Detection reagent and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a detection reagent and application thereof. The invention provides a detection reagent which comprises the following components: 10 to 200 mM of a buffer solution, 5 to 20 g / L of polyvinylpyrrolidone K30 or K90, 0.5 to 10 g / L of 5-bromo-4-chloro-3-indolyl-alpha-D-N-acetylneuraminic acid sodium salt or 5-bromo-4-chloro-3-indolyl-alpha-D-N-acetylneuraminic acid, and 0.1 to 5 g / L of diazonium salt. According to the sialidase detection test paper provided by the invention, after a sample is dropwise added into the test paper, direct color development can be realized, an independently packaged joint detection color development solution is not needed, and the test paper is simple and convenient to operate and easy to popularize.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to detection reagents and application thereof. BACKGROUND

[0002] Vaginitis is inflammation of the mucosa and submucosal connective tissue of the vagina, and is a common disease in gynecological clinics. Due to the anatomical and biochemical characteristics, the vagina has a natural defense function against pathogen invasion. When the natural defense function of the vagina is destroyed, pathogenic bacteria are easy to invade, leading to inflammation of the vagina.

[0003] Common vaginitis includes bacterial vaginitis (BV / AV), fungal vaginitis (VVC) and trichomonas vaginitis (TV). Among them, female bacterial vaginitis can be divided into two types, one is caused by anaerobic bacteria and facultative anaerobic bacteria, clinically known as bacterial vaginosis (BV), and the other is caused by aerobic bacteria, clinically known as aerobic vaginitis (AV). The pathogen of the two types of bacterial vaginitis is different, and the clinical symptoms, treatment plan and outcome are also different. It is of great significance to distinguish the two types of bacterial vaginitis in the diagnosis and treatment of vaginal infection.

[0004] Sialidase, also known as neuraminidase, is an enzyme protein present in lysosomes, cytoplasm and cell membrane. The enzyme activity is similar to the virulence factor, which can hydrolyze various substrates with non-reducing terminal sialic acid, such as glycoprotein, glycolipid, ganglioside and polysaccharide, destroy mucin, enhance bacterial adhesion, and resist other virulence factors by damaging the immunoreactivity of IgA. Sialidase is produced by anaerobic bacteria such as Prevotella and Bacteroides in vaginal flora, and its activity is considered to be positively correlated with the level of BV infection. The activity of sialidase in BV (1.35±1.87 nmol / mL min) is significantly higher than that in women without BV (0.03±0.14 nmol / mL min).

[0005] At present, most of the detection test papers for detecting sialidase are two-step methods, and the general operation method is as follows: a certain amount of sample is dropped on the test paper, and after incubation at 37±1℃ for 1 minute, a drop of color developing solution is added, and after 9 minutes, the result is judged by comparing with the color scale within 1 minute. The operation needs to add the color developing solution at a specific time, which has the limitation of complicated operation. The detection principle of this method is that the sialidase substrate is artificially synthesized, and the different colors displayed after the enzyme and the substrate react are used to diagnose BV. In view of the problems in the prior art, the present application provides a sialidase detection test paper, which can directly develop color after dropping the sample, without additional drop of color developing solution, and the operation is simple and convenient, and is suitable for promotion. SUMMARY

[0006] Therefore, the application provides a detection reagent and application thereof.

[0007] In order to achieve the above-mentioned purposes, the application provides the following technical solutions.

[0008] The application provides a detection reagent, comprising:

[0009]

[0010] In some embodiments of the application, the diazonium salt comprises 4-methoxybenzenediazonium tetrafluoroborate.

[0011] In some embodiments of the application, the concentration of the diazonium salt is 0.1 g / L, 0.5 g / L or 5 g / L.

[0012] In some embodiments of the application, the concentration of the polyvinylpyrrolidone K30 is 5 g / L, 10 g / L or 20 g / L.

[0013] In some embodiments of the application, the concentration of the 5-bromo-4-chloro-3-indolyl-alpha-D-N-acetylneuraminic acid sodium salt is 0.5 g / L, 1 g / L or 10 g / L.

