Dye and application thereof, syphilis detection reagent or kit comprising dye and preparation method and application of syphilis detection reagent or kit

Abstract

The invention discloses a dye and application thereof, a syphilis detection reagent or kit comprising the dye, and a preparation method and application of the syphilis detection reagent or kit. The syphilis detection reagent comprises a solution containing a composite titanium red dye and a VDRL antigen. According to the scheme, the lipid microspheres are prepared from the composite titanium red dye serving as an indicator and VDRL and used for detecting syphilis; the composite titanium red is a plastic dye and has controllable particle size and color stability, the color is not influenced by the pH value of a solution and a chemical reagent, the chromaticity is controllable, and the color is basically consistent with the color presented by toluidine red, the composite titanium red is used as an indicator for preparing the syphilis detection reagent for syphilis detection, indicator material particles are more uniform, and the syphilis detection efficiency is improved. The method has the advantages of good batch-to-batch difference control, difficult influence by sample pH, improvement of the product sensitivity, strong agglutination reaction after particle collision, easy observation of the negative and positive properties of the detection result, high sensitivity and specificity, and realization of accurate detection of syphilis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a dye and application thereof, and a syphilis detection reagent or kit comprising the dye and a preparation method and application thereof. Background Art

[0002] Syphilis was the most daunting sexually transmitted disease before the emergence of AIDS. Syphilis is a chronic infectious disease. Syphilis is a systemic infection at the beginning, with a slow course, and invades various organs and tissues of the human body during development. Syphilis can also remain dormant for many years or even for a lifetime without clinical manifestations. At present, the incidence of syphilis is increasing year by year. Therefore, it is necessary to screen out syphilis patients in an early and comprehensive manner to enable early diagnosis and treatment to reduce the spread of the disease.

[0003] The serological test for syphilis is an important part of the diagnosis of syphilis and an indispensable screening method. According to the different antigens used in the test, syphilis serological tests are divided into two categories: one is the non-syphilis treponema serological test (also known as the syphilis non-specific antibody test); the other is the syphilis treponema serological test (also known as the syphilis specific antibody test). The rapid plasma reagin test (RPR) and the toluidine red unheated serum test (TRUST) are currently the most commonly used methods for detecting non-specific antibodies for syphilis. However, RPR and TRUST may have false positives or false negatives. False positives can be affected by more than 60 physiological or pathological factors such as malignant tumors, dermatitis, cerebral infarction, systemic lupus erythematosus, old age and pregnancy, and cannot be avoided even through repeated tests. In addition, there is a false negative caused by the prozone phenomenon, and a very small number of late syphilis may also appear negative.

[0004] The detection of non-specific antibodies to syphilis in the related art mainly uses the syphilis toluidine red unheated serum test diagnostic reagent, which is a non-syphilis treponema pallidum antigen serological test. It is an improved method based on the Venereal Disease Research Laboratory Antigen (VDRL), the inactivated serum reagin test (USR) and the rapid plasma reagin test (RPR), that is, a certain proportion of cardiolipin, lecithin and cholesterol complexes are labeled into antigen liquid by toluidine red. The antigen liquid reacts with the anti-lipid antigen antibody (reagin) produced in the body of syphilis patients to produce a red flocculent agglutination reaction visible to the naked eye. The higher the reagin titer, the stronger the intensity of the agglutination reaction. After further diluting the serum in multiples, it reacts with the toluidine red antigen reagent to detect the antibody titer. However, it uses toluidine red as an indicator. The color development of toluidine red at different pH values ​​is quite different. The color will be different due to the difference in samples, and the adsorption of toluidine red is very poor. It needs to be coated with lecithin to make it color. Therefore, the detection specificity and sensitivity are poor, and a new reagent for detecting syphilis is urgently needed. Summary of the invention

[0005] The present invention aims to solve the problem of poor detection specificity and sensitivity of reagents for detecting syphilis in the prior art. To this end, the present invention proposes the use of composite titanium red dye in the preparation of an indicator for syphilis detection. By using the composite titanium red dye as a syphilis detection indicator, the defects of the toluidine red indicator can be overcome and the syphilis detection can be efficiently used.

