to the detection hole, the control hole and the sample adding hole of the detection card respectively; detection is performed at any time. Compared with the prior art, the sodium tungstate is added into the detection card, so that the blocking effect is achieved, DTNB in the detection hole is not prone to being oxidized and reduced, and the false positive probability is reduced.

Description

Technical Field

[0001] The invention relates to the field of free thiol detection technology, in particular to a dry cervical cancer free thiol detection test paper processing technology and a kit. Background Art

[0002] The existing technology includes the reaction of the detection reagent with the thiol group on the test paper, and the observation result is positive and blue, such as patent CN202230856663.9; or the reaction of the detection reagent solution with the thiol group, and the observation result is positive and blue; or the reaction of the detection reagent solution with the thiol group, and the observation result is positive and yellow, and the test data result requires an ultraviolet spectrophotometer.

[0003] The principle of this timely detection: Histochemistry proves that the number of thiol groups in normal cervical tissue is significantly higher than that in normal tissues of other parts. After the cervical tissue becomes cancerous, the thiol groups generated and shed are relatively more. Therefore, the thiol content in the urine of cervical cancer patients is significantly higher than that of other cancer patients, and the degree of quantitative change is proportional to the number of cancer cells. Based on the characteristics that a large number of thiol groups are discharged in the urine of patients with cervical cancer and precancerous lesions, the thiol groups in urine are determined by the principle that the thiol group makes the kit develop color. The higher the thiol content, the darker the color. This is used to check whether there is a possibility of cervical cancer and precancerous lesions, so as to achieve the purpose of early detection, early diagnosis, early treatment and detection of cervical cancer and precancerous lesions. As a screening method for cervical cancer, the detection of thiol groups in urine has many advantages, such as safety and effectiveness, easy sampling, non-invasiveness, fast and convenient detection, no need for instruments and equipment, low price, no cross-contamination of inspection, and can be used for all women with sexual experience.

[0004] Other detection methods use chemical reagents that react with thiol groups to produce a blue color, such as the mercuric chloride contained in patent CN115541570A, which is a heavy metal pollution; or contain corrosive chemicals; or the test results are unclear and inconvenient to distinguish with the naked eye, such as the solution reagent detection method in which the positive is yellow and blue; or are prone to leakage and uneven color after color development, such as the two-hole flat card shell of the patent CN202230856663.9 test kit. Secondly, this test card is easily oxidized and reduced to blue, affecting the experimental results and causing false positives. Summary of the invention

[0005] The present invention provides a dry cervical cancer free thiol detection test paper processing technology and a kit, which can make DTNB in ​​the detection hole less likely to be oxidized and reduced, thereby reducing the probability of false positives.

[0006] The technical solution of the present invention is achieved as follows: A dry cervical cancer free thiol detection test paper processing technology comprises the following steps:

[0007] Step 1, prepare a dissolving buffer solution capable of dissolving DTNB, divide the solution into two parts, A and B, add a solution containing 0.05-10% DTNB to the A component solution as a detection group for detecting thiol groups, and do not add a solution containing DTNB to the B component solution as a control group; prepare a 0.1-20% sodium tungstate solution as a sample addition group;

[0008] Step 2, select the same specific absorbent paper and divide it into three parts, soak the three parts of absorbent paper in the solutions of the test group, the control group and the sample group respectively, directly filter the water from the soaked absorbent paper and put it into the oven, bake it at 40-50°C to become test paper, and set it aside for later use;

[0009] Step 3, cutting the dried test paper so that it is suitable for each installation slot in the test kit, each installation slot corresponds to the detection hole, control hole and sample addition hole of the test card;

[0010] Step 4, add the test strips of the detection group, control group and sample addition group to the corresponding positions of the card case respectively, and cover the lid;

[0011] Step 5, put the prepared test card and desiccant into a light-proof sealed bag. You can configure a urine cup as needed and use it for testing at any time.

[0012] Preferably, in step 1, the DTNB, i.e., 5,5-dithiobis-(2-nitrobenzoic acid) (5,5-Dithiobis-(2-nitrobenzoic acid, DTNB)) dissolution buffer solution, specifically comprises 1-50 mm phosphate buffer, 1-20 mm disodium EDTA, 0.01-0.5% hexadecyltrimethylethylammonium bromide, 0.1-10% dimethyl sulfoxide and 0.01-10% trifonx-100.