[0014] In some embodiments of the application, the concentration of the 5-bromo-4-chloro-3-indolyl-alpha-D-N-acetylneuraminic acid is 1 g / L.

[0015] In some embodiments of the application, the buffer comprises any one of a citric acid-sodium citrate buffer solution, an acetic acid-sodium acetate buffer solution, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution and a borax-boric acid buffer solution.

[0016] In some embodiments of the application, the buffer is a 10 mM or 100 mM citric acid-sodium citrate buffer solution or a 200 mM borax-boric acid buffer solution.

[0017] In some embodiments of the application, the pH value of the buffer is 4.0-8.0.

[0018] The application also provides application of the detection reagent in preparation of a product for detecting sialidase by one-step method.

[0019] In some embodiments of the application, the one-step method does not comprise a step of adding a color developing solution.

[0020] In some embodiments of the present invention, the above-described application includes the following steps: adding the sample to be tested to the product, allowing it to stand, and obtaining the test result.

[0021] In some embodiments of the present invention, in the above applications, the settling temperature is 36-38°C and the time is 10 minutes.

[0022] The present invention also provides a sialidase test strip, comprising: a reaction pad coated with the above-mentioned detection reagent.

[0023] The present invention also provides a method for preparing the above-mentioned sialylase test strip, wherein the test reagent is coated onto a reaction pad, dried, and assembled to obtain the sialylase test strip.

[0024] In some embodiments of the present invention, in the above preparation method, the drying time is 20-25 min and the temperature is 50-60°C.

[0025] The present invention also provides a method for detecting sialidase, comprising the following steps:

[0026] S1: Obtain the sample solution;

[0027] S2: Add the sample solution to the above-mentioned sialylase test strip or the sialylase test strip prepared by the above method;

[0028] S3: Incubate at 36-38℃ for 10 minutes, and compare the result with the color mark within 1 minute.

[0029] This invention provides a detection reagent, comprising:

[0030]

[0031] This invention provides a sialidase test strip. The test strip can directly develop color after the sample is added, without the need for additional color developing solution. It is easy to operate and suitable for widespread application. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0033] Figure 1 The colorimetric comparison between the one-step test strip prepared in Example 2 and a commercially available two-step product is shown; where: the left side shows the result of Example 2; the right side shows the result of the two-step comparison product.

[0034] Figure 2The color development comparison between the one-step test strip prepared in Example 3 and the commercially available two-step product is shown; the left side shows the result of Example 3; the right side shows the result of the two-step comparison product.

[0035] Figure 3 The left side shows the color development comparison between the one-step test strip prepared in Example 4 and the commercially available two-step product; the left side shows the result of Example 3; the right side shows the result of the two-step comparison product.

[0036] Figure 4 The left side shows the color development comparison between the one-step test strip prepared in Example 5 and a commercially available two-step product; the left side shows the result of Example 3; the right side shows the result of the two-step comparison product.

[0037] Figure 5 The color development effects of different diazonium salts are shown; from left to right: 4-methoxyphenyldiazotetrafluoroborate, Solid Blue B salt, Solid Red B salt, and Solid Violet B salt.

[0038] Figure 6 The color development effect is shown under different pH conditions; from left to right: pH3, pH4, pH5, pH6, pH7, pH8, pH9. Detailed Implementation

[0039] This invention discloses the detection reagents and their applications.

[0040] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0041] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0042] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0043] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0044] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0045] This invention provides a one-step detection dry chemistry test strip for sialidase and its preparation method, the formula of which is as follows:

[0046] Table 1

[0047]

[0048] A one-step method for preparing and detecting sialidase using dry chemical test strips includes the following steps:

[0049] Step 1: Immerse the filter paper completely in the soaking solution and dry it at 50-60℃ for 20-25 minutes;

[0050] Step 2: Cut the dried filter paper and fix it with the substrate. After rolling and cutting, prepare a one-step sialidase test strip.