[0006] The invention also provides a syphilis detection reagent.

[0007] The present invention also provides a kit.

[0008] The present invention also provides a method for preparing the syphilis detection reagent.

[0009] The present invention also provides an application of the syphilis detection reagent.

[0010] According to one aspect of the present invention, the use of a composite titanium red dye in preparing an indicator for syphilis detection is proposed.

[0011] In some embodiments of the present invention, the composite titanium red dye is composite titanium red.

[0012] According to a second aspect of the present invention, a syphilis detection reagent is provided, the syphilis detection reagent comprising: a solution containing lipid microspheres; the lipid microspheres comprising a composite titanium red dye and a VDRL antigen.

[0013] In some embodiments of the present invention, the VDRL antigen is prepared by dissolving cardiolipin, phosphatidylcholine and cholesterol in an organic solvent.

[0014] In some embodiments of the present invention, the lecithin includes soybean lecithin or egg yolk lecithin.

[0015] In some embodiments of the present invention, the mass addition ratio of cardiolipin, lecithin and cholesterol is 1:(2-4):(6-8).

[0016] In some embodiments of the present invention, the mass addition ratio of cardiolipin, lecithin and cholesterol is 1:3:7.

[0017] In some embodiments of the present invention, the organic solvent comprises ethanol.

[0018] In some embodiments of the present invention, the mass volume ratio of the composite titanium red dye and the VDRL antigen is (3-7):(3-5) (mg / mL).

[0019] In some embodiments of the present invention, the mass volume ratio of the composite titanium red dye and the VDRL antigen is 5:4 (mg / mL).

[0020] In some embodiments of the present invention, the mass percentage of the lipid microspheres in the solution containing lipid microspheres is 1% to 5%.

[0021] In some embodiments of the present invention, the mass percentage of the lipid microspheres in the solution containing lipid microspheres is 2% to 4%.

[0022] In some embodiments of the present invention, the mass percentage of the lipid microspheres in the solution containing lipid microspheres is 3%.

[0023] In some embodiments of the present invention, the solvent of the solution containing lipid microspheres is VDRL resuspension; the VDRL resuspension is a PBS solution containing 4-5 g / L EDTA and 140-160 g / L choline chloride.

[0024] According to a third aspect of the present invention, a kit is provided, which comprises the above-mentioned syphilis detection reagent.

[0025] In some embodiments of the present invention, the kit further comprises a negative control serum, a positive control serum, a test-specific card, and a test-specific dropper and pipette.

[0026] According to the fourth aspect of the present invention, a method for preparing the above-mentioned syphilis detection reagent is proposed, and the method comprises the following steps: adding an organic solvent and a VDRL antigen to a composite titanium red dye to mix to obtain a mixture, and drying the mixture to obtain lipid microspheres; and redissolving the lipid microspheres to obtain.

[0027] In some embodiments of the present invention, the organic solvent comprises ethanol.

[0028] In some embodiments of the present invention, the mass volume ratio of the composite titanium red dye to the organic solvent is 1:(1-3) (mg / mL).

[0029] In some embodiments of the present invention, the mass volume ratio of the composite titanium red dye and the VDRL antigen is (3-7):(3-5) (mg / mL).

[0030] In some embodiments of the present invention, the mass volume ratio of the composite titanium red dye and the VDRL antigen is 5:4 (mg / mL).

[0031] In some embodiments of the present invention, the drying method is baking.

[0032] In some embodiments of the present invention, the drying temperature is 45-55°C.

[0033] In some embodiments of the present invention, the drying process further includes the step of repeatedly mixing the mixture.

[0034] In some embodiments of the present invention, the re-dissolving solvent is a VDRL resuspension solution; the VDRL resuspension solution is a PBS solution containing 4-5 g / L EDTA and 140-160 g / L choline chloride.

[0035] According to a fifth aspect of the present invention, an application of the above-mentioned syphilis reagent is proposed, wherein the application is an application in preparing a product for detecting syphilis.