[0013] Preferably, the dissolution buffer solution comprises 1 mM phosphate buffer, 5 mM EDTA-disodium, 0.2% hexadecyltrimethylethylammonium bromide, 5% dimethyl sulfoxide and 0.5% TritonX-100.

[0014] Preferably, the concentration of DTNB is 0.5%, and the concentration of sodium tungstate is 0.5%. Sodium tungstate combines with vitamin C and uric acid in urine, so that DTNB in ​​the detection hole is not easily oxidized and reduced.

[0015] Preferably, in step 2, the absorbent paper after soaking is directly drained and put into an oven, and dried at 45° C. to become a test paper.

[0016] Preferably, in step 4, the test strips of the detection well and the control well are extended and placed at the bottom of the test strip of the sample adding well, so that the detection test strip and the control test strip each have an extra section placed at the bottom of the sample adding well, so that they can be chromatographed to the detection well and the control well when the sample is added.

[0017] A reagent kit corresponding to the dry cervical cancer free thiol detection test paper processing technology, the reagent kit comprises a base plate and a cover plate, the middle of the base plate is provided with a card slot for installing the test paper, including a detection slot, a control slot and a sample addition slot, corresponding to the test papers of the detection group, the control group and the sample addition group.

[0018] Preferably, the installation slots in the kit are arranged in an inverted herringbone shape, the detection slot and the control slot are symmetrically arranged and isolated by a baffle, and the sample loading slot is located below the middle of the detection slot and the control slot and is connected to both through a test paper.

[0019] Preferably, the cover plate is provided with three corresponding circular holes, namely, a detection hole, a control hole and a sample addition hole, which are arranged in an inverted herringbone shape and correspond to the detection slot, the control slot and the sample addition slot of the bottom plate respectively.

[0020] Preferably, a sealed baffle is provided between the detection slot and the control slot on the bottom plate, and a slot cooperating with the baffle is provided on the cover plate.

[0021] Compared with the prior art, the advantages of the present invention are as follows: sodium tungstate is added to the test card in the present application, which plays a blocking role (can react with some proteins, uric acid, vitamin C, etc. in urine), making the DTNB in ​​the test hole less likely to be oxidized and reduced, thereby reducing the probability of false positives; in addition, a sealed baffle is provided between the test slot and the control slot on the bottom plate, as well as a card slot corresponding to the baffle, which can isolate the mutual influence between the test card and the control card, making the detection more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of a free thiol detection card of the present invention without adding an L-cysteine ​​aqueous solution standard;

[0023] Figure 2 This is a schematic diagram of a test card containing different concentrations of L-cysteine ​​aqueous solution standard products of the present invention;

[0024] Figure 3 This is a schematic diagram of the test card for sample test results of the present invention;

[0025] Figure 4 This is a comparative schematic diagram of the free sulfur group detection card of the present invention;

[0026] Figure 5 It is a structural schematic diagram of the bottom plate part of the kit of the present invention;

[0027] Figure 6 It is a schematic structural diagram of the cover plate portion of the test kit of the present invention;

[0028] Figure 7 It is a schematic diagram of the overall structure of the kit of the present invention.

[0029] In the figure: 1. bottom plate; 2. cover plate;

[0030] 11. Detection slot; 12. Control slot; 13. Sample adding slot; 14. Baffle;

[0031] 21. Detection well; 22. Control well; 23. Sample addition well. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In the description of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0034] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0035] The present invention is based on the characteristic that a large amount of thiol is excreted in the urine of patients with cervical cancer and precancerous lesions, and utilizes the principle that thiol makes DTNB in ​​the kit appear yellow to determine the free thiol contained in the urine. The higher the free thiol content, the darker the color. This is used to check whether the patient may have cervical cancer and precancerous lesions, so as to achieve the purpose of early detection, early diagnosis, early treatment and detection of cervical cancer and precancerous lesions.