[0051] Step 3: Add the sample to the test strip without adding the colorimetric reagent. The result can be read in 10 minutes. This invention features simple reagent preparation and convenient use, eliminating the need for external colorimetric reagent and achieving a one-step operation for sialidase, making it suitable for widespread application.

[0052] In Examples 1 to 7 and the comparative examples of this invention, all raw materials and reagents used can be purchased from the market.

[0053] The present invention will be further illustrated below with reference to the embodiments:

[0054] Example 1

[0055] Preparation of the infusion solution: 1000mL purified water, 10mM citric acid-sodium citrate buffer solution (pH4), 5g polyvinylpyrrolidone K30, 0.5g 5-bromo-4-chloro-3-indolyl-α-DN-acetylneuraminic acid sodium salt, 0.1g 4-methoxyphenyldiazotetrafluoroborate.

[0056] A method for preparing a one-step dry chemical test strip for detecting sialidase includes the following steps:

[0057] Step 1: Immerse the filter paper completely in the soaking solution and dry it at 50-60℃ for 20-25 minutes;

[0058] Step 2: Cut the dried filter paper and fix it with the substrate. After rolling and cutting, prepare a one-step sialidase test strip.

[0059] Performance Testing: Comparison with commercially available two-step method products (manufacturer: AVE Technology Co., Ltd.; product name: Vaginitis Combined Detection Kit (Dry Chemical Enzymatic Method), model AVE-11A). Sialidase standard solutions with different enzyme activities were prepared and diluted with physiological saline.

[0060] The detection method for commercially available two-step products is as follows: Take 10 μL of standard solution and add it to the dry chemical test paper of the commercially available two-step product. After incubating at 37±1℃ for 1 minute, add one drop of colorimetric solution. After 9 minutes, compare the result with the color standard within 1 minute to interpret the result.

[0061] The detection method of the one-step product of this invention is as follows: take 10 μL of standard solution and add it to the sialidase detection dry chemical test paper obtained in step two, incubate at 37±1℃ for 10 minutes, and compare the result with the color standard within 1 minute.

[0062] A light red, red, or dark red color indicates a positive result; no color development indicates a negative result. - indicates a negative result, ± indicates a weak positive result, and + indicates a positive result.

[0063] Table 2

[0064] Standard liquid enzyme activity (U / mL) Example 1 Comparative product 0 - - 0.02 ± ± 0.1 + +

[0065] The results of Example 1 show that both the one-step and two-step test strips can detect sialidase. Sialidase causes the test strip to turn pink, and the pink color deepens with increasing sialidase concentration. The one-step test strip in Example 1 shows more uniform color development and a more obvious color gradient than the two-step test strip, and it does not require an external colorimetric reagent, making it convenient to use and suitable for widespread application.

[0066] Example 2

[0067] Preparation of the infusion solution: 1000mL purified water, 200mM borax-boric acid buffer solution (pH 8), 20g polyvinylpyrrolidone K30, 10g 5-bromo-4-chloro-3-indolyl-α-DN-acetylneuraminic acid sodium salt, and 5.0g 4-methoxyphenyldiazotetrafluoroborate.

[0068] The test strip preparation method and performance testing method are the same as in Example 1.

[0069] Table 3

[0070] Standard liquid enzyme activity (U / mL) Example 2 Comparative product 0 - - 0.02 ± ± 0.1 + +

[0071] The results of Example 2 show that both the one-step and two-step test strips can detect sialidase. Sialidase causes the test strip to turn pink, and the pink color deepens with increasing sialidase concentration. The one-step test strip in Example 2 shows more uniform color development and a more obvious color gradient than the two-step test strip, and it does not require an external colorimetric reagent, making it convenient to use and suitable for widespread application.

[0072] Example 3

[0073] Preparation of the infusion solution: 1000mL purified water, 100mM citric acid-sodium citrate buffer solution (pH 6), 10g polyvinylpyrrolidone K30, 1g 5-bromo-4-chloro-3-indolyl-α-DN-acetylneuraminic acid sodium salt, 0.5g 4-methoxyphenyldiazotetrafluoroborate.