[0036] In some embodiments of the present invention, the product includes at least one of a detection plate, a test kit, and a detection chip.

[0037] In some embodiments of the present invention, the detection step comprises: using the above-mentioned syphilis detection reagent to detect the sample to be tested.

[0038] In some embodiments of the present invention, the detection specifically includes the following steps: adding the sample to be tested into a special test card; then adding the above-mentioned syphilis detection reagent into the special test card to react with the sample to be tested, thereby obtaining.

[0039] In some embodiments of the present invention, the added amount of the sample to be tested is 30-60 μL.

[0040] In some embodiments of the present invention, the amount of the syphilis detection reagent added is 16-25 μL.

[0041] In some embodiments of the present invention, the reaction conditions are 100-120 rpm and the reaction time is 6-10 min.

[0042] In some embodiments of the present invention, the application is application in drug screening, and the drug is used to treat and / or prevent syphilis.

[0043] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention uses composite titanium red dye as an indicator and VDRL antigen to prepare lipid microspheres for detecting syphilis; composite titanium red is a plastic dye with controllable particle size, color stability, and color that is not affected by solution pH and chemical reagents, and controllable chromaticity, and the color is basically consistent with the color presented by toluidine red. The present invention uses it as an indicator to prepare reagents for detecting syphilis, and for syphilis detection, the indicator material particles are more uniform, the batch difference is well controlled, and it is not easily affected by sample pH, which can better improve product sensitivity, the agglutination reaction after collision between particles is stronger, and it is easier to observe the positivity of the test results, and it has high sensitivity and specificity, and can achieve accurate detection of syphilis. DETAILED DESCRIPTION

[0044] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0045] Example 1

[0046] In this embodiment, a syphilis detection reagent is prepared. The syphilis detection reagent is a suspension containing 3% (2% to 4%) lipid microspheres.

[0047] The specific preparation process of the suspension containing 3% lipid microspheres is as follows:

[0048] 1. Preparation of VDRL antigen stock solution:

[0049] Weigh 3 mg of cardiolipin, 9 mg of soybean lecithin (or egg yolk lecithin), and 21 mg of cholesterol and dissolve them in 10 mL of anhydrous ethanol to obtain the VDRL antigen stock solution.

[0050] 2. Preparation of VDRL resuspension:

[0051] EDTA (ethylenediaminetetraacetic acid) and choline chloride were dissolved in 0.1 M PBS to make the EDTA content 4.653 g / L and the choline chloride content 150 g / L.

[0052] 3. Preparation of VDRL suspension:

[0053] (1) 5 mg of a red composite titanium red dye (also referred to as "composite titanium red dye" in the present invention) with an average particle size of about 2 μm (average particle size ≤ 2 μm) is placed in a 10 mL centrifuge tube (the red composite titanium red dye occupies about 1 mL), 9.5 mL of anhydrous ethanol is added to the centrifuge tube, and then 4 mL of VDRL antigen stock solution is added and mixed to obtain a mixture;

[0054] (2) placing the mixture in a stainless steel plate, with the thickness of the liquid spread not exceeding 5 mm, and placing the stainless steel plate in a drying oven at 50° C. for drying. During this period, the mixture needs to be repeatedly mixed so that the red composite titanium red dye is evenly coated on cardiolipin, lecithin and cholesterol, thereby forming lipid microspheres coated with the red composite titanium red dye;

[0055] (3) After the lipid microspheres are dried, a stainless steel scraper is used to collect the completely dried lipid microspheres and place them in a container, and then the lipid microspheres are re-dissolved using a VDRL resuspension solution to prepare a solution containing lipid microspheres;

[0056] (4) The solution containing lipid microspheres prepared in step (3) is centrifuged at 2000 rpm (1000-3000 rpm) for 8 min (6-10 min), and the upper liquid is retained and the precipitate is discarded. The operation is repeated 4 times to remove impurities and take an appropriate amount of VDRL to resuspend the solution so that the concentration is a suspension containing 3 wt% lipid microspheres.