[0036] Example:

[0037] Step 1, prepare a dissolving buffer solution capable of dissolving DTNB, divide the solution into two parts, A and B, add a solution dissolving 0.5% DTNB to the A component solution as a detection group for detecting thiol groups, and do not add a solution of DTNB to the B component solution as a control group; prepare a 0.5% sodium tungstate solution as a sample addition group; sodium tungstate combines with vitamin C and uric acid in urine, so that DTNB in ​​the detection hole 21 is not easily oxidized and reduced;

[0038] Step 2, select the same specific absorbent paper and divide it into three parts, soak the three parts of absorbent paper in the solutions of the test group, the control group and the sample group respectively, directly filter the water from the soaked absorbent paper and put it into the oven, bake it at 40-50°C to become test paper, and set it aside for later use;

[0039] Step 3, cutting the dried test paper so that it is suitable for each installation slot in the test kit, each installation slot corresponds to the detection hole 21, the control hole 22 and the sample addition hole 23 of the test card;

[0040] Step 4, add the test strips of the detection group, control group and sample addition group to the corresponding positions of the card case respectively, and cover the lid;

[0041] Step 5, put the prepared test card and desiccant into a light-proof sealed bag. You can configure a urine cup as needed and use it for testing at any time.

[0042] See also Figure 1 , Figure 2 and Figure 3 The test kit includes a base plate 1 and a cover plate 2. The base plate 1 is provided with a card slot for installing test paper in the middle, including a detection slot 11, a control slot 12 and a sample adding slot 13, which respectively correspond to the test papers of the detection group, the control group and the sample adding group. The various installation slots in the test kit are arranged in an inverted Chinese-character shape. The detection slot 11 and the control slot 12 are symmetrically arranged and isolated by a baffle 14. The sample adding slot 13 is located below the middle of the detection slot 11 and the control slot 12, and is connected to the two through the test paper. The cover plate 2 is provided with three corresponding circular holes, namely the detection hole 21, the control hole 22 and the sample adding hole 23, which are arranged in an inverted Chinese-character shape and correspond to the detection slot 11, the control slot 12 and the sample adding slot 13 of the base plate 1. A sealed baffle 14 is provided between the detection slot 11 and the control slot 12 on the base plate 1, and a card slot matching the baffle 14 is provided on the cover plate 2.

[0043] The immunohistochemical color development principle is adopted in which 5,5-dithiobis-(2-nitrobenzoic acid) (5,5-Dithiobis-(2-nitrobenzoic acid, DTNB) reacts with thiol compounds to be converted into yellow 5-thiol-2-nitrobenzoic acid. The content of thiol in abnormal cells of exfoliated tissue in urine is proportional to the depth of color change. If the abnormal exfoliated tissue cells in urine will change color, it is judged as a thiol-positive result. If the exfoliated tissue cells in urine do not change color, it is judged as a thiol-negative result. The kit of this product is a three-hole detection with a three-dimensional structure, namely, a sample addition hole 23, a detection hole 21 and a control hole. The chromatography principle is used to develop the color of the test paper. It can make the color development uniform, without leakage, and can filter dark impurities in urine so that they do not affect the test results.

[0044] Positive result: detection well 21 appears yellow.

[0045] Negative result: There is basically no difference in color between the test well 21 and the control well 22.

[0046] Qualitative test experiment (standard product test)

[0047] Five aqueous L-cysteine ​​solutions with different concentrations were prepared, from low to high, 0mmol / L, 0.25mmol / L, 0.5mmol / L, 1mmol / L, 5mmol / L and 10mmol / L.

[0048] The prepared solutions were added into the sample well 23 of the free thiol detection card in sequence, and the color change was observed after 5 minutes. The results are shown in the table:

[0049] Group A 0.05% DTNB, test results

[0050]

[0051] Group A 0.5% DTNB, test results

[0052]

[0053] Group A 1% DTNB, test results

[0054]

[0055] Group A 10% DTNB, test results

[0056]

[0057] After comprehensive comparison, when 0.5% DTNB corresponds to each concentration of L-cysteine ​​aqueous solution, the color change is the most uniform and clear, so 0.5% DTNB is the best test concentration.

[0058] Group C 0.1% sodium tungstate, test results

[0059]

[0060]

[0061] Group C 0.5% sodium tungstate, test results

[0062]

[0063] Group C 1% sodium tungstate, test results

[0064]

[0065]

[0066] Group C 5% sodium tungstate, test results

[0067]

[0068] Group C 20% sodium tungstate, test results

[0069]

[0070] After comprehensive comparison, when 0.5% sodium tungstate corresponds to each concentration of L-cysteine ​​aqueous solution, the color change is the most uniform and clear, so 0.5% sodium tungstate is the best test concentration.

[0071] Figure 1 This is a free thiol test card without the addition of L-cysteine ​​aqueous solution standard.