[0074] The test strip preparation method and performance testing method are the same as in Example 1.

[0075] Table 4

[0076] Standard liquid enzyme activity (U / mL) Example 3 Comparative product 0 - - 0.02 ± ± 0.1 + +

[0077] The results of Example 3 show that both the one-step and two-step test strips can detect sialidase. Sialidase causes the test strip to turn pink, and the pink color deepens with increasing sialidase concentration. The one-step test strip in Example 3 shows more uniform color development and a more obvious color gradient than the two-step test strip, and it does not require an external colorimetric reagent, making it convenient to use and worthy of promotion.

[0078] Example 4

[0079] Preparation of the infusion solution: 1000mL purified water, 100mM citric acid-sodium citrate buffer solution (pH 6), 10g polyvinylpyrrolidone K30, 1g 5-bromo-4-chloro-3-indolyl-α-DN-acetylneuraminic acid, 0.5g 4-methoxyphenyldiazotetrafluoroborate.

[0080] The test strip preparation method and performance testing method are the same as in Example 1.

[0081] Table 5

[0082] Standard liquid enzyme activity (U / mL) Example 4 Comparative product 0 - - 0.02 ± ± 0.1 + +

[0083] The results of Example 4 show that both the one-step and two-step test strips can detect sialidase. Sialidase causes the test strip to turn pink, and the pink color deepens with increasing sialidase concentration. The one-step test strip in Example 4 shows more uniform color development and a more obvious color gradient than the two-step test strip, and it does not require an external colorimetric reagent, making it convenient to use and suitable for widespread application.

[0084] Example 5

[0085] Preparation of the infusion solution: 1000mL purified water, 100mM acetate-sodium acetate buffer solution (pH 6), 10g polyvinylpyrrolidone K90, 8g 5-bromo-4-chloro-3-indolyl-α-DN-acetylneuraminic acid, 5g 4-methoxyphenyldiazotetrafluoroborate.

[0086] The test strip preparation method and performance testing method are the same as in Example 1.

[0087] Table 6

[0088] Standard liquid enzyme activity (U / mL) Example 5 Comparative product 0 - - 0.02 ± ± 0.1 + +

[0089] The results of Example 5 show that both the one-step and two-step test strips can detect sialidase. Sialidase causes the test strip to turn pink, and the pink color deepens with increasing sialidase concentration. The one-step test strip in Example 5 shows more uniform color development and a more obvious color gradient than the two-step test strip, and it does not require an external colorimetric reagent, making it convenient to use and suitable for widespread application.

[0090] Example 6

[0091] Preparation of the infusion solution: 1000mL purified water, 200mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 6), 10g polyvinylpyrrolidone K90, 5g 5-bromo-4-chloro-3-indolyl-α-DN-acetylneuraminic acid, 2g 4-methoxyphenyldiazotetrafluoroborate.

[0092] The test strip preparation method and performance testing method are the same as in Example 1.

[0093] Table 7

[0094] Standard liquid enzyme activity (U / mL) Example 6 Comparative product 0 - - 0.02 ± ± 0.1 + +

[0095] The results of Example 6 show that both the one-step and two-step test strips can detect sialidase. Sialidase causes the test strip to turn pink, and the pink color deepens with increasing sialidase concentration. The one-step test strip in Example 6 shows more uniform color development and a more obvious color gradient than the two-step test strip, and it does not require an external colorimetric reagent, making it convenient to use and suitable for widespread application.

[0096] Example 7

[0097] The one-step sialidase test strips obtained in Examples 1-6 and commercially available two-step products were used to test 20 clinical vaginal secretion samples. Vaginal secretion samples were collected using sterile flocked swabs and placed in soft test tubes. 1.0 mL of cell preservation solution was added to each sample, and the swabs were repeatedly rinsed to ensure that the sample on the swabs was fully eluted, thus obtaining the sample solution.