[0057] Example 2

[0058] This example prepares a syphilis detection kit, which contains the syphilis detection reagent prepared in Example 1, negative control serum, positive control serum, a test-specific card, and a test-specific dropper and pipette.

[0059] The storage temperature of the reagent is: 2 ~ 8 ℃.

[0060] Example 3

[0061] The kit prepared in Example 2 was used to detect syphilis, and the specific method was the paper strip method. The experiment was as follows:

[0062] (1) Take 50 μL of the gradient dilutions of the negative control and syphilis positive control (original, 2×, 4×, 8×, 16×, 32×) and evenly spread them in the circle on the paper card.

[0063] (2) In the experimental group, one drop (about 20.25 μL) of the syphilis detection reagent prepared in Example 1 was vertically dripped into the circle on the above paper using a special dropper and a pipette.

[0064] For the control group, one drop of TRUST reagent (about 20.25 μL) was vertically dropped into the circle on the above paper using a special dropper and pipette.

[0065] (3) Shake at 115 rpm for 8 min and observe the results with the naked eye.

[0066] Result judgment:

[0067] 1. Positive reaction (+++(3+) to ++++(4+)): Medium or large red agglutinates are visible.

[0068] 2. Weak positive reaction (+(1+)~++(2+)): Smaller red agglutinates can be seen.

[0069] 3. Negative reaction (-): Uniform antigen particles are visible without agglutination.

[0070] Table 1

[0071]

[0072]

[0073] The results are shown in Table 1. It can be seen from Table 1 that the sensitivity of the syphilis detection reagent prepared by the scheme of the present invention is better than the existing reagent of the control group.

[0074] Example 4

[0075] In this example, a kit containing a suspension of lipid microspheres of different concentrations (1%, 2%, 3%, 4%, 5%) was used to detect syphilis. The lipid microsphere preparation method was the same as that in Example 1. The specific experiment was as follows:

[0076] (1) Pipette 50 μL of the gradient dilutions of the negative control and syphilis positive control (original, 2×, 4×, 8×, 16×, 32×) and evenly spread them in the circles on the paper card.

[0077] (2) Use a special dropper and pipette to vertically add one drop (about 20.25 μL) of the syphilis detection reagent of the kit containing a suspension of lipid microspheres of different concentrations (1%, 2%, 3%, 4%, 5%) into the circle on the above paper card.

[0078] (3) Shake at 115 rpm for 8 min and observe the results with the naked eye.

[0079] Result judgment:

[0080] 1. Positive reaction (+++(3+) to ++++(4+)): Medium or large red agglutinates are visible.

[0081] 2. Weak positive reaction (+(1+)~++(2+)): Smaller red agglutinates can be seen.

[0082] 3. Negative reaction (-): Uniform antigen particles are visible without agglutination.

[0083] Table 2

[0084] Group Positive Control Original times 2 times 4 times 8 times 16 times 32 times Negative control 1％ 4+ 3+ 3+ 3+ 3+ 2+ 1+ - 2％ 4+ 3+ 4+ 4+ 3+ 2+ 1+ - 3％ 4+ 3+ 4+ 4+ 3+ 2+ 1+ - 4％ 4+ 3+ 4+ 4+ 3+ 2+ 1+ - 5％ 4+ 3+ 4+ 3+ 2+ 1+ - -

[0085] The results are shown in Table 2. It can be seen from Table 2 that the sensitivity of the syphilis detection reagent prepared by the scheme of the present invention is the best when the reagent contains 2% to 4% lipid microspheres.

[0086] Example 5

[0087] This example uses a reagent containing a suspension of lipid microspheres with different mass volume ratios (3:4, 4:4, 5:4, 6:4, 7:4) of composite titanium red dye and VDRL antigen to detect syphilis. The preparation method of lipid microspheres is the same as that of Example 1. The specific experiment is as follows:

[0088] (1) Pipette 50 μL of the gradient dilutions of the negative control and syphilis positive control (original, 2×, 4×, 8×, 16×, 32×) and evenly spread them in the circles on the paper card.