[0072] Figure 2 An aqueous solution of L-cysteine ​​standard was added with concentrations of 0 mmol / L, 0.25 mmol / L, 0.5 mmol / L, 1 mmol / L, 5 mmol / L and 10 mmol / L, respectively.

[0073] Figure 4 This is a comparison diagram of the free sulfur detection card. The concentration of free sulfur groups can be determined based on the depth of the color on the comparison card (actually yellow, replaced by gray).

[0074] Sample test experiment

[0075] 0.5% DTNB and 0.5% sodium tungstate were selected as reagents of the detection group and the sample addition group, respectively, and the test paper was soaked and the detection card was made for this test experiment.

[0076] 2.1 Collect urine from patients with cervical cancer and precancerous lesions, as well as urine from normal people.

[0077] 2.2 Take about 6 drops of the collected urine and add them to the sample well 23 of the free thiol detection card. Observe the color change after 5 minutes. The results are shown in the table: (corresponding to Figure 3 )

[0078]

[0079] 1. Collect urine from patients with cervical cancer and precancerous lesions, as well as normal people. Note that the urine should be collected on an empty stomach in the morning. The precautions for collecting morning urine are as follows:

[0080] 1.1 Do not drink or eat for 8 hours starting from the night before the test. Collect the first morning urine ≥ 2ml in a clean urine cup the next morning.

[0081] 1.2 This product is only used for in vitro diagnostic testing. It is a single-use product and must not be used repeatedly. Keep it out of reach of children.

[0082] 1.3 For those who are color blind or color weak, please ask someone who is not color blind or color weak to help observe the correct results.

[0083] 1.4 For the accuracy of the results, please do the following 3 days in advance or on the day of the test:

[0084] 1.4.1 During the non-menstrual period, the blood in the sample to be tested will affect the determination of the color development result;

[0085] 1.4.2 Try to avoid using vitamin C to avoid affecting the color development results;

[0086] 1.4.3 Body temperature and liver function are normal (urine during the jaundice period will affect the color discrimination and color development results).

[0087] 1.5 It is contraindicated for patients with proteinuria to ensure the simplicity of detecting free thiol groups.

[0088] 1.6 Collecting the correct sample and correct operation are crucial to the performance of this test. Incorrect operation will lead to incorrect result interpretation.

[0089] 2. Take about 6 drops (150uL) of the collected urine and add them to the sample well 23 of the free thiol detection card. Observe the color change after 5 minutes. The results are shown in the figure:

[0090] 3. Judgment method:

[0091] Positive result: detection well 21 appears yellow.

[0092] Negative result: There is basically no difference in color between the test well 21 and the control well 22.

[0093] 4. Determination of experimental results: For patients 1 and 2, the color of the detection hole 21 was light yellow, and the result was determined to be weakly positive; for patients 3 and 4, the color of the detection hole 21 was yellow, and the result was determined to be positive; the color of the normal detection hole 21 and the control hole 22 were both colorless, and the result was determined to be negative.

[0094] Figure 5 , Figure 6 and Figure 7 In the present invention, a reagent kit corresponding to a dry cervical cancer free thiol detection test paper processing technology is provided. The reagent kit includes a base plate 1 and a cover plate 2. A card slot for installing a test paper is provided in the middle of the base plate 1, including a detection slot 11, a control slot 12 and a sample adding slot 13, which respectively correspond to the test papers of the detection group, the control group and the sample adding group. Each installation slot in the reagent kit is arranged in an inverted Chinese-character shape. The detection slot 11 and the control slot 12 are symmetrically arranged and isolated by a baffle 14. The sample adding slot 13 is located below the middle of the detection slot 11 and the control slot 12, and is connected to the two through the test paper. The cover plate 2 is provided with three corresponding circular holes, namely, the detection hole 21, the control hole 22 and the sample adding hole 23, which are arranged in an inverted Chinese-character shape and correspond to the detection slot 11, the control slot 12 and the sample adding slot 13 of the base plate 1. A sealed baffle 14 is provided between the detection slot 11 and the control slot 12 on the base plate 1, and a card slot matching the baffle 14 is provided on the cover plate 2.