[0098] The performance testing method is the same as in Example 1. The test results are as follows:

[0099] Table 8

[0100]

[0101]

[0102] The results of Example 7 show that both the one-step and two-step test strips can detect sialidase in vaginal secretion samples. Sialidase causes the test strip to turn pink, and the pink color deepens with increasing sialidase concentration. The one-step test strip in Example 7 shows more uniform color development and a more obvious color gradient than the two-step test strip, and it does not require an external colorimetric reagent, making it convenient to use and suitable for widespread application.

[0103] Example 8: One-step method for testing the stability of test strips

[0104] The one-step sialidase test strip obtained in Example 1 was stored in a sealed refrigerator at 2-8℃. The color development was tested monthly using sialidase standard solution, and the results are shown in Table 9.

[0105] Table 9

[0106]

[0107]

[0108] The results of Example 8 show that the one-step sialidase test strip prepared in this invention can be stably stored for 12 months at 2-8℃.

[0109] Comparative Example

[0110] Existing sialidase test strips require a two-step process. The specific procedure is as follows: Take a diluted sample, add one drop to the test strip, incubate at 37±1℃ for 1 minute, then add one drop of chromogenic solution. After 9 minutes, compare the result with the color standard within 1 minute to interpret the result. The color change of the sialidase test strip can be observed; a change from white to pink indicates a positive result for sialidase. The chromogenic solution is generally a diazonium salt.

[0111] The reason why conventional sialic anhydrase test strips are made using a two-step method instead of a one-step method is that a one-step method requires fixing both the substrate and the chromogenic agent onto a single strip, which could lead to incompatibility between the substrate and the chromogenic agent. Furthermore, the fixing process requires heating and drying at 50–60°C, at which temperature diazonium salt chromogenic agents are prone to deactivation, resulting in test strips that fail to develop color or cannot be stably stored.

[0112] Therefore, it is necessary to screen various colorimetric agents. Several commonly used diazonium salts were tested, such as: 1) 4-methoxyphenyldiazotetrafluoroborate; 2) Solid Blue B salt; 3) Solid Blue BB; and 4) Solid Violet B.

[0113] Only 4-methoxyphenyldiazotetrafluoroborate showed a normal gradient, and its colorimetric results were as follows: Figure 5 As shown:

[0114] In addition, the pH of the buffer solution in the formulation also needs to be controlled. A pH that is too low or too high will result in poor or no color development. The suitable pH range is 4–8. Color development effects under different pH conditions are shown below. Figure 6 As shown.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A detection reagent, characterized in that, include:

2. The detection reagent as described in claim 1, characterized in that, The diazonium salt includes: 4-methoxyphenyldiazotetrafluoroborate.

3. The detection reagent as described in claim 1 or 2, characterized in that, The buffer solution includes any one of the following: citrate-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and borax-boric acid buffer solution.

4. The detection reagent according to any one of claims 1 to 3, characterized in that, The pH value of the buffer solution is 4.0 to 8.

0.

5. The use of the detection reagent according to any one of claims 1 to 4 in the preparation of a product for one-step detection of sialidase.

6. The application as described in claim 5, characterized in that, The detection includes the following steps: adding the sample to be tested to the product, letting it stand, and obtaining the test results.

7. A sialidase test strip, characterized in that, include: The reaction pad is coated with the detection reagent as described in any one of claims 1 to 4.

8. The method for preparing the sialidase test strip as described in claim 7, characterized in that, The test reagent is coated onto the reaction pad, dried, and assembled to obtain the sialidase test strip.

9. The preparation method according to claim 8, characterized in that, The drying time is 20-25 minutes, and the temperature is 50-60℃.

10. A method for detecting sialidase, characterized in that, Includes the following steps: S1: Obtain the sample solution; S2: Add the sample solution to the sialyl sialidase test strip as described in claim 7 or the sialyl sialidase test strip prepared by the method described in claim 8 or 9; S3: Incubate at 36-38℃ for 10 minutes, and compare the result with the color mark within 1 minute.

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