[0089] (2) Use a special dropper and pipette to vertically add one drop (about 20.25 μL) of the syphilis detection reagent of the kit containing a suspension of lipid microspheres with different mass volume ratios (3:4, 4:4, 5:4, 6:4, 7:4) of composite titanium red dye and VDRL antigen into the circle on the above paper card.

[0090] (3) Shake at 115 rpm for 8 min and observe the results with the naked eye.

[0091] Result judgment:

[0092] 1. Positive reaction (+++(3+) to ++++(4+)): Medium or large red agglutinates are visible.

[0093] 2. Weak positive reaction (+(1+)~++(2+)): Smaller red agglutinates can be seen.

[0094] 3. Negative reaction (-): Uniform antigen particles are visible without agglutination.

[0095] Table 3

[0096] Group Positive Control Original times 2 times 4 times 8 times 16 times 32 times Negative control 3:4 4+ 3+ 4+ 3+ 1+ 1+ - - 4:4 4+ 3+ 4+ 3+ 1+ 1+ - - 5:4 4+ 3+ 4+ 4+ 3+ 2+ 1+ - 6:4 4+ 3+ 4+ 3+ 1+ 1+ - - 7:4 4+ 3+ 4+ 3+ 1+ 1+ - -

[0097] The results are shown in Table 3. It can be seen from Table 3 that the sensitivity of the syphilis detection reagent prepared by the scheme of the present invention is the best when the reagent contains lipid microspheres with a mass volume ratio of composite titanium red dye and VDRL antigen of 5:4.

[0098] Example 6

[0099] The syphilis detection kit prepared in Example 2 was used for sensitivity testing, and the specific method was the paper strip method. The experiment was as follows:

[0100] Acquisition of biological samples: A total of 140 samples were used in this embodiment, including 10 primary syphilis sera, 40 secondary syphilis sera, 10 latent syphilis sera, and 80 negative controls.

[0101] (1) Pipette 50 μL of negative control, primary syphilis serum, secondary syphilis serum and latent syphilis serum respectively and spread them evenly in the circles on the paper card.

[0102] (2) In the experimental group, one drop (about 20.25 μL) of the syphilis detection reagent prepared in Example 1 was vertically added to the above circles using a special dropper and a pipette.

[0103] For the control group, one drop of TRUST reagent (about 20.25 μL) was dripped vertically into the above circles using a special dropper and needle.

[0104] (3) Shake at 115 rpm for 8 min and observe the results with the naked eye.

[0105] Result judgment:

[0106] 1. Positive reaction (+++(3+) to ++++(4+)): Medium or large red agglutinates are visible.

[0107] 2. Weak positive reaction (+(1+)~++(2+)): Smaller red agglutinates can be seen.

[0108] 3. Negative reaction (-): Uniform antigen particles are visible without agglutination.

[0109] Table 4

[0110] Group Negative control (number of cases) Phase I (number of cases) Phase II (number of cases) Incubation period (number of cases) Control group 82 9 40 9 Experimental Group 80 10 40 10

[0111] The results are shown in Table 4. It can be seen from Table 4 that the sensitivity of the syphilis detection reagent prepared by the scheme of the present invention is better than that of the control commercially available product.

[0112] Example 7

[0113] In this example, a kit containing suspensions of lipid microspheres of different concentrations (1%, 2%, 3%, 4%, 5%) was used for sensitivity testing. The preparation method of the lipid microspheres was the same as that in Example 1, and the specific method was the paper disc method. The experiment was as follows:

[0114] Acquisition of biological samples: A total of 140 samples were used in this embodiment, including 10 primary syphilis sera, 40 secondary syphilis sera, 10 latent syphilis sera, and 80 negative controls.

[0115] (1) Pipette 50 μL of negative control, primary syphilis serum, secondary syphilis serum and latent syphilis serum respectively and spread them evenly in the circles on the paper card.

[0116] (2) Use a special dropper and pipette to vertically add one drop (about 20.25 μL) of the detection reagent containing a suspension of lipid microspheres of different concentrations (1%, 2%, 3%, 4%, 5%) into the above circles.