[0095] Sodium tungstate is added to the test card in the present application, which plays a blocking role (it can react with some proteins, uric acid, vitamin C, etc. in urine) so that the DTNB in ​​the detection hole 21 is not easily oxidized and reduced, thereby reducing the probability of false positives; in addition, a sealed baffle 14 is provided between the detection slot 11 and the control slot 12 on the bottom plate 1, and a card slot corresponding to the baffle 14 can isolate the mutual influence between the test card and the control card, making the detection more accurate.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dry cervical cancer free thiol detection test strip processing technology, characterized in that: The following steps are involved: Step 1, prepare a dissolving buffer solution capable of dissolving DTNB, divide the solution into two parts, A and B, add a solution containing 0.05-10% DTNB to the A component solution as a detection group for detecting thiol groups, and do not add a solution containing DTNB to the B component solution as a control group; prepare a 0.1-20% sodium tungstate solution as a sample addition group; Step 2, select the same specific absorbent paper and divide it into three parts, soak the three parts of absorbent paper in the solutions of the test group, the control group and the sample group respectively, directly filter the water from the soaked absorbent paper and put it into the oven, bake it at 40-50°C to become test paper, and set it aside for later use; Step 3, cutting the dried test paper so that it is suitable for each installation slot in the test kit, each installation slot corresponds to the detection hole, control hole and sample addition hole of the test card; Step 4, add the test strips of the detection group, control group and sample addition group to the corresponding positions of the card case respectively, and cover the lid; Step 5, put the prepared test card and desiccant into a light-proof sealed bag. You can configure a urine cup as needed and use it for testing at any time.

2. A dry cervical cancer free thiol detection test strip processing technology according to claim 1, characterized in that: In the step 1, DTNB, i.e., 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) dissolution buffer solution, specifically includes 1-50 mm phosphate buffer, 1-20 mm disodium EDTA, 0.01-0.5% hexadecyltrimethylethylammonium bromide, 0.1-10% dimethyl sulfoxide and 0.01-10% trifonx-100.

3. A dry cervical cancer free thiol detection test strip processing technology according to claim 2, characterized in that: The lysis buffer solution includes 1 mM phosphate buffer, 5 mM EDTA-disodium, 0.2% hexadecyltrimethylethylammonium bromide, 5% dimethyl sulfoxide and 0.5% TritonX-100.

4. A dry cervical cancer free thiol detection test strip processing technology according to claim 3, characterized in that: The concentration of DTNB is selected to be 0.5%, and the concentration of sodium tungstate is selected to be 0.5%. Sodium tungstate combines with vitamin C and uric acid in urine, so that the DTNB in ​​the detection hole is not easily oxidized and reduced.

5. A dry cervical cancer free thiol detection test strip processing technology according to claim 3, characterized in that: In step 2, the soaked absorbent paper is directly filtered and put into an oven, and dried at 45° C. to become a test paper.

6. A dry cervical cancer free thiol detection test strip processing technology according to claim 5, characterized in that: In step 4, the test strips of the detection well and the control well are extended and placed at the bottom of the sample well test strip, so that the detection test strip and the control test strip each have an extra section placed at the bottom of the sample well, so that they can be chromatographed to the detection well and the control well when the sample is added.

7. A kit corresponding to the dry cervical cancer free thiol detection test strip processing technology according to any one of claims 1 to 6, characterized in that: The test kit comprises a bottom plate and a cover plate. A card slot for installing test papers is arranged in the middle of the bottom plate, including a detection slot, a control slot and a sample addition slot, which respectively correspond to the test papers of the detection group, the control group and the sample addition group.

8. The kit corresponding to the dry cervical cancer free thiol detection test strip processing technology according to claim 7, characterized in that: The installation slots in the test kit are arranged in an inverted herringbone shape. The detection slot and the control slot are symmetrically arranged and isolated by a baffle. The sample addition slot is located below the middle of the detection slot and the control slot and is connected to the two slots through a test paper.

9. The kit corresponding to the dry cervical cancer free thiol detection test strip processing technology according to claim 8, characterized in that: The cover plate is provided with three corresponding circular holes, namely, a detection hole, a control hole and a sample addition hole, which are arranged in an inverted herringbone shape and correspond to the detection slot, the control slot and the sample addition slot of the bottom plate respectively.

10. The kit corresponding to the dry cervical cancer free thiol detection test strip processing technology according to claim 9, characterized in that: A sealed baffle is arranged between the detection slot and the control slot on the bottom plate, and a clamping slot matched with the baffle is arranged on the cover plate.

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