[0117] (3) Shake at 115 rpm for 8 min and observe the results with the naked eye.

[0118] Result judgment:

[0119] 1. Positive reaction (+++(3+) to ++++(4+)): Medium or large red agglutinates are visible.

[0120] 2. Weak positive reaction (+(1+)~++(2+)): Smaller red agglutinates can be seen.

[0121] 3. Negative reaction (-): Uniform antigen particles are visible without agglutination.

[0122] Table 5

[0123] Group Negative control (number of cases) Phase I (number of cases) Phase II (number of cases) Incubation period (number of cases) 1％ 75 14 40 11 2％ 82 9 40 9 3％ 80 10 40 10 4％ 82 9 40 9 5％ 83 8 40 9

[0124] The results are shown in Table 5. It can be seen from Table 5 that the sensitivity of the syphilis detection reagent prepared by the scheme of the present invention is the best when the reagent contains 3% lipid microspheres, and the detection effect on the negative control, the first stage, the second stage and the latent period is the most stable.

[0125] Example 8

[0126] This example uses a kit containing a suspension of lipid microspheres with different mass volume ratios (3:4, 4:4, 5:4, 6:4, 7:4) of composite titanium red dye and VDRL antigen for sensitivity detection. The preparation method of lipid microspheres is the same as that of Example 1, and the specific method is the paper strip method. The experiment is as follows:

[0127] Acquisition of biological samples: A total of 140 samples were used in this embodiment, including 10 primary syphilis sera, 40 secondary syphilis sera, 10 latent syphilis sera, and 80 negative controls.

[0128] (1) Pipette 50 μL of negative control, primary syphilis serum, secondary syphilis serum and latent syphilis serum respectively and spread them evenly in the circles on the paper card.

[0129] (2) Use a special dropper and pipette to vertically add one drop (about 20.25 μL) of the detection reagent containing a suspension of lipid microspheres with different mass volume ratios (3:4, 4:4, 5:4, 6:4, 7:4) of composite titanium red dye and VDRL antigen into the above circles.

[0130] (3) Shake at 115 rpm for 8 min and observe the results with the naked eye.

[0131] Result judgment:

[0132] 1. Positive reaction (+++(3+) to ++++(4+)): Medium or large red agglutinates are visible.

[0133] 2. Weak positive reaction (+(1+)~++(2+)): Smaller red agglutinates can be seen.

[0134] 3. Negative reaction (-): Uniform antigen particles are visible without agglutination.

[0135] Table 6

[0136] Group Negative control (number of cases) Phase I (number of cases) Phase II (number of cases) Incubation period (number of cases) 3:4 83 8 40 9 4:4 82 9 40 9 5:4 80 10 40 10 6:4 82 9 40 9 7:4 84 8 39 9

[0137] The results are shown in Table 6. It can be seen from Table 6 that the sensitivity of the syphilis detection reagent prepared by the scheme of the present invention is optimal when the reagent contains lipid microspheres with a mass volume ratio of composite titanium red dye and VDRL antigen of 5:4.

[0138] Example 9

[0139] The kit prepared in Example 2 was used for specific detection.

[0140] Specific test samples include: serum and plasma samples are suitable for this test. The best selected samples should be clear and transparent, without particulate matter, so as not to interfere with the result judgment. Serum and plasma samples with bacterial contamination, hemolysis, chyle or turbidity may affect the test results.

[0141] Sample collection requirements:

[0142] Serum: According to the test requirements, collect 3-5 mL of venous blood in a clean vacuum non-anticoagulant or procoagulant test tube.

[0143] Plasma: According to the test requirements, collect 3-5 mL of venous blood in a clean vacuum anticoagulant tube. The anticoagulant can be the commonly used EDTA, heparin and oxalic acid anticoagulant.

[0144] Experimental methods:

[0145] 1. All samples, reagents, reference materials, and quality control materials taken out from a low-temperature environment should be placed at room temperature for at least 30 minutes, and experiments can only be performed after they are restored to room temperature.

[0146] 2. Pipette 50 μL of the serum sample to be tested, reference substance and quality control substance respectively, and place each sample in a circle on the card without overflowing the circle.

[0147] 3. Apply the sample with the angle between the pipette tip and the card close to 30°, filling the entire reaction circle with the sample. Be careful to avoid the pipette tip scratching the waterproof coating on the surface of the card.

[0148] 4. Before adding the suspension containing 3% lipid microspheres (the syphilis detection reagent prepared in Example 1), rotate the reagent bottle horizontally to mix the suspension containing 3% lipid microspheres. Use a dropper to blow and suck gently and repeatedly for several times, but not violently, until it is fully suspended and sucked into the dropper.

[0149] 5. Keep the syringe vertical when adding the suspension containing 3% lipid microspheres. Discard the first drop of reagent in the syringe in a free-falling manner, and add 1 drop (17 μL) of antigen to each sample circle starting from the second drop.

[0150] 6. After adding the suspension containing 3% lipid microspheres, pick up the card immediately, tilt it about 30 degrees and rotate it twice to fully mix the syphilis test reagent and the sample.

[0151] 7. For qualitative observation, if any agglutination reaction occurs in the sample, the sample should be diluted with saline in multiple ratios and the operation steps 1 to 6 should be performed.

[0152] Experimental results:

[0153] 1) Negative and positive compliance rate, total compliance rate and 95% confidence interval:

[0154] Positive coincidence rate = 58 / 60 × 100% = 96.67%, 95% CI: [99.22%, 100.00%];

[0155] Negative coincidence rate = 80 / 80 × 100% = 100%, 5% CI: [97.01%, 99.54%];

[0156] Overall compliance rate = (58 + 80) / 140 × 100% = 98.57%, 95% CI: [98.76%, 99.81%].

[0157] Kappa consistency analysis of the above data showed K=0.98997, indicating that the test results of the experimental group and the control group were very consistent; P<0.05, indicating that the consistency test between the two was statistically significant. The results showed that the syphilis detection kit prepared by the scheme of the present invention can be accurately used for syphilis detection and has good specificity.

[0158] In summary, the scheme of the present invention has been screened and verified to use composite titanium red instead of toluidine red for syphilis detection. Composite titanium red is a plastic dye with controllable particle size and color stability. The color is not affected by the pH value of the solution and chemical reagents, and the chromaticity is controllable. The color is basically consistent with the color presented by toluidine red.

[0159] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of composite titanium red dye in the preparation of indicators for syphilis detection.

2. A syphilis detection reagent, characterized in that: The syphilis detection reagent comprises: a solution containing lipid microspheres; the lipid microspheres comprise a composite titanium red dye and a VDRL antigen.

3. The syphilis detection reagent according to claim 2, characterized in that: The VDRL antigen is prepared by dissolving cardiolipin, phosphatidylcholine and cholesterol in an organic solvent.

4. The syphilis detection reagent according to claim 2, characterized in that: The mass addition ratio of cardiolipin, lecithin and cholesterol is 1:(2-4):(6-8).

5. The syphilis detection reagent according to claim 1, characterized in that: The mass percentage of the lipid microspheres in the solution containing the lipid microspheres is 1% to 5%.

6. A kit, characterized in that: The kit comprises the syphilis detection reagent according to any one of claims 2 to 5.

7. A method for preparing a syphilis detection reagent according to any one of claims 2 to 5, characterized in that: The method comprises the following steps: adding an organic solvent and a VDRL antigen to a composite titanium red dye to obtain a mixture, drying the mixture to obtain lipid microspheres; and redissolving the lipid microspheres to obtain the product.

8. The preparation method according to claim 7, characterized in that: The organic solvent includes ethanol; And / or, the re-dissolving solvent is a VDRL resuspension solution; the VDRL resuspension solution is a PBS solution containing 4-5 g / L EDTA and 140-160 g / L choline chloride.

9. Use of the syphilis detection reagent according to any one of claims 2 to 5 in preparing products for detecting syphilis.

10. The use according to claim 9, characterized in that: The product includes at least one of a detection plate, a test kit, and a detection chip.